Production and Biological Activities of Exopolysaccharides Synthesized by Thermophilic Bacilli Isolated from Hot Springs in Türkiye

Production and Biological Activities of Exopolysaccharides Synthesized by Thermophilic Bacilli Isolated from Hot Springs in Türkiye | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Production and Biological Activities of Exopolysaccharides Synthesized by Thermophilic Bacilli Isolated from Hot Springs in Türkiye Mehmet Aytar, Deniz Aktaş Uygun, Gamze Başbülbül This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4679730/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Sep, 2024 Read the published version in International Microbiology → Version 1 posted 9 You are reading this latest preprint version Abstract A total of one hundred seven thermophilic bacteria were screened for their ability to produce exopolysaccharides. Nine isolates belonging to Geobacillus , Parageobacillus , Aeribacillus and Anoxybacillus genera with highest EPS quantities were chosen and purified EPSs used for biological activity studies. EPS yields of selected thermophilic bacteria ranged between 117–419 mg/L. Among the tested EPSs, 61, 106 and 261 showed antibacterial effect against E. faecalis JH2-2 at a concentration of 15 mg/mL. EPS samples had significant antioxidant capacity, especially EPS 134, with highest DPPH radical scavenging activity of 100% at a concentration of 5 mg/mL and strongest reducing power. EPS 20, showed highest lipid peroxidation inhibition effect at a rate of 31%. EPSs displayed weak alpha amylase inhibition activity when compared with standart acarbose. The prebiotic indices of EPSs 20, 61, 76, 89, 134 and 261 were found to be higher than that of inulin, a representative prebiotic carbohydrate for all tested lactic acid bacteria in the study. All examined EPSs inhibited the biofilms formed by various bacteria depending on the test strain. Results indicated that thermophilic EPSs had remarkable antioxidant, prebiotic, and antibiofilm activities. Therefore, EPSs characterized in this study may have technological applications in health and food fields. Thermophilic Bacteria Exopolysaccharide Purification Biological Activity Characterization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Microbial exopolysaccharides (EPSs) are high molecular weight polymers produced by microalgae, yeast, mold and bacteria. They can accumulate outside the cell as capsular exopolysaccharides or secreted to the environment (Karadayi et al. 2021 ). EPSs promote adhesion to surfaces as well as formation of cellular aggregates and biofilm (Nwodo et al., 2012 ). In addition to these functions, they protect cells against microbial predators, as well as provide the host cell with advantages in drought and nutrient starved environmental conditions. Their molecular weights are ranging from 100–2000 kDa (Suresh Kumar et al. 2007 ). EPSs are classified into two groups either homopolysaccharides (HoPS) or heteropolysaccharides (HePS) depending on the composition of the repeating units (Mende et al., 2016 ). Homopolysaccharides (HoPS) are composed of a single type of monosaccharide while heteropolysaccharides (HePS) are composed of either linear or branched repeating units (Ruas-Madiedo et al., 2002 ). Pentoses like D-arabinose, D-ribose, D-xylose, hexoses (i.e. D-fructose, D-glucose, D-mannose and D-galactose), deoxy-hexoses (L-rhamnose and L-fucose), uronic acids (D-glucuronic acid, and D-galacturonic acid), D-glucosamine and D-galactosamine, which are known as amino sugars, could be structural monomeric units of exopolysaccharides (Joulak et al. 2019 ). In a number of instances, EPS may contain non-carbohydrate moieties like proteins, nucleic acids, fats, phosphates, sulfates, lactates attached to the polysaccharide backbone chain. Depending on the monomeric ratio and additive degree, they can have cationic, neutral or anionic nature. On the other hand, straight or branched chain conformation determines their hydrophilic or hydrophobic structure. So, there can exist many different types of EPSs with their unique functional and physicochemical properties (López-Ortega et al. 2021 ). Over the past three to four decades, microbial polysaccharides such as xanthan, dextran, alginate, gellan gum, and curdlan have found application in many industrial areas (Radchenkova et al., 2013 ). However, some mesophilic EPS producers are well-known human pathogens. Isolation of non-pathogenic producers could enlarge the area of application in novel biotechnological processes, such as drug delivery, medical diagnosis, new biodegradable plastics, etc. Despite mesophilic EPS producers that can be pathogenic, extremophiles are not disease-causing microorganisms and most have biotechnological advantages due to their short fermentation time (Wang et al. 2017 ). Thermophilic microorganisms are capable of growing from 55 to 80°C and a few members of thermophilic bacteria had been revealed as EPSs producers previously. Among the thermophilic Gram positive bacilli, some members of the genera Geobacillus , Aeribacillus , Anoxybacillus have been examined for their EPS producing capabilities (Arena et al. 2009; Radchenkova et al. 2013 ; Yasar Yildiz et al. 2014 ; Zhao et al. 2014 ; Wang et al. 2017 ; Panosyan et al. 2018 ; Genc et al. 2021 ; Karadayi et al. 2021 ). Thermus aquaticus YT-1 (Lin et al. 2011 ) and Rhodothermus marinus (Sardari et al. 2017 ) are both Gram negative thermophilic bacteria isolated from hot springs and have also been reported as EPSs producers. Whole genome based analysis has provided new insights regarding EPSs biosynthesis capabilities of thermophilic Brevibacillus thermoruber 423 (Yildiz et al. 2015 ), Geobacillus sp. WSUCF1 (Wang et al. 2019 ) and Parageobacillus thermantarcticus M1 (Yasar Yildiz et al. 2022 ). In addition to their faster growth and lack of pathogenicity, the advantages of using thermophilic bacteria as cell factories to obtain EPS include thermostability of metabolites, resistance to contamination by mesophiles, and decreased viscosity of broth medium with increasing temperature (Staudt et al. 2004 ; Malang et al2015). It has also been revealed that thermophilic EPSs have important biological activities including anti-cancer, anti-oxidant, anti-viral and immunostimulant effects without any cytotoxic potential (Setlow, 2006 ; Minana-Galbis et al. 2010 ; Goh et al. 2013 ; Panosyan et al., 2018 ). When considering the potential of unexplored and rich sources of EPSs, thermophiles can reveal new and promising exopolysaccharides with useful properties. The present study focused on production and characterization of EPSs from thermophilic bacteria. For this purpose, we screened a total of 107 isolates and selected most yielding nine strains, which belongs to the genera Anoxybacillus , Geobacillus , Parageobacillus and Aeribacillus according to 16S rRNA sequencing results. Furthermore, we aimed to evaulate biological activity of purified exopolysaccharides by searching their cytotoxic, antibacterial, antibiofilm, fibrinolytic, antioxidant, prebiotic and antidiabetic properties. To our knowledge, this is the first report regarding antidiabetic, fibrinolytic and prebiotic activities of EPSs produced by thermophilic endospore forming bacteria. Morever, EPS production by A. suryakundensis, A. flavithermus , and P.thermoglucosidasius has not been studied before. 2. Material and methods 2.1. Reagent and chemicals Yeast Extract (Biokar), TSA (Biokar), TSB (Biokar), MgSO 4 (Biokar), thiamine (Sigma), KCl (Tekkim), diammonium hydrogen phosphate (NH 4 ) 2 HPO 4 (Carlo Erba), Sucrose (Merck), Starch (Edukim), Acarbose (Biosynth), Inulin (Tito), Linoleic acid (Alpha Aesar), Gallic acid (Riedel-deHaen), 2,2-Diphenyl-1-picrylhydrazyl ( DPPH) (Sigma), Butyl hydroxy toluene (BHT) (Sigma), Congo Red (Isolab), 3,5 dinitrosalicylic acid (DNS) (Boston USA) Chemistry), Gentamicin (Bioanalyse), Fluconazole (Bioanalyse), [Taq Polymerase, dNTP, MgCl 2 , PCR Buffer] (Thermo Scientific) and distilled water. Other reagents, solvents, or chemicals are also pure, molecular biology or HPLC grade. 2.2. Bacterial isolates A total of one hundred seven thermophilic endospore forming bacteria previously isolated by Dr. Gamze Başbülbül from hot springs or soil samples in and around Aydin province were used for EPS screening (Başbülbül Özdemir and Biyik, 2012 ). Escherichia coli (35218), Staphylococcus aureus (RN4220), Enterococcus faecalis (JH2-2), Pseudomonas aeruginosa (PAO1), Proteus vulgaris (591), Candida albicans (4) strains were used for antimicrobial activity tests. Lactobacillus acidophilus 66, L. bulgaricus 118 and L. plantarum DSM 20174 were used as probiotic bacteria, while Escherichia coli DH10B was selected as an enteric representative for prebiotic activity assay. 2.3. Culture conditions Thermophilic isolates were taken from the stock cultures and inoculated in TSA plates for re-growth. After 24–48 hours incubation at 55°C colonies were transferred to TSB medium and incubated at 55°C and 180 rpm for 18–24 hours. For EPS production, thermophilic bacteria were inoculated from TSB medium into modified basal medium 2 (B2 broth) containing % 0.1 (NH 4 ) 2 HPO 4 , % 0.01 MgSO 4 , % 1 yeast extract, % 0.02 KCl, % 0.00001 thiamine, % 2 sucrose at pH 7.2. 2.4. Molecular identification of bacterial isolates Genomic DNA samples were isolated from 107 thermophilic bacteria, using easyDNA genomic DNA isolation kit (R-Tech, Türkiye) according to manufacturer instructions. 20F (5ˈ-AGAGTTTGATCCTGGCTCAG-3ˈ) and 1390R (5ˈ-GACGGGCGGTGTGTACAA-3ˈ) primers were used to amplify16S rDNA genes using PCR. Amplicons were sequenced by a company (MEDSANTEK) and after alignment with the sequences from NCBI database closest phylogenetic relatives of isolates were determined ( http://www.ncbi.nlm.nih.gov ). The sequence data were deposited in GenBank, under accession numbers OR896922.1.- OR897028.1. 2.5. Isolation and purification of EPSs For preincubation in TSB medium, thermophilic bacteria were incubated overnight at 55°C, 180 rpm in a shaking incubator for 18–24 hours. The preincubated culture was subsequently inoculated into 5% of the modified basal 2 medium with the absorbance adjusted to 0.6 Abs at OD540 nm, and incubated for 18–24 hours at 55°C (Wang et al. 2021 ). Overnight culture supernatant was taken and autoclaved and then evaporated in an incubator at 70°C to reduce the volume (Radchenkova et al. 2013 ; Li et al. 2015 ; Patwal and Baranwal, 2021 ). Trichloroacetic acid (TCA) solution was added to the 10 mL of concentrated supernatant with a final concentration of 4% and incubated in a mixer at room temperature for 30 minutes. The culture was then centrifuged at 2500 x g for 10 minutes. Pure alcohol, twice its volume, was added to the supernatant and kept at + 4°C for 24 hours for precipitation. It was then centrifuged at 2500 x g for 10 minutes. The pellet was dissolved with distilled water and lyophilized and stored at -80°C for analysis (Wang et al. 2015 ; Bajpai et al. 2016 ). To determine the sugar content of EPSs phenol sulfuric acid method was used. For this purpose, 250 µL of EPS, 750 µL of H 2 SO 4 , 150 µL of phenol solution (5%) were mixed in a 1.5 mL microcentrifuge tube. The tubes were kept in a thermal shaker heated to 90°C for 5 minutes. Samples from tubes with color change were placed in 1 mL cuvettes to measure the absorbance at 491 nm in the UV-vis spectrophotometer. Glucose was used as standard. Empty medium was used as a blank solution (Masuko et al. 2005 ). Bradford method was used to determine the protein concentration of crude EPSs. Bovine Serum Albumin (BSA) standard was prepared. Then, 10 µL of protein sample was added into 190 µL of dye solution into the microplate wells, and after mixing, it was kept at room temperature for 10 minutes. Distilled water was added to the dye instead of protein as a blank. The absorbance was measured in a spectrophotometer at 595 nm (Noble and Bailey, 2009 ). EPS purification was first performed by ion exchange chromatography column (1.2cm x 50cm) with Diethylaminoethyl (DEAE)-Cellulose (30477) Sigma-Aldrich. One mL of EPS (40 mg/mL) was fractionated. Twenty-five mL of solutions were passed through the column as NaCl gradient (0.1, 0.25, 0.5, 0.75, 1M) at a flow rate 1mL/min. Secondly, both size separation and desalting were performed by a gel filtration chromatography column (1.2cm x 30cm) with Sephadex G-100 at a flow rate 0.5 mL/min. The column was loaded with 15mL EPS obtained from ion exchange chromatography. Then, 25 mL of distilled water was passed through the column. Samples were collected in 5 mL fractions. Carbohydrate content was determined by the phenol sulfuric acid method (Ye et al. 2012 ; Hu et al. 2021 ). Purified exopolysaccharides were used for biological activity examinations. 2.6. Biological characterization 2.6.1. Cytotoxicity determination by hemolytic potential test Hemolytic activity was first evaluated qualitatively on blood agar. Briefly, the medium was prepared by adding 5 mL of sheep blood sample to 100 mL of basal medium and poured into petri dishes. After agar solidified, 8 mm wells were cut by sterilized cork borer and then 100µL of EPS solutions (0.025, 0.50, 1.0, 2.5 and 5.0 mg/mL) were filled into the wells. Plates were incubated at 37°C for 24 hours and the presence of zones around the wells was evaluated (Abinaya et al. 2018 ; Filik and Kubilay, 2020 ). To determine the quantitative hemolytic activity, a method applied by Abinaya et al. (Abinaya et al., 2018 ) was used. EPS solutions of different final concentrations (0.025, 0.50, 1.0, 2.5 and 5.0 mg/mL), were added to each test tube and mixed by gently inverting. Supernatants were separated by centrifugation and taken into a new 96-well microplate. Then absorbance was measured at 540 nm. The percentage of hemolysis was calculated according to the formula (%) hemolysis = (A sample-A blank)/ A control x 100 (A sample; EPS, A blank; saline, A control; distilled water). The experiment was repeated three times (Guezennec et al. 2012 ; Li et al. 2018 ). 2.6.2. Antimicrobial activity The agar well diffusion method was used to examine the antimicrobial activity. Suspensions of test microorganisms ( Escherichia coli 35218, Staphylococcus aureus RN4220, Enterococcus faecalis JH2-2, Pseudomonas aeruginosa PAO1, Proteus vulgaris 591, Candida albicans 4) were prepared at 0.5 McFarland turbidity and 100 µL of microorganism suspension was spread on Mueller Hinton Agar plates by a sterile cotton swab. Gentamicin (10 µg) and fluconazole (25 µg) discs were used as control antibiotics for bacteria and yeast, respectively. A hundred microliter of three different concentrations (1, 5 and 15 mg/mL) of isolated exopolysaccharides, were added to the agar wells. After 24 hours of incubation inhibition zones around the wells were measured (El Essawy et al. 2016 ). 2.6.3. Antioxidant activity Determination of DPPH radical scavenging activity was made according to Cao et al. (Cao et al. 2020 ). Zero (Control), 25, 50, 250, 1000, 4000, 5000 µg/mL of the extract solutions (0.5 mL) were put into eppendorf tubes and were mixed with 1 mL methanol. Then, 0.1 mL of the 0.4 mM solution of DPPH prepared in methanol was added onto the samples and mixed with vortexing and incubated for 30 minutes in the dark. The absorbances of the solutions were read at 517 nm. Trolox, gallic acid and ascorbic acid were used as standard antioxidants. The percent inhibition value was calculated from the formula % inhibition = (A control -A sample )/A control x 100 (A control : Absorbance of control, A sample : Absorbance of sample/standard antioxidant), and the IC 50 values were calculated. Determination of total antioxidant activity by ferric thiocyanate (FTC) method described by Saha et al. (Saha et al., 2004 ) was performed. For this, 1.0 milliliter of EPS (2 mg/mL) mixed with 1.025 mL of 2.5% linoleic acid emulsion (in ethyl alcohol). Then, 2.0 milliliters of 0.04 M phosphate buffer (pH 7.4) and 975 µL of distilled water were added. The mixture was incubated at 40°C, and measurements were made every 24 hours. For measurement, 50 µL of the solution was taken and was mixed with 4.85 mL of pure ethyl alcohol, 50 µL of NH 4 SCN, and 50 µL of FeCl 2 . The absorbance of the complex was measured at 500 nm by using a spectrophotometer (Saha et al. 2004 ). Determination of reducing power, previously described by Lin et al. was performed (Lin et al. 2012 ). Five hundred µL solutions were prepared from the stock EPS solution (20 mg/mL) in a 15 mL centrifuge tube with a final concentration of 25, 50, 250, 1000, 4000, 5000 µg/mL. Subsequently, 1.25 mL of 0.2 M PBS (pH 6.6) was added to each solution. Then, 1.25 mL of potassium ferricyanide K 3 Fe(CN) 6 was added and the mixtures were kept at 50°C for 20 minutes. After incubation in a water bath, 1.25 mL of 10% TCA was added to the mixture, and vortexed. Finally, 1.25 mL of the solution was taken into another 15 mL centrifuge tube, mixed with 1.25 mL of distilled water and 250 µL of 0.1% FeCl 3, and incubated for ten minutes and measured against distilled water at 700 nm. Ascorbic acid was used as a standard. 2.6.4. Alpha-amylase enzyme inhibition assay To evaluate the anti-diabetic potentials of EPSs, alpha-amylase enzyme inhibition method was used. Two different concentrations of EPSs (100 µg/mL and 800 µg/mL) and standard acarbose (1 mg/mL) solution in dimethyl sulfoxide were prepared. EPSs and acarbose solutions (500 µL) were mixed with 500 µL of amylase (0.5 mg/mL) and then mixtures were incubated for 10 min at room temperature. After that, 500 µL of starch solution (1%) was added to each tube and incubated for another 10 minutes. At the end of the incubation period, 1 mL of DNS reagent was added to the reaction mixture and kept in a water bath at 90°C for 5 minutes. After cooling to room temperature, mixtures were diluted with 10 mL of distilled water. Absorbance was measured at 540 nm against a reactive blank. Results are expressed as percent inhibition of α-amylase and calculated according to the formula, % inhibition = (A control -A test )/A control x 100 (A control : acarbose, A test : EPS) (Dilna et al. 2015 ). 2.6.5. Prebiotic activity test Lactobacillus acidophilus 66, L. bulgaricus 118 and L. plantarum DSM 20174 were used as probiotic bacteria, while Escherichia coli DH10B was selected as an enteric representative (El Essawy et al. 2016 ). TY broth media (tryptone 10 g/L, yeast extract 5 g/L, Tween 80 1 g/L, L-cysteine HCl monohydrate 0.1 g/L, pH 6.8 ± 0.2) supplemented with glucose, inulin or EPS solutions (2%) were inoculated with bacteria and the microplates were incubated at 37°C for 24 hours anaerobically. Samples were taken from bacterial cultures at the beginning of the experiment and after 24 hours to count the bacterial cells. The prebiotic activity was evaluated using the formula below and the effectiveness of EPSs as prebiotics was compared with that of inulin (Huebner et al. 2007 ). $$\text{P}\text{r}\text{e}\text{b}\text{i}\text{o}\text{t}\text{i}\text{c} \text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}= \frac{Probiotic log cfu mL-1 \left(24. hours- 0. hours\right) with prebiotic }{Probiotic log cfu mL-1\left(24. hours- 0. hours\right) with glucose} – \frac{Enteric log cfu mL-1 \left(24. hours- 0. hours\right) with prebiotic}{Enteric log cfu mL-1\left(24. hours- 0. hours\right) with glucose}$$ 2.6.6. Fibrinolytic activity test The fibrinolytic activity of EPSs was investigated by modifying the USP 28-NF 23 pharmacopeia (2005) method. Briefly, for each test tube, 0.8 mL of saline solution (0.89% w/v), 1 mL of sheep blood, and 0.2 mL of calcium chloride solution (1% w/v) were mixed, and tubes were placed in a water bath at 37°C. After the coagulation was completed, the tubes were divided into three. Each of the three tubes is grouped into negative control, standard, and test. Then, 1 mL of saline solution, Actilyse (2 mg/tube) and EPS (2 mg/tube) samples were added to each tube group, respectively. Blood clot lysis was visually detected after 24 hours at 37°C. Evaluation of fibrinolytic activity level was made by the naked eye and identified at 5 levels (+ 1, + 2, +3, + 4 and + 5). The fifth level is defined as a complete lysis (Al-Nahas et al. 2011 ). 2.6.7. Biofilm inhibition assay Antibiofilm activity was tested on biofilm-forming bacteria Escherichia coli 3055, Klebsiella pneumoniae 5108, Pseudomonas aeruginosa PAO1 and Staphylococcus aureus RN4220. Overnight cultures of bacteria were diluted at 1:100 ratio with glucose TSB broth. A final volume of 200 µL from diluted cultures and EPS samples at different final concentrations (0.1, 0.5, 1 and 2 mg/mL) were added in wells on microplates. Plates were then incubated at 37°C for 24 hours for biofilm production. After incubation, bacteria that could not adhere to the wells were removed by washing three times with distilled water. Adhered bacteria were fixed by drying at 60°C for 1 hour (Stepanović et al., 2007 ). Bacteria attached to the wells were stained with 1% (w/v) crystal violet solution for 45 minutes. Excess dye was removed, and the wells were washed three times with distilled water and air-dried for 45 minutes. The stained biofilms were dissolved with 99% ethanol. The stained biofilm mass was measured spectrophotometrically at 595 nm (Li et al., 2014 ). The reduction in biofilm formation calculated with the following formula (the percentage of antibiofilm = 1 - A sample /A control ). 2.6.8. Statistical analysis Experimental results were expressed as the means of three parallel experiments ± standard deviation (S.D.). Data were statistically analyzed by one-way analysis of variance (ANOVA) using SPSS 23.0 software. p < 0.01 value was used to identify statistically significant differences. 3. Results 3.1. Identification and EPS production of thermophilic bacteria According to 16S rDNA sequence analysis results, isolates showed the highest homology ratios with the following species: Parageobacillus toebii (n = 32), Geobacillus thermodenitrificans (n = 29), Parageobacillus thermoglucosidasius (n = 18), Aeribacillus pallidus (n = 17), Anoxybacillus flavithermus (n = 7), Anoxybacillus suryakundensis (n = 2), Anoxybacillus gonensis (n = 1), Anoxybacillus kestanbolensis (n = 1). Molecular identification results of nine thermophilic isolates are given in (Table 1 ). It was determined that 99 of the isolates were EPS producers under tested culture conditions and nine of them with highest yields (117–419 mg/L) were selected for EPS characterization and biological activity studies. The amounts of total protein in EPS solutions ranged between 0,4-5.2 mg/L. Table 1 16S rDNA analysis results of thermophilic isolates Isolate Highest homology Similarity rate HBB 20 Geobacillus thermodenitrificans subsp. calidus strain AS16 (Accession number: MH371332) 99.66% HBB 61 Anoxybacillus gonensis strain GSB-U4 (Accession number: MH333214) 99.83% HBB 74 Anoxybacillus suryakundensis strain JS5 (Accession number: KF772607) 100% HBB 76 Anoxybacillus flavithermus strain K-1 (Accession number: MK418420) 99.83% HBB 78 Parageobacillus toebii NBRC 107807 strain DSM 14590 (Accession number: CP049703) 99.66% HBB 89 Aeribacillus pallidus strain SHJP4 (Accession number: MK296526) 98.97% HBB 106 Parageobacillus thermoglucosidasius strain TM242 (Accession number: CP016916) 99.21% HBB 134 Anoxybacillus kestanbolensis strain ACT14 (Accession number: GQ265909) 100% HBB 261 Geobacillus thermodenitrificans strain T12 (Accession number: CP020030) 99.48% 3.2. EPS purification The EPS produced by the bacteria given in (Table 1 ) were named (EPS**) using identical numbering, and the results of their purification by ion exchange and gel filtration chromatography are given in (Fig. 1 ). Purification percentages of crude EPSs in our study for ion exchange chromatography was 79.7%-82.3%-33.9%-32.1%-48.4%-53.0%-53.8%-48.6%-41.4%, respectively. For gel filtration chromatography, they were 100%-67.6%-100%-89.3%-100%-76.9%-100%-100%-86.2%, respectively. 3. Cytotoxicity of EPSs Cytotoxic effects of the nine purified exopolysaccharides were evaluated by hemolytic activity test. None of the EPSs had shown to possess hemolytic activity with the qualitative cytotoxicity method (Fig. 2 ). Quantitative cytotoxicity tests of EPSs were evaluated according to their hemolytic potential in red blood cells. The results are given in (Table 3 ) and (Fig. 3 ). Table 3 Hemolysis percentages of EPSs. The presented data are means of three replicates, with standard errors shown. Hemolysis (%) EPS Concentration EPS 20 EPS 61 EPS 74 EPS 76 EPS 78 EPS 89 EPS 106 EPS 134 EPS 261 0.025 mg/mL 0 ± 0 0.1 ± 0 0.1 ± 0.08 0 ± 0.04 0 ± 0 0.1 ± 0.04 0 ± 0.04 0 ± 0.04 0 ± 0 0.5 mg/mL 0 ± 0 0 ± 0 0.2 ± 0.23 0.1 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0.04 0 ± 0 1 mg/mL 0 ± 0 0 ± 0 1.3 ± 0.47 0.1 ± 0.04 0 ± 0 0.2 ± 0.04 0 ± 0 0 ± 0.04 0 ± 0 2.5 mg/mL 0.2 ± 0 0.1 ± 0 2.3 ± 0.12 0 ± 0 0.1 ± 0 0.8 ± 0.13 0.1 ± 0 0 ± 0 0 ± 0 5 mg/mL 2.5 ± 0.77 0.3 ± 0 11.1 ± 0.84 0.2 ± 0 0.1 ± 0 3.9 ± 0.32 0.1 ± 0 0 ± 0 0 ± 0 3.3. Antimicrobial activity of EPSs The results of antimicrobial activity tests of purified EPSs are given in (Table 4 ). EPS 61, 106 and 261 showed antibacterial effect against E. faecalis JH2-2 at a concentration of 15 mg/mL. None of the other EPSs was found to be inhibitory to the tested bacteria and C. albicans . Table 4 Antimicrobial activity test results (mm) of EPSs (15mg/mL). Values are means of three replicates with standard deviations. Tested microorganisms EPS 20 EPS 61 EPS 74 EPS 76 EPS 78 EPS 89 EPS 106 EPS 134 EPS 261 Gentamicin (10mcg) Fluconazol (25mcg) E. coli 35218 - - - - - - - - - 17 ± 0 - E. faecalis JH2-2 - 18 ± 1.2 - - - - 21 ± 0.6 - 20 ± 0 15 ± 0 - P.aeruginosa PAO1 - - - - - - - - - 20 ± 0 - S. aureus RN4220 - - - - - - - - - 15 ± 0 - P. vulgaris 591 - - - - - - - - - 14 ± 0 - C. albicans 4 - - - - - - - - - - 30 ± 0 3.4. Antioxidant properties In the determination of antioxidant activities of EPSs, the following methods based on the electron transfer principle were used to examine the reducing capacity of DPPH radical with DPPH radical scavenging activity determination, the reduction capacity to Fe 3+ →Fe 2+ with reducing power determination, and the capacity to prevent peroxidation of linoleic acid by ferric thiocyanate method (Yavaşer, 2011 ). The DPPH radical scavenging activities of the exopolysaccharides were evaluated. It was observed that EPSs showed varying degrees of DPPH activity. (Fig. 4 A). In terms of DPPH radical scavenging activities, EPS 134 showed the highest activity as 100% at 5000 µg/mL concentration. It was determined that EPS 89 with the lowest capacity showed 45% activity at 5000 µg/mL. FTC antioxidant activities of exopolysaccharides were also investigated. The antioxidant activities of EPSs were compared with standard antioxidants such as BHT and ascorbic acid (Fig. 4 B). Total antioxidant activity determination by ferric thiocyanate method works with the logic of inhibiting lipid peroxidation. In our study, EPS (20, 61, 74, 76, 78, and 134) showed activity at 2 mg/mL concentration, respectively, at a rate of 31-28-38-38-35-28%. The reducing power of exopolysaccharides was studied as an indicator of antioxidant activity and determined spectrophotometrically at 700 nm (Fig. 4 C). Within the results obtained, the highest reducing power was observed by EPS 134, while EPS 78 showed lowest reducing power activity. In the reducing power analysis, the color change that occurs with the reduction of Fe 3+ to Fe 2+ is examined as an indicator of antioxidant activity. 3.5. Alpha amylase inhibitory activity The α-amylase inhibition activity of exopolysaccharides was measured at two different concentrations (100 µg/mL and 800 µg/mL). The determination of α-amylase inhibition activity was performed to evaluate the potential of EPSs for use in the antidiabetic field. While acarbose, used as a standard, showed 37% activity at 1 mg/mL concentration, EPS 74 showed the highest inhibition activity (10%) among the tested EPSs at 100 µg/mL (Fig. 5 ). 3.6. Prebiotic activities The prebiotic activities of EPSs were investigated using LAB strains and E. coli as an enteric bacterium. Prebiotic index relates the growth rates of the probiotics with that of the pathogens like E. coli and Clostridium difficile as they were grown on the same EPS. EPSs were used at a final concentration of 2 mg/mL to evaluate the growth intensities of L. bulgaricus 118, L. acidophilus and L. plantarum DSM20174 and the results are shown in (Fig. 6 ). The prebiotic indices of purified EPSs 20, 61, 76, 89, 134 and 261 were higher than that of inulin, a representative prebiotic carbohydrate for all tested LAB. EPS 74, 89 and 106 showed lowest prebiotic indices when they were utilized by L. bulgaricus 118. EPS 76 had the highest prebiotic activity between 12.4–14.9 for all Lactobacilli species tested in the experiment with almost three-fold activity of inulin. These results indicated that EPSs produced by thermophilic strains HBB-20, HBB-61, HBB-76, HBB-89, HBB-134 and HBB-261 have high growth promotion activities on probiotic strains but not of pathogens such as E. coli . 3.7. Fibrinolytic activities The fibrinolytic activities of EPSs in the study were investigated to determine their capacity to dissolve blood clots and thus their potential usage as drugs. After 24 hours of incubation at 37 ºC, and the lysis of the clot became evident with the darkening of the color. Evaluation of fibrinolytic activity results was made by considering the degree of clot lysis between (+ 1 and + 5) grades. The results are given in (Table 5 ) and (Fig. 7 ). Table 5 24-hour fibrinolytic activity results of EPSs. P.C: Positive control, Actilyse 50 mg, N.C: Negative control, Saline). The presented data are means of three replicates, with standard errors shown. EPS 20 EPS 61 EPS 74 EPS 76 EPS 78 EPS 89 EPS 106 EPS 134 EPS 261 P.C. N.C Clot lysis grade + 2 ± 0 + 2 ± 0 + 2 ± 0 + 2 ± 0 + 2 ± 0 + 2 ± 0 + 1 ± 0 + 1 ± 0 + 2 ± 0 + 3 ± 0 + 1 ± 0 3.8. Inhibition of bacterial biofilms Antibiofilm activity results of nine exopolysaccharides selected in the study are given in (Table 6 ). It has been determined that exopolysaccharides have varying degrees of antibiofilm effects and antibiofilm activities were not concentration-dependent between 100 and 2000 µg mL − 1 . All the tested EPSs showed negative effect on biofilm formation of E. faecalis JH2-2 and also, highest biofilm inhibition values were recorded against the biofilm formed by the same bacteria. EPS 134 was found to be the most active (56%) against biofilm produced by E. facealis JH2-2 at a concentration of 100 µg/mL. Biofilms formed by S. aureus RN4220 and E.coli 3055 were mostly inhibited by exopolysaccharide isolated from HBB 261 at the concentration of 2000 µg/mL and 1000 µg/mL, respectively. EPS 74, 76 and 89 were found to effective against biofilm formed by K. pneumoniae , even at low (100 µg/mL) concentration, while EPS 61 (1000 µg/mL) had the highest effect with the inhibition ratio of 14% against P. aeruginosa PA01. Table 6 Antibiofilm activity of EPSs on A) E. coli 3055, B) S. aureus RN4220, C) K. pneumoniae 5108, D) P. aeruginosa PAO1, E) E. faecalis JH2-2. Values are means of three replicates with standard errors shown. Concentration of EPS (µg mL − 1 ) Inhibition ratio of different bacterial antibiofilm (%) E. coli S. aureus K. pneumoniae P. aeruginosa E. faecalis EPS 20 100 0 ± 0 0 ± 0 4 ± 2 4 ± 0 42 ± 10 500 6 ± 1 0 ± 0 10 ± 4 6 ± 1 25 ± 11 1000 0 ± 0 0 ± 0 10 ± 1 11 ± 3 41 ± 12 2000 0 ± 0 0 ± 0 7 ± 4 6 ± 3 37 ± 10 EPS 61 100 1 ± 0 0 ± 0 11 ± 2 0 ± 0 31 ± 7 500 0 ± 0 0 ± 0 8 ± 3 7 ± 3 26 ± 9 1000 0 ± 0 0 ± 0 7 ± 2 14 ± 3 25 ± 11 2000 0 ± 0 0 ± 0 5 ± 3 5 ± 3 35 ± 4 EPS 74 100 0 ± 0 0 ± 0 12 ± 5 0 ± 0 26 ± 8 500 0 ± 0 0 ± 0 11 ± 5 4 ± 1 28 ± 3 1000 0 ± 0 0 ± 0 8 ± 1 5 ± 4 12 ± 0 2000 0 ± 0 0 ± 0 6 ± 4 6 ± 3 38 ± 11 EPS 76 100 5 ± 0 0 ± 0 12 ± 3 0 ± 0 42 ± 10 500 7 ± 3 0 ± 0 5 ± 1 6 ± 1 31 ± 5 1000 1 ± 0 0 ± 0 5 ± 1 0 ± 2 29 ± 6 2000 0 ± 0 0 ± 0 3 ± 0 6 ± 1 33 ± 1 EPS 78 100 0 ± 1 0 ± 0 7 ± 2 0 ± 0 36 ± 6 500 8 ± 2 0 ± 0 7 ± 3 0 ± 0 31 ± 11 1000 0 ± 0 0 ± 0 3 ± 1 6 ± 2 18 ± 4 2000 0 ± 0 7 ± 3 5 ± 1 2 ± 1 29 ± 7 EPS 89 100 7 ± 4 0 ± 2 12 ± 5 0 ± 0 42 ± 9 500 0 ± 2 0 ± 0 4 ± 0 0 ± 0 33 ± 8 1000 6 ± 3 0 ± 0 1 ± 0 1 ± 1 25 ± 8 2000 0 ± 0 8 ± 0 3 ± 3 0 ± 1 46 ± 7 EPS 106 100 4 ± 0 2 ± 1 1 ± 0 0 ± 0 35 ± 2 500 21 ± 4 0 ± 0 2 ± 0 0 ± 0 0 ± 0 1000 1 ± 1 0 ± 0 0 ± 1 5 ± 1 34 ± 9 2000 0 ± 0 10 ± 3 0 ± 0 0 ± 1 40 ± 9 EPS 134 100 14 ± 1 6 ± 3 3 ± 3 0 ± 1 56 ± 11 500 19 ± 3 3 ± 2 0 ± 1 5 ± 1 17 ± 3 1000 8 ± 0 3 ± 0 0 ± 1 1 ± 1 29 ± 9 2000 0 ± 1 23 ± 5 0 ± 0 5 ± 1 26 ± 6 EPS 261 100 7 ± 1 8 ± 4 7 ± 3 6 ± 1 40 ± 12 500 16 ± 2 8 ± 2 0 ± 0 0 ± 0 34 ± 8 1000 23 ± 5 9 ± 3 1 ± 1 12 ± 0 33 ± 8 2000 0 ± 0 24 ± 9 0 ± 0 4 ± 0 35 ± 14 4. Discussion There are limited number of studies regarding EPS production by thermophilic bacteria which include G. thermodenitrificans , A. gonensis, A. pushchinoensis, P. toebii, A. pallidus, Brevibacillus thermoruber and A. kestanboliensis species (Nicolaus et al., 2000 ; Arena et al. 2009; Minana-Galbis et al. 2010 ; Radchenkova et al. 2013 ; Yasar Yildiz et al. 2014 ; A.I. Ahmed, 2018; Panosyan et al. 2018 ; Genc et al. 2021 ; Karadayi et al. 2021 ). To the best of our knowledge, strains belonging to A. suryakundensis, A. flavithermus, P.thermoglucosidasius species are the first to examine for their EPS production and characterization in our report. EPSs producing thermophilic bacilli, crude EPS, and protein amounts produced by themselves are summarized in (Table 6 ). Table 6 Thermophilic Gram positive bacilli with EPS production levels Bacteria Isolation source Crude EPS yield (mg/L) Protein amount (mg/L) References Geobacillus thermodenitrificans HBB-20 Soil, Ortakçı, Aydın 419.4 1,3 This study Anoxybacillus gonensis HBB-61 Thermal pool, Alangüllü, Aydın 116.9 1,8 This study Anoxybacillus suryakundensis HBB-74 Soil, Alangüllü, Aydın 269.7 3,8 This study Anoxybacillus flavithermus HBB-76 Soil, Alangüllü, Aydın 186.3 4,6 This study Parageobacillus toebii HBB-78 Thermal mud, Alangüllü, Aydın 224.9 3,8 This study Aeribacillus pallidus HBB-89 Thermal mud, Davutlar, Aydın 187.9 3,6 This study Parageobacillus thermoglucosidasius HBB-106 Thermal mud, Gümüşköy, Aydın 352.2 5,2 This study Anoxybacillus kestanbolensis HBB-134 Hot spring, Alangüllü, Aydın 141.6 0,4 This study Geobacillus thermodenitrificans HBB-261 Sediment, Yenice, Denizli 357.6 4,3 This study Geobacillus sp. strain WSUCF1 Compost facility, USA 404 3.3% (Wang et al. 2021 ) Geobacillus thermodenitrificans ArzA-6 Arzakan geothermal spring, Armenia 76 6.1% (Panosyan et al. 2018 ) Geobacillus toebii ArzA-8 Arzakan geothermal spring, Armenia 80 7% (Panosyan et al. 2018 ) Brevibacillus thermoruber 423 Gradechnista hot spring, Bulgaria 863 8.7% (Yasar Yildiz et al. 2014 ) Anoxybacillus sp. R4-33 Radioactive radon hot spring, China 1083 - (Zhao et al. 2014 ) Aeribacillus pallidus 418 Rupi hot spring, Bulgaria 80 19% (Radchenkova et al. 2013 ) Geobacillus sp. 4004 Hydrothermal vent, Italy 60 (Poli et al. 2010 ) Geobacillus ( Parageobacillus ) thermantarcticus Crater of Mount Melbourne, Antarctica 400 - (Aliyu et al. 2016 ) Geobacillus stearothermophilus 1A60 Hydrothermal vent, Italy < 60 (Gugliandolo et al. 2012 ) Geobacillus thermodenitrifcans B3-7 Hydrothermal vent, Italy 70 - (Arena et al. 2009) Anoxybacillus tepidamans V264 Velingrad hot spring, Bulgaria 111.4 1.8% (Kambourova et al. 2009 ; Coorevits et al. 2012 ) Anoxybacillus sp. R4-33, which is isolated from radioactive radon hot spring was the most EPS productive (1083 mg/L) thermophilic bacilli so far (Zhao et al. 2014 ). Geobacillus sp. WSUCF1 and G. thermantarcticus were also reported as thermophiles with high EPS yields of 404 mg/L and 400 mg/L, respectively. Geobacillus sp. TS3-9, (87 mg/L), G . tepidanmans V264 (111.4 mg/L), G. thermodenitrifcans ArzA-6 (76 mg/L), G. toebii ArzA-8 (80 mg/L), Aeribacillus pallidus (53 mg/L), Geobacillus toebii (50 mg/L) and Anoxybacillus kestanbolensis (25.3 mg/L) showed productivity to a lesser extent (Radchenkova et al. 2013 ; Wang et al. 2017 ; Panosyan et al. 2018 ). EPS yields of our thermophilic isolates ranged between 117–419 mg/L which means that their production capacities are comparable with those of other thermophiles reported, previously. Hu et al. obtained 4 fractions by ion exchange chromatography (DEAE-52) (EPS 1, EPS 2, EPS 3, EPS 4) with the yields % of 21.2-25.0-18.8-20.0%, respectively. These four fractions were then purified by gel filtration chromatography (Sephadex G-100). It was found to have 88.0-90.1-89.1-87.9 percent purification, respectively (Hu et al., 2021 ). Schiano Moriello et al. purified EPS of 4004 coded thermophilic bacteria belonging to the genus Geobacillus , gel filtration chromatography (Sephadex G-50; 2.5 x 50cm) and ion exchange chromatography (Sepharose DEAE CL-6B; 1.5 x 40cm) were applied, respectively. Total EPS purification percentage were 80% and 70%, respectively (Schiano Moriello et al. 2003 ). It was observed that our study demonstrated higher purification yields than that of above-mentioned studies. It has been reported that the allowable level of hemolysis for biomaterials is 5%. The percentages of hemolysis for our EPSs at the highest concentration (5000µg/mL) were in the range of 0-0.3% for EPS 61, 76, 78, 106, 134, 261, while EPS 20 and EPS 89 showed hemolysis potential of 2.5% and 3.9%, respectively. Among the EPSs purified from nine thermophilic bacteria, only EPS 74 was evaluated as cytotoxic due to its high (11.1%) hemolytic percentage. It was observed that eight of the EPSs obtained from nine different bacteria used in our study were not cytotoxic. Abinaya et al. reported that EPS obtained from Bacillus licheniformis Dahb1 had a cytotoxicity of 1.2% at a concentration of 5000µg/mL (Abinaya et al. 2018 ). Genc et al., isolated EPS from A. pushchinoensis G11 and revealed that EPS had a dose-independent cytotoxic effect on A-549, Caco-2 and HT-29 cell lines (Genc et al., 2021 ). In another study, Arena et al. tested the cytotoxic effect of EPS from G. thermodenitrificans B3-72 on human peripheral blood mononuclear normal cells and observed dose-dependent inhibition on healthy cells (Arena et al. 2009). As a result of cytotoxicity assay, Wang et al., showed no significant effect on HEK-293 cell viability when they tested EPS-1 and EPS-2 from Geobacillus sp. WSUCF1 even at high concentrations (2 mg/mL of EPS-1 and 3 mg/mL of EPS-2) (Wang et al., 2021 ). Although the antibacterial activity tests of EPSs produced by lactic bacteria were reported many times in the literature, works on those produced by thermophiles are very rare. Tuşar et al., tested the antibacterial effects of EPSs by thermophilic Bacillus zhangzhounesis 2CA and Bacillus licheniformis 2CS strains against pathogenic E. coli , S. aureus , K. pneumoniae and P. aeruginosa . EPS produced by B. licheniformis 2CS showed the highest antibacterial activity against E. coli (with 16 mm zone diameter) when grown in M3 medium (0.2% yeast extract + 1% sucrose) (Tuşar et al. 2022 ). Additionally, Genç examined the antimicrobial activity of EPS by A. pushchinoensis G11 against Salmonella enteritidis ATCC13076, Aeromonas hydrophila ATCC 35654, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumonia ATCC 13883 and Candida albicans ATCC 10231 with final densities of 0.01, 0.25, 1, 1.25 and 2.5 g/L of freeze-dried sample. According to the test results, authors stated that antibacterial and antifungal activities were not detected (Genc et al. 2021 ). Among the studies by mesophilic EPSs, Rani et al., tested EPS from Lactobacillus gasseri FR4, and they reported antibacterial activity against E. faecalis at a concentration of 10 mg/mL similarly to our results (Rani et al., 2018 ). In the study conducted by Ghalem, EPS obtained from the yogurt starter bacteria mixture exhibited antimicrobial activity against E. coli and C. albicans with an inhibitory zone of 13 mm and 9 mm, respectively (Ghalem, 2017 ). The EPSs formed by L. plantarum 47FE, and L. pentosus 68FE showed inhibitory effect against representatives of both Gram-positive and Gram-negative bacteria and E. coli , S. typhimurium , and S. aureus were found to have the highest sensitivities in their study (Saif and Sakr, 2020 ). The mechanism that enables exopolysaccharides to show antimicrobial activity is thought to be the functional groups of EPS (Abdalla et al. 2021 ). It is known that negatively charged EPSs which contain sulfate groups interact better with Gram-positive bacteria that have higher positive charge on their cell walls (Saif and Sakr, 2020 ). This result may be due to the negative charge of thermophilic EPSs, and the sulfate groups they may contain affect bacterial cell surface communication. The DPPH radical scavenging activity of EPSs has been investigated in many studies and generally positive results have been demonstrated. It was reported that EPS of the thermophilic bacterium Geobacillus sp. showed high antioxidant activity at a concentration higher than 8 mg/mL (Wang et al., 2017 ). The EPS produced by a thermo-halophilic bacteria Halomonas nitroreducens strain WB1 had antioxidant properties at the concentration of 5.0 mg/ml. The EPS and ascorbic acid showed 83.3% and 90.2% of DPPH radical scavenging activity, respectively (Chikkanna et al. 2018 ). In our study, EPS (76 and 134) obtained from thermophilic bacteria were found to have 86% and 91% activity at 1 mg/mL concentration, respectively. Additionally, at higher concentration the activity increased. EPS (76, 106, 134 and 261) showed 90-92-99-90% DPPH radical scavenging activity at 4 mg/mL concentration, respectively. It has been reported that the use of a compost fermented with thermophiles as feed prevents lipid peroxidation in the liver in rats. Under these conditions, antioxidants were not decreased in the livers of rats fed compost extract (Miyamoto et al., 2013 ). EPSs of Paenibacillus polymyxa , EPS 1 and EPS 2 showed 41.45% and 50.43% lipid peroxidation inhibition activity at 4 mg/mL concentration, respectively (Liu et al., 2010 ). L. paracasei subsp. paracasei and L. plantarum EPS have been reported to have linoleic acid peroxidation inhibiting activities of 43.48%-27.57%, respectively, at 10 mg/mL (Liu et al. 2011 ). In a previously published study, antioxidant activity was determined in L. acidophilus EPS (2 mg/mL) by reducing power analysis at an absorbance value of 1.047 (Amiri et al., 2019 ). In the study conducted with EPS (5 mg/mL) of L. rhamnosus , it was observed that the absorbance value of the reducing power was between the highest (0.2–0.3). In addition, it has been reported that the fact that the reducing power antioxidant activity is higher than the others is due to the excess sulfate content in the structure of EPS and the low molecular weight (Hu et al., 2021 ). It was determined that L. plantarum EPS has a reducing power of 1.38 at a concentration of 2 mg/mL (Dilna et al., 2015 ). In our study, EPS (76 and 134) absorbance values of 0.578–1.176 were measured at 1 mg/mL concentration, respectively. Concentration-dependent antioxidant activity increased. EPS (76, 106, 134, 261) at 5 mg/mL concentration and absorbance values of 2.128-1.026-4.603-0.621, respectively, and reducing power and antioxidant activity were determined. Dilna et al. found 40% inhibition of alpha-amylase for L. plantarum RJF4 EPS (0.8 mg/mL) and 98% of acarbose at the same concentration (Dilna et al. 2015 ). Jiang et al. studied α-amylase inhibition of EPS (2 mg/mL) of Lactobacillus plantarum . As a result, inhibition of α-amylase was determined as 37.4% (Jiang et al., 2021 ). Xu et al. studied α-amylase inhibition of two EPSs of Bacillus licheniformis , BL-P1 and BL-P2 at 150 µg/mL. According to the findings, EPS BL-P1 and BL-P2 showed 67.24% and 75.63% inhibition levels, respectively, while positive control, acarbose, had an inhibition percentage of around 90% at the same concentration (Xu et al. 2019 ). It can be concluded that alpha-amylase inhibition levels exhibited by our EPSs were relatively lower than those reported by other authors. L. delbrueckii bulgaricus EPS tested for prebiotic activity determination and the index was found to be between 7.9 and 10.1 (Hussein et al. 2015 ). Lee et al. investigated the prebiotic activity of L. paracasei EPS at a concentration of 20 mg/mL and determined that the prebiotic index was between 15–25 (Lee et al. 2022 ). EPS produced by Enterobacter sp. ACD2 was reported as non-active because the prebiotic index was less than 1 when it was tested at concentration of 15 mg/mL (Almutairi and Helal, 2021 ). In our study, higher prebiotic activity results were obtained compared to the literature. It was reported by Al-Nahas et al. that EPS (2 mg/tube) from Pseudoalteromonas sp. AM exhibited a fibrinolytic activity score of + 3 (Al-Nahas et al., 2011 ). Almutairi and Helal studied the fibrinolytic activity of EPS (2 mg/mL) of Enterobacter sp. ACD2. Hemoclar (2 mg/mL) was used as standard. Activity results were determined as 100% and 75% lysis, respectively (Almutairi and Helal, 2021 ). Saif and Sakr investigated the fibrinolytic activity of EPSs of L. plantarum 47FE, and L. pentosus 68FE in their study. They reported that both EPSs (10 mg/mL) showed + 5 fibrinolytic activity (Saif and Sakr, 2020 ). It can be concluded that the blood clot lytic activity of thermophilic EPSs is low. In the study conducted by Genç et al, EPS of Anoxybacillus pushchinoensis showed antibiofilm activity at a concentration of 2000 µg/mL. It inhibited biofilm formation of S. aureus , E. coli and K. pneumoniae, B. subtilis, C. albicans and S. enteritidis with inhibition rates between 4.29–10.46% (Genc et al. 2021 ). Zammuto et al., investigated the antibiofilm and antiadhesive effects of the EPS B3-15, produced by a thermophilic strain of Bacillus licheniformis (B3-15), on different surfaces such as, a polyvinyl-chloride medical device, polystyrene microplates and human epithelial nasal cells. The EPS was effective on bacterial adhesion of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 at a concentration of 300 µg/mL but had no activity on mature biofilms. EPS B3-15 also reduced the adhesion of P. aeruginosa and S. aureus (five logs-scale and one log, respectively) on human nasal epithelial cells (Zammuto et al. 2023 ). A novel thermophilic EPS, named as EPS1-T14 produced by B. licheniformis strain T14 was evaluated for its effects on biofilm formation by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae clinical strains. The EPS1-T14 was found to active on biofilms formed by all the target pathogenic bacteria without antibacterial effects and it showed a dose-dependent inhibitory effect depend on the strain tested (Spanò et al. 2016 ). Since only EPS261 was found to be effective against E. faecalis JH2-2 during antibacterial activity assays, antibiofilm ability of other exopolysaccharides could not be caused by growth inhibition. It is thought that the antibiofilm activity occurs by the EPS preventing the attachment of pathogenic bacteria and thus the formation of biofilm. EPS inhibits the initial attachment and association of bacteria. It does this by reducing cell-cell surface communication and weakening cell surface modifications (Kim and Kim, 2009 ). Thus it can be proposed that EPSs, except for produced by HBB 261, might interfere with the steps of biofilm formation. 5. Conclusion In conclusion, exopolysaccharides produced by thermophilic strains belonging to species of A. suryakundensis, A. flavithermus, P. thermoglucosidasius have been isolated and characterized for the first time in this report. Beside of this, a lot of biological activity tests were performed for EPSs, which have been rarely studied for thermophilic ones. Nine thermophilic EPSs were highly purified by gel filtration and ion exchange chromatography. According to our results, most remarkable biological characteristics of EPSs are prebiotic and antioxidant activities and their lack of cytotoxicity. EPSs produced by nine thermophilic isolates were also found to have antimicrobial, anti-diabetic and fibrinolytic properties. Therefore, EPSs of thermophilic origin from our study have a high potential for use in health and food fields. Future investigations on chemical structures of EPSs will reveal new insights into thermophilic exopolysaccharides. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Aydın Adnan Menderes University (Türkiye) Research fund, by the project FEF-20026. Mehmet AYTAR received funding from Higher Education Council of Türkiye (YOK) as a participant of the 100/2000 PhD scholarship. Author Contribution M.A., wrote the first draft, prepared Figures 2-7 and Tables 1-5.G. B. wrote the main manucsript text, prepared Table 6. D.A.U. prepared Figure 1, edited language. 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Xu Z, Chen G, Xue L, Zhang H, Wang J, Xiang H, Li J, Zheng K (2019) Isolation, structural characterizations and bioactivities of exopolysaccharides produced by Bacillus licheniformis . Int J Biol Macromol 141; 298–306. https://doi.org/10.1016/j.ijbiomac.2019.08.217 . Yasar Yildiz S, Anzelmo G, Ozer T, Radchenkova N, Genç S, Di Donato P, Nicolaus B, Toksoy Oner E, Kambourova M (2014) Brevibacillus themoruber : a promising microbial cell factory for exopolysaccharide production. J Appl Microbiol 116; 314–324. https://doi.org/10.1111/jam.12362 . Yasar Yildiz S, Finore I, Leone L, Romano I, Lama L, Kasavi C, Nicolaus B, Toksoy Oner E, Poli A (2022) Genomic analysis provides new insights into biotechnological and industrial potential of Parageobacillus thermantarcticus M1. Front microbiol 13; 923038. https://doi.org/10.3389/fmicb.2022.923038 . Yavaşer R (2011) Comparison of Antioxidant Capacities of Natural And Synthetic Antioxidant Compounds. MSc Thesis, Aydın Adnan Menderes University, Institute of Science, p. 124. Ye S, Liu F, Wang J, Wang H, Zhang M (2012) Antioxidant activities of an exopolysaccharide isolated and purified from marine Pseudomonas PF-6. Carbohydr Polym 87; 764–770. https://doi.org/10.1016/j.carbpol.2011.08.057 . Yildiz SY, Radchenkova N, Arga KY, Kambourova M, Toksoy Oner E (2015) Genomic analysis of Brevibacillus thermoruber 423 reveals its biotechnological and industrial potential. Appl Microbiol Biotechnol 99; 2277–2289. https://doi.org/10.1007/s00253-015-6388-5 . Zammuto V, Spanò A, Agostino E, Macrì A, De Pasquale C, Ferlazzo G, Rizzo MG, Nicolò M., Guglielmino S, Gugliandolo C (2023) Anti-bacterial adhesion on abiotic and biotic surfaces of the exopolysaccharide from the marine Bacillus licheniformis B3-15. Mar Drugs 21; 313. https://doi.org/10.3390/md21050313 . Zhao S, Cao., Zhang H, Zhang L, Zhang F, Liang X (2014) Structural characterization and biosorption of exopolysaccharides from Anoxybacillus sp. R4-33 isolated from radioactive radon hot spring. Appl Biochem Biotechnol 172; 2732–2746. https://doi.org/10.1007/s12010-013-0680-6 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Sep, 2024 Read the published version in International Microbiology → Version 1 posted Editorial decision: Revision requested 06 Aug, 2024 Reviews received at journal 03 Aug, 2024 Reviews received at journal 02 Aug, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers agreed at journal 15 Jul, 2024 Reviewers invited by journal 15 Jul, 2024 Editor assigned by journal 05 Jul, 2024 Submission checks completed at journal 04 Jul, 2024 First submitted to journal 03 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4679730","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330792291,"identity":"e00266ba-785f-4311-b3d2-f6c16b3a098d","order_by":0,"name":"Mehmet Aytar","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Aytar","suffix":""},{"id":330792292,"identity":"dd60df95-3cc3-4f9d-a2f1-1ef5acda61b3","order_by":1,"name":"Deniz Aktaş Uygun","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Deniz","middleName":"Aktaş","lastName":"Uygun","suffix":""},{"id":330792293,"identity":"9e68fa5f-5f38-43ad-99b6-2168989d878a","order_by":2,"name":"Gamze Başbülbül","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCQYGZgYGiwQGduYDDDwgkQPEaZFIYGBmS4BpYWwgUguPAXFa5KObH38ubJPIMzjM8/HD2zYGOb4bCeyPK/BoMbxzzEx6ZptEscFh3s2Sc9sYjCVvJDA2nsGnZUaCGTNvm0TihsO8G6R52xgSN4C04HOZ4Yz0z58hWnge/wZqqSeoRV4ix0AaqoUNZEuCASEtBjJnyqRnnJMoljzMZmY555yE4cwzDxtn4rVldvvmzwVlNnl8x5sf33hTZiPPdzz5wEe8thxA5QOjiVBMyuOXHgWjYBSMglEABAAYsVAJhzWWxAAAAABJRU5ErkJggg==","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":true,"prefix":"","firstName":"Gamze","middleName":"","lastName":"Başbülbül","suffix":""}],"badges":[],"createdAt":"2024-07-03 10:36:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4679730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4679730/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10123-024-00588-6","type":"published","date":"2024-09-09T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61343917,"identity":"70377a5d-0380-4ccd-af35-bc54b86ce329","added_by":"auto","created_at":"2024-07-29 17:32:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1044491,"visible":true,"origin":"","legend":"\u003cp\u003ePurification of exopolysaccharides (20, 61, 74, 76, 78, 89, 106, 134, 261) by ion exchange chromatography (A1, B1, C1, D1, E1, F1, G1, H1, I1) and gel filtration chromatography (A2, B2, C2, D2, E2, F2, G2, H2, I2), respectively\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/ecf88fd49b348321b15462d7.jpeg"},{"id":61343418,"identity":"c5ae5b0f-3a34-448f-af09-9c5e738024ed","added_by":"auto","created_at":"2024-07-29 17:24:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398488,"visible":true,"origin":"","legend":"\u003cp\u003eQualitative hemolytic potential test (A: Purified EPS 20 with five different concentrations and three repeats, B: S. pyogenes as positive control with three repeats)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/20f92eb945012988fa73c945.jpeg"},{"id":61343416,"identity":"367cf2e3-a0f2-492b-8125-0f223edf12a5","added_by":"auto","created_at":"2024-07-29 17:24:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4514,"visible":true,"origin":"","legend":"\u003cp\u003eHemolysis percentages of EPSs. The presented data are means of three replicates, with standard errors shown.\u003c/p\u003e","description":"","filename":"Onlinedrawingimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/24a52a1f375e1988ff99a101.png"},{"id":61343420,"identity":"660733e2-cb97-47b4-a164-53dcc5dd56dd","added_by":"auto","created_at":"2024-07-29 17:24:40","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146495,"visible":true,"origin":"","legend":"\u003cp\u003eA) Determination of DPPH radical scavenging activity, B) Total Antioxidant Activity Percent Inhibition Rates by Ferric Thiocyanate (FTC) Method C) Determination of Reduction Power.Values are means of three replicates with standard error bars shown.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/26752a515d609863951f75e9.jpeg"},{"id":61343421,"identity":"8e3fb941-ba67-4eac-b158-8daa4d7ae05a","added_by":"auto","created_at":"2024-07-29 17:24:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6019,"visible":true,"origin":"","legend":"\u003cp\u003eα-amylase inhibition activity of EPSs. The presented data are means of three replicates, with standard errors shown.\u003c/p\u003e","description":"","filename":"Onlinedrawingimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/d60045402b21cf62cfc43b98.png"},{"id":61343422,"identity":"df43be08-6f37-45c8-b555-08b98ab7a50a","added_by":"auto","created_at":"2024-07-29 17:24:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5371,"visible":true,"origin":"","legend":"\u003cp\u003ePrebiotic activity of EPSs. Values are means of three replicates with standard error bars shown.\u003c/p\u003e","description":"","filename":"Onlinedrawingimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/3fa6866bee759d2d9a40e76a.png"},{"id":61343918,"identity":"a1553d93-c5c8-4e6a-a7f4-e7299753f56f","added_by":"auto","created_at":"2024-07-29 17:32:40","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":189602,"visible":true,"origin":"","legend":"\u003cp\u003eFibrinolytic activity of EPS 74 after 24 hours (A) Negative control, (B) Positive control, (C) EPS 74\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/0de52ed46a84ff7c68d82696.jpeg"},{"id":64619544,"identity":"071e7bfb-4343-405e-9f29-736fced60f79","added_by":"auto","created_at":"2024-09-16 16:15:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2996791,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679730/v1/d24b9fbe-e6ac-4053-9dfa-ab8648567789.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Production and Biological Activities of Exopolysaccharides Synthesized by Thermophilic Bacilli Isolated from Hot Springs in Türkiye","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicrobial exopolysaccharides (EPSs) are high molecular weight polymers produced by microalgae, yeast, mold and bacteria. They can accumulate outside the cell as capsular exopolysaccharides or secreted to the environment (Karadayi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). EPSs promote adhesion to surfaces as well as formation of cellular aggregates and biofilm (Nwodo et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition to these functions, they protect cells against microbial predators, as well as provide the host cell with advantages in drought and nutrient starved environmental conditions. Their molecular weights are ranging from 100\u0026ndash;2000 kDa (Suresh Kumar et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEPSs are classified into two groups either homopolysaccharides (HoPS) or heteropolysaccharides (HePS) depending on the composition of the repeating units (Mende et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Homopolysaccharides (HoPS) are composed of a single type of monosaccharide while heteropolysaccharides (HePS) are composed of either linear or branched repeating units (Ruas-Madiedo et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Pentoses like D-arabinose, D-ribose, D-xylose, hexoses (i.e. D-fructose, D-glucose, D-mannose and D-galactose), deoxy-hexoses (L-rhamnose and L-fucose), uronic acids (D-glucuronic acid, and D-galacturonic acid), D-glucosamine and D-galactosamine, which are known as amino sugars, could be structural monomeric units of exopolysaccharides (Joulak et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a number of instances, EPS may contain non-carbohydrate moieties like proteins, nucleic acids, fats, phosphates, sulfates, lactates attached to the polysaccharide backbone chain. Depending on the monomeric ratio and additive degree, they can have cationic, neutral or anionic nature. On the other hand, straight or branched chain conformation determines their hydrophilic or hydrophobic structure. So, there can exist many different types of EPSs with their unique functional and physicochemical properties (L\u0026oacute;pez-Ortega et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOver the past three to four decades, microbial polysaccharides such as xanthan, dextran, alginate, gellan gum, and curdlan have found application in many industrial areas (Radchenkova et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, some mesophilic EPS producers are well-known human pathogens. Isolation of non-pathogenic producers could enlarge the area of application in novel biotechnological processes, such as drug delivery, medical diagnosis, new biodegradable plastics, etc. Despite mesophilic EPS producers that can be pathogenic, extremophiles are not disease-causing microorganisms and most have biotechnological advantages due to their short fermentation time (Wang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThermophilic microorganisms are capable of growing from 55 to 80\u0026deg;C and a few members of thermophilic bacteria had been revealed as EPSs producers previously. Among the thermophilic Gram positive bacilli, some members of the genera \u003cem\u003eGeobacillus\u003c/em\u003e, \u003cem\u003eAeribacillus\u003c/em\u003e, \u003cem\u003eAnoxybacillus\u003c/em\u003e have been examined for their EPS producing capabilities (Arena et al. 2009; Radchenkova et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yasar Yildiz et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Panosyan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Genc et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Karadayi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eThermus aquaticus\u003c/em\u003e YT-1 (Lin et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and \u003cem\u003eRhodothermus marinus\u003c/em\u003e (Sardari et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) are both Gram negative thermophilic bacteria isolated from hot springs and have also been reported as EPSs producers. Whole genome based analysis has provided new insights regarding EPSs biosynthesis capabilities of thermophilic \u003cem\u003eBrevibacillus thermoruber\u003c/em\u003e 423 (Yildiz et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eGeobacillus\u003c/em\u003e sp. WSUCF1 (Wang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and \u003cem\u003eParageobacillus thermantarcticus\u003c/em\u003e M1 (Yasar Yildiz et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to their faster growth and lack of pathogenicity, the advantages of using thermophilic bacteria as cell factories to obtain EPS include thermostability of metabolites, resistance to contamination by mesophiles, and decreased viscosity of broth medium with increasing temperature (Staudt et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Malang et al2015). It has also been revealed that thermophilic EPSs have important biological activities including anti-cancer, anti-oxidant, anti-viral and immunostimulant effects without any cytotoxic potential (Setlow, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Minana-Galbis et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Goh et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Panosyan et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). When considering the potential of unexplored and rich sources of EPSs, thermophiles can reveal new and promising exopolysaccharides with useful properties.\u003c/p\u003e \u003cp\u003eThe present study focused on production and characterization of EPSs from thermophilic bacteria. For this purpose, we screened a total of 107 isolates and selected most yielding nine strains, which belongs to the genera \u003cem\u003eAnoxybacillus\u003c/em\u003e, \u003cem\u003eGeobacillus\u003c/em\u003e, \u003cem\u003eParageobacillus\u003c/em\u003e and \u003cem\u003eAeribacillus\u003c/em\u003e according to 16S rRNA sequencing results. Furthermore, we aimed to evaulate biological activity of purified exopolysaccharides by searching their cytotoxic, antibacterial, antibiofilm, fibrinolytic, antioxidant, prebiotic and antidiabetic properties. To our knowledge, this is the first report regarding antidiabetic, fibrinolytic and prebiotic activities of EPSs produced by thermophilic endospore forming bacteria. Morever, EPS production by \u003cem\u003eA. suryakundensis, A. flavithermus\u003c/em\u003e, and \u003cem\u003eP.thermoglucosidasius\u003c/em\u003e has not been studied before.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagent and chemicals\u003c/h2\u003e \u003cp\u003eYeast Extract (Biokar), TSA (Biokar), TSB (Biokar), MgSO\u003csub\u003e4\u003c/sub\u003e (Biokar), thiamine (Sigma), KCl (Tekkim), diammonium hydrogen phosphate (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (Carlo Erba), Sucrose (Merck), Starch (Edukim), Acarbose (Biosynth), Inulin (Tito), Linoleic acid (Alpha Aesar), Gallic acid (Riedel-deHaen), 2,2-Diphenyl-1-picrylhydrazyl ( DPPH) (Sigma), Butyl hydroxy toluene (BHT) (Sigma), Congo Red (Isolab), 3,5 dinitrosalicylic acid (DNS) (Boston USA) Chemistry), Gentamicin (Bioanalyse), Fluconazole (Bioanalyse), [Taq Polymerase, dNTP, MgCl\u003csub\u003e2\u003c/sub\u003e, PCR Buffer] (Thermo Scientific) and distilled water. Other reagents, solvents, or chemicals are also pure, molecular biology or HPLC grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Bacterial isolates\u003c/h2\u003e \u003cp\u003eA total of one hundred seven thermophilic endospore forming bacteria previously isolated by Dr. Gamze Başb\u0026uuml;lb\u0026uuml;l from hot springs or soil samples in and around Aydin province were used for EPS screening (Başb\u0026uuml;lb\u0026uuml;l \u0026Ouml;zdemir and Biyik, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eEscherichia coli\u003c/em\u003e (35218), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (RN4220), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (JH2-2), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PAO1), \u003cem\u003eProteus vulgaris\u003c/em\u003e (591), \u003cem\u003eCandida albicans\u003c/em\u003e (4) strains were used for antimicrobial activity tests. \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e 66, \u003cem\u003eL. bulgaricus\u003c/em\u003e 118 and \u003cem\u003eL. plantarum\u003c/em\u003e DSM 20174 were used as probiotic bacteria, while \u003cem\u003eEscherichia coli\u003c/em\u003e DH10B was selected as an enteric representative for prebiotic activity assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Culture conditions\u003c/h2\u003e \u003cp\u003eThermophilic isolates were taken from the stock cultures and inoculated in TSA plates for re-growth. After 24\u0026ndash;48 hours incubation at 55\u0026deg;C colonies were transferred to TSB medium and incubated at 55\u0026deg;C and 180 rpm for 18\u0026ndash;24 hours. For EPS production, thermophilic bacteria were inoculated from TSB medium into modified basal medium 2 (B2 broth) containing % 0.1 (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, % 0.01 MgSO\u003csub\u003e4\u003c/sub\u003e, % 1 yeast extract, % 0.02 KCl, % 0.00001 thiamine, % 2 sucrose at pH 7.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Molecular identification of bacterial isolates\u003c/h2\u003e \u003cp\u003eGenomic DNA samples were isolated from 107 thermophilic bacteria, using easyDNA genomic DNA isolation kit (R-Tech, T\u0026uuml;rkiye) according to manufacturer instructions. 20F (5ˈ-AGAGTTTGATCCTGGCTCAG-3ˈ) and 1390R (5ˈ-GACGGGCGGTGTGTACAA-3ˈ) primers were used to amplify16S rDNA genes using PCR. Amplicons were sequenced by a company (MEDSANTEK) and after alignment with the sequences from NCBI database closest phylogenetic relatives of isolates were determined (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The sequence data were deposited in GenBank, under accession numbers OR896922.1.- OR897028.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Isolation and purification of EPSs\u003c/h2\u003e \u003cp\u003eFor preincubation in TSB medium, thermophilic bacteria were incubated overnight at 55\u0026deg;C, 180 rpm in a shaking incubator for 18\u0026ndash;24 hours. The preincubated culture was subsequently inoculated into 5% of the modified basal 2 medium with the absorbance adjusted to 0.6 Abs at OD540 nm, and incubated for 18\u0026ndash;24 hours at 55\u0026deg;C (Wang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overnight culture supernatant was taken and autoclaved and then evaporated in an incubator at 70\u0026deg;C to reduce the volume (Radchenkova et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Patwal and Baranwal, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Trichloroacetic acid (TCA) solution was added to the 10 mL of concentrated supernatant with a final concentration of 4% and incubated in a mixer at room temperature for 30 minutes. The culture was then centrifuged at 2500 x g for 10 minutes. Pure alcohol, twice its volume, was added to the supernatant and kept at +\u0026thinsp;4\u0026deg;C for 24 hours for precipitation. It was then centrifuged at 2500 x g for 10 minutes. The pellet was dissolved with distilled water and lyophilized and stored at -80\u0026deg;C for analysis (Wang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bajpai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine the sugar content of EPSs phenol sulfuric acid method was used. For this purpose, 250 \u0026micro;L of EPS, 750 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 150 \u0026micro;L of phenol solution (5%) were mixed in a 1.5 mL microcentrifuge tube. The tubes were kept in a thermal shaker heated to 90\u0026deg;C for 5 minutes. Samples from tubes with color change were placed in 1 mL cuvettes to measure the absorbance at 491 nm in the UV-vis spectrophotometer. Glucose was used as standard. Empty medium was used as a blank solution (Masuko et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBradford method was used to determine the protein concentration of crude EPSs. Bovine Serum Albumin (BSA) standard was prepared. Then, 10 \u0026micro;L of protein sample was added into 190 \u0026micro;L of dye solution into the microplate wells, and after mixing, it was kept at room temperature for 10 minutes. Distilled water was added to the dye instead of protein as a blank. The absorbance was measured in a spectrophotometer at 595 nm (Noble and Bailey, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEPS purification was first performed by ion exchange chromatography column (1.2cm x 50cm) with Diethylaminoethyl (DEAE)-Cellulose (30477) Sigma-Aldrich. One mL of EPS (40 mg/mL) was fractionated. Twenty-five mL of solutions were passed through the column as NaCl gradient (0.1, 0.25, 0.5, 0.75, 1M) at a flow rate 1mL/min. Secondly, both size separation and desalting were performed by a gel filtration chromatography column (1.2cm x 30cm) with Sephadex G-100 at a flow rate 0.5 mL/min. The column was loaded with 15mL EPS obtained from ion exchange chromatography. Then, 25 mL of distilled water was passed through the column. Samples were collected in 5 mL fractions. Carbohydrate content was determined by the phenol sulfuric acid method (Ye et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Purified exopolysaccharides were used for biological activity examinations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biological characterization\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Cytotoxicity determination by hemolytic potential test\u003c/h2\u003e \u003cp\u003eHemolytic activity was first evaluated qualitatively on blood agar. Briefly, the medium was prepared by adding 5 mL of sheep blood sample to 100 mL of basal medium and poured into petri dishes. After agar solidified, 8 mm wells were cut by sterilized cork borer and then 100\u0026micro;L of EPS solutions (0.025, 0.50, 1.0, 2.5 and 5.0 mg/mL) were filled into the wells. Plates were incubated at 37\u0026deg;C for 24 hours and the presence of zones around the wells was evaluated (Abinaya et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Filik and Kubilay, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine the quantitative hemolytic activity, a method applied by Abinaya et al. (Abinaya et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was used. EPS solutions of different final concentrations (0.025, 0.50, 1.0, 2.5 and 5.0 mg/mL), were added to each test tube and mixed by gently inverting. Supernatants were separated by centrifugation and taken into a new 96-well microplate. Then absorbance was measured at 540 nm. The percentage of hemolysis was calculated according to the formula (%) hemolysis = (A sample-A blank)/ A control x 100 (A sample; EPS, A blank; saline, A control; distilled water). The experiment was repeated three times (Guezennec et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Antimicrobial activity\u003c/h2\u003e \u003cp\u003eThe agar well diffusion method was used to examine the antimicrobial activity. Suspensions of test microorganisms (\u003cem\u003eEscherichia coli\u003c/em\u003e 35218, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e RN4220, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e JH2-2, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1, \u003cem\u003eProteus vulgaris\u003c/em\u003e 591, \u003cem\u003eCandida albicans\u003c/em\u003e 4) were prepared at 0.5 McFarland turbidity and 100 \u0026micro;L of microorganism suspension was spread on Mueller Hinton Agar plates by a sterile cotton swab. Gentamicin (10 \u0026micro;g) and fluconazole (25 \u0026micro;g) discs were used as control antibiotics for bacteria and yeast, respectively. A hundred microliter of three different concentrations (1, 5 and 15 mg/mL) of isolated exopolysaccharides, were added to the agar wells. After 24 hours of incubation inhibition zones around the wells were measured (El Essawy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Antioxidant activity\u003c/h2\u003e \u003cp\u003eDetermination of DPPH radical scavenging activity was made according to Cao et al. (Cao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Zero (Control), 25, 50, 250, 1000, 4000, 5000 \u0026micro;g/mL of the extract solutions (0.5 mL) were put into eppendorf tubes and were mixed with 1 mL methanol. Then, 0.1 mL of the 0.4 mM solution of DPPH prepared in methanol was added onto the samples and mixed with vortexing and incubated for 30 minutes in the dark. The absorbances of the solutions were read at 517 nm. Trolox, gallic acid and ascorbic acid were used as standard antioxidants. The percent inhibition value was calculated from the formula % inhibition = (A\u003csub\u003econtrol\u003c/sub\u003e -A\u003csub\u003esample\u003c/sub\u003e)/A\u003csub\u003econtrol\u003c/sub\u003e x 100 (A\u003csub\u003econtrol\u003c/sub\u003e: Absorbance of control, A\u003csub\u003esample\u003c/sub\u003e: Absorbance of sample/standard antioxidant), and the IC\u003csub\u003e50\u003c/sub\u003e values were calculated.\u003c/p\u003e \u003cp\u003eDetermination of total antioxidant activity by ferric thiocyanate (FTC) method described by Saha et al. (Saha et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) was performed. For this, 1.0 milliliter of EPS (2 mg/mL) mixed with 1.025 mL of 2.5% linoleic acid emulsion (in ethyl alcohol). Then, 2.0 milliliters of 0.04 M phosphate buffer (pH 7.4) and 975 \u0026micro;L of distilled water were added. The mixture was incubated at 40\u0026deg;C, and measurements were made every 24 hours. For measurement, 50 \u0026micro;L of the solution was taken and was mixed with 4.85 mL of pure ethyl alcohol, 50 \u0026micro;L of NH\u003csub\u003e4\u003c/sub\u003eSCN, and 50 \u0026micro;L of FeCl\u003csub\u003e2\u003c/sub\u003e. The absorbance of the complex was measured at 500 nm by using a spectrophotometer (Saha et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDetermination of reducing power, previously described by Lin et al. was performed (Lin et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Five hundred \u0026micro;L solutions were prepared from the stock EPS solution (20 mg/mL) in a 15 mL centrifuge tube with a final concentration of 25, 50, 250, 1000, 4000, 5000 \u0026micro;g/mL. Subsequently, 1.25 mL of 0.2 M PBS (pH 6.6) was added to each solution. Then, 1.25 mL of potassium ferricyanide K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e was added and the mixtures were kept at 50\u0026deg;C for 20 minutes. After incubation in a water bath, 1.25 mL of 10% TCA was added to the mixture, and vortexed. Finally, 1.25 mL of the solution was taken into another 15 mL centrifuge tube, mixed with 1.25 mL of distilled water and 250 \u0026micro;L of 0.1% FeCl\u003csub\u003e3,\u003c/sub\u003e and incubated for ten minutes and measured against distilled water at 700 nm. Ascorbic acid was used as a standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4. Alpha-amylase enzyme inhibition assay\u003c/h2\u003e \u003cp\u003eTo evaluate the anti-diabetic potentials of EPSs, alpha-amylase enzyme inhibition method was used. Two different concentrations of EPSs (100 \u0026micro;g/mL and 800 \u0026micro;g/mL) and standard acarbose (1 mg/mL) solution in dimethyl sulfoxide were prepared. EPSs and acarbose solutions (500 \u0026micro;L) were mixed with 500 \u0026micro;L of amylase (0.5 mg/mL) and then mixtures were incubated for 10 min at room temperature. After that, 500 \u0026micro;L of starch solution (1%) was added to each tube and incubated for another 10 minutes. At the end of the incubation period, 1 mL of DNS reagent was added to the reaction mixture and kept in a water bath at 90\u0026deg;C for 5 minutes. After cooling to room temperature, mixtures were diluted with 10 mL of distilled water. Absorbance was measured at 540 nm against a reactive blank. Results are expressed as percent inhibition of α-amylase and calculated according to the formula, % inhibition = (A\u003csub\u003econtrol\u003c/sub\u003e -A\u003csub\u003etest\u003c/sub\u003e)/A\u003csub\u003econtrol\u003c/sub\u003e x 100 (A\u003csub\u003econtrol\u003c/sub\u003e: acarbose, A\u003csub\u003etest\u003c/sub\u003e: EPS) (Dilna et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.5. Prebiotic activity test\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e 66, \u003cem\u003eL. bulgaricus\u003c/em\u003e 118 and \u003cem\u003eL. plantarum\u003c/em\u003e DSM 20174 were used as probiotic bacteria, while \u003cem\u003eEscherichia coli\u003c/em\u003e DH10B was selected as an enteric representative (El Essawy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). TY broth media (tryptone 10 g/L, yeast extract 5 g/L, Tween 80 1 g/L, L-cysteine HCl monohydrate 0.1 g/L, pH 6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) supplemented with glucose, inulin or EPS solutions (2%) were inoculated with bacteria and the microplates were incubated at 37\u0026deg;C for 24 hours anaerobically. Samples were taken from bacterial cultures at the beginning of the experiment and after 24 hours to count the bacterial cells. The prebiotic activity was evaluated using the formula below and the effectiveness of EPSs as prebiotics was compared with that of inulin (Huebner et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{P}\\text{r}\\text{e}\\text{b}\\text{i}\\text{o}\\text{t}\\text{i}\\text{c} \\text{A}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}= \\frac{Probiotic log cfu mL-1 \\left(24. hours- 0. hours\\right) with prebiotic }{Probiotic log cfu mL-1\\left(24. hours- 0. hours\\right) with glucose} \u0026ndash; \\frac{Enteric log cfu mL-1 \\left(24. hours- 0. hours\\right) with prebiotic}{Enteric log cfu mL-1\\left(24. hours- 0. hours\\right) with glucose}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.6. Fibrinolytic activity test\u003c/h2\u003e \u003cp\u003eThe fibrinolytic activity of EPSs was investigated by modifying the USP 28-NF 23 pharmacopeia (2005) method. Briefly, for each test tube, 0.8 mL of saline solution (0.89% w/v), 1 mL of sheep blood, and 0.2 mL of calcium chloride solution (1% w/v) were mixed, and tubes were placed in a water bath at 37\u0026deg;C. After the coagulation was completed, the tubes were divided into three. Each of the three tubes is grouped into negative control, standard, and test. Then, 1 mL of saline solution, Actilyse (2 mg/tube) and EPS (2 mg/tube) samples were added to each tube group, respectively. Blood clot lysis was visually detected after 24 hours at 37\u0026deg;C. Evaluation of fibrinolytic activity level was made by the naked eye and identified at 5 levels (+\u0026thinsp;1, +\u0026thinsp;2, +3, +\u0026thinsp;4 and +\u0026thinsp;5). The fifth level is defined as a complete lysis (Al-Nahas et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.7. Biofilm inhibition assay\u003c/h2\u003e \u003cp\u003eAntibiofilm activity was tested on biofilm-forming bacteria \u003cem\u003eEscherichia coli\u003c/em\u003e 3055, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e 5108, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e RN4220. Overnight cultures of bacteria were diluted at 1:100 ratio with glucose TSB broth. A final volume of 200 \u0026micro;L from diluted cultures and EPS samples at different final concentrations (0.1, 0.5, 1 and 2 mg/mL) were added in wells on microplates. Plates were then incubated at 37\u0026deg;C for 24 hours for biofilm production.\u003c/p\u003e \u003cp\u003eAfter incubation, bacteria that could not adhere to the wells were removed by washing three times with distilled water. Adhered bacteria were fixed by drying at 60\u0026deg;C for 1 hour (Stepanović et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Bacteria attached to the wells were stained with 1% (w/v) crystal violet solution for 45 minutes. Excess dye was removed, and the wells were washed three times with distilled water and air-dried for 45 minutes. The stained biofilms were dissolved with 99% ethanol. The stained biofilm mass was measured spectrophotometrically at 595 nm (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The reduction in biofilm formation calculated with the following formula (the percentage of antibiofilm\u0026thinsp;=\u0026thinsp;1 - A\u003csub\u003esample\u003c/sub\u003e/A\u003csub\u003econtrol\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eExperimental results were expressed as the means of three parallel experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D.). Data were statistically analyzed by one-way analysis of variance (ANOVA) using SPSS 23.0 software. \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e value was used to identify statistically significant differences.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Identification and EPS production of thermophilic bacteria\u003c/h2\u003e \u003cp\u003eAccording to 16S rDNA sequence analysis results, isolates showed the highest homology ratios with the following species: \u003cem\u003eParageobacillus toebii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;32), \u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;29), \u003cem\u003eParageobacillus thermoglucosidasius\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;18), \u003cem\u003eAeribacillus pallidus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;17), \u003cem\u003eAnoxybacillus flavithermus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7), \u003cem\u003eAnoxybacillus suryakundensis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eAnoxybacillus gonensis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eAnoxybacillus kestanbolensis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1). Molecular identification results of nine thermophilic isolates are given in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was determined that 99 of the isolates were EPS producers under tested culture conditions and nine of them with highest yields (117\u0026ndash;419 mg/L) were selected for EPS characterization and biological activity studies. The amounts of total protein in EPS solutions ranged between 0,4-5.2 mg/L.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e16S rDNA analysis results of thermophilic isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHighest homology\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSimilarity rate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e subsp. \u003cem\u003ecalidus\u003c/em\u003e strain AS16 (Accession number: MH371332)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.66%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus gonensis\u003c/em\u003e strain GSB-U4 (Accession number: MH333214)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.83%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus suryakundensis\u003c/em\u003e strain JS5 (Accession number: KF772607)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus flavithermus\u003c/em\u003e strain K-1 (Accession number: MK418420)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.83%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eParageobacillus toebii\u003c/em\u003e NBRC 107807 strain DSM 14590 (Accession number: CP049703)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.66%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAeribacillus pallidus\u003c/em\u003e strain SHJP4 (Accession number: MK296526)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.97%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eParageobacillus thermoglucosidasius\u003c/em\u003e strain TM242 (Accession number: CP016916)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.21%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus kestanbolensis\u003c/em\u003e strain ACT14 (Accession number: GQ265909)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBB 261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e strain T12 (Accession number: CP020030)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.48%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. EPS purification\u003c/h2\u003e \u003cp\u003eThe EPS produced by the bacteria given in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were named (EPS**) using identical numbering, and the results of their purification by ion exchange and gel filtration chromatography are given in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Purification percentages of crude EPSs in our study for ion exchange chromatography was 79.7%-82.3%-33.9%-32.1%-48.4%-53.0%-53.8%-48.6%-41.4%, respectively. For gel filtration chromatography, they were 100%-67.6%-100%-89.3%-100%-76.9%-100%-100%-86.2%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3. Cytotoxicity of EPSs\u003c/h3\u003e\n\u003cp\u003eCytotoxic effects of the nine purified exopolysaccharides were evaluated by hemolytic activity test. None of the EPSs had shown to possess hemolytic activity with the qualitative cytotoxicity method (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative cytotoxicity tests of EPSs were evaluated according to their hemolytic potential in red blood cells. The results are given in (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHemolysis percentages of EPSs. The presented data are means of three replicates, with standard errors shown.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"9\" nameend=\"c10\" namest=\"c2\"\u003e \u003cp\u003eHemolysis (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEPS Concentration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEPS 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEPS 61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEPS 74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEPS 76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEPS 78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEPS 89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEPS 106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEPS 134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEPS 261\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.025 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antimicrobial activity of EPSs\u003c/h2\u003e \u003cp\u003eThe results of antimicrobial activity tests of purified EPSs are given in (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). EPS 61, 106 and 261 showed antibacterial effect against \u003cem\u003eE. faecalis\u003c/em\u003e JH2-2 at a concentration of 15 mg/mL. None of the other EPSs was found to be inhibitory to the tested bacteria and \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial activity test results (mm) of EPSs (15mg/mL). Values are means of three replicates with standard deviations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTested microorganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEPS 20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEPS\u003c/p\u003e \u003cp\u003e61\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEPS 74\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEPS 76\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEPS 78\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEPS 89\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEPS\u003c/p\u003e \u003cp\u003e106\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEPS 134\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEPS\u003c/p\u003e \u003cp\u003e261\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003cp\u003e(10mcg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eFluconazol (25mcg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e 35218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e JH2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP.aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e RN4220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e 591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antioxidant properties\u003c/h2\u003e \u003cp\u003eIn the determination of antioxidant activities of EPSs, the following methods based on the electron transfer principle were used to examine the reducing capacity of DPPH radical with DPPH radical scavenging activity determination, the reduction capacity to Fe\u003csup\u003e3+\u003c/sup\u003e\u0026rarr;Fe\u003csup\u003e2+\u003c/sup\u003e with reducing power determination, and the capacity to prevent peroxidation of linoleic acid by ferric thiocyanate method (Yavaşer, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe DPPH radical scavenging activities of the exopolysaccharides were evaluated. It was observed that EPSs showed varying degrees of DPPH activity. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In terms of DPPH radical scavenging activities, EPS 134 showed the highest activity as 100% at 5000 \u0026micro;g/mL concentration. It was determined that EPS 89 with the lowest capacity showed 45% activity at 5000 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eFTC antioxidant activities of exopolysaccharides were also investigated. The antioxidant activities of EPSs were compared with standard antioxidants such as BHT and ascorbic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Total antioxidant activity determination by ferric thiocyanate method works with the logic of inhibiting lipid peroxidation. In our study, EPS (20, 61, 74, 76, 78, and 134) showed activity at 2 mg/mL concentration, respectively, at a rate of 31-28-38-38-35-28%.\u003c/p\u003e \u003cp\u003eThe reducing power of exopolysaccharides was studied as an indicator of antioxidant activity and determined spectrophotometrically at 700 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Within the results obtained, the highest reducing power was observed by EPS 134, while EPS 78 showed lowest reducing power activity. In the reducing power analysis, the color change that occurs with the reduction of Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e is examined as an indicator of antioxidant activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Alpha amylase inhibitory activity\u003c/h2\u003e \u003cp\u003eThe α-amylase inhibition activity of exopolysaccharides was measured at two different concentrations (100 \u0026micro;g/mL and 800 \u0026micro;g/mL). The determination of α-amylase inhibition activity was performed to evaluate the potential of EPSs for use in the antidiabetic field. While acarbose, used as a standard, showed 37% activity at 1 mg/mL concentration, EPS 74 showed the highest inhibition activity (10%) among the tested EPSs at 100 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Prebiotic activities\u003c/h2\u003e \u003cp\u003eThe prebiotic activities of EPSs were investigated using LAB strains and \u003cem\u003eE. coli\u003c/em\u003e as an enteric bacterium. Prebiotic index relates the growth rates of the probiotics with that of the pathogens like \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eClostridium difficile\u003c/em\u003e as they were grown on the same EPS. EPSs were used at a final concentration of 2 mg/mL to evaluate the growth intensities of \u003cem\u003eL. bulgaricus\u003c/em\u003e 118, \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e DSM20174 and the results are shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe prebiotic indices of purified EPSs 20, 61, 76, 89, 134 and 261 were higher than that of inulin, a representative prebiotic carbohydrate for all tested LAB. EPS 74, 89 and 106 showed lowest prebiotic indices when they were utilized by \u003cem\u003eL. bulgaricus\u003c/em\u003e 118. EPS 76 had the highest prebiotic activity between 12.4\u0026ndash;14.9 for all \u003cem\u003eLactobacilli\u003c/em\u003e species tested in the experiment with almost three-fold activity of inulin. These results indicated that EPSs produced by thermophilic strains HBB-20, HBB-61, HBB-76, HBB-89, HBB-134 and HBB-261 have high growth promotion activities on probiotic strains but not of pathogens such as \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Fibrinolytic activities\u003c/h2\u003e \u003cp\u003eThe fibrinolytic activities of EPSs in the study were investigated to determine their capacity to dissolve blood clots and thus their potential usage as drugs. After 24 hours of incubation at 37 \u0026ordm;C, and the lysis of the clot became evident with the darkening of the color. Evaluation of fibrinolytic activity results was made by considering the degree of clot lysis between (+\u0026thinsp;1 and +\u0026thinsp;5) grades. The results are given in (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e24-hour fibrinolytic activity results of EPSs. P.C: Positive control, Actilyse 50 mg, N.C: Negative control, Saline). The presented data are means of three replicates, with standard errors shown.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEPS 20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEPS 61\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEPS 74\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEPS 76\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEPS 78\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEPS 89\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEPS 106\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEPS 134\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEPS 261\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eP.C.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN.C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClot lysis grade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u0026thinsp;1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e+\u0026thinsp;2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e+\u0026thinsp;3\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e+\u0026thinsp;1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Inhibition of bacterial biofilms\u003c/h2\u003e \u003cp\u003eAntibiofilm activity results of nine exopolysaccharides selected in the study are given in (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It has been determined that exopolysaccharides have varying degrees of antibiofilm effects and antibiofilm activities were not concentration-dependent between 100 and 2000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All the tested EPSs showed negative effect on biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e JH2-2 and also, highest biofilm inhibition values were recorded against the biofilm formed by the same bacteria. EPS 134 was found to be the most active (56%) against biofilm produced by \u003cem\u003eE. facealis\u003c/em\u003e JH2-2 at a concentration of 100 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eBiofilms formed by \u003cem\u003eS. aureus\u003c/em\u003e RN4220 and \u003cem\u003eE.coli\u003c/em\u003e 3055 were mostly inhibited by exopolysaccharide isolated from HBB 261 at the concentration of 2000 \u0026micro;g/mL and 1000 \u0026micro;g/mL, respectively. EPS 74, 76 and 89 were found to effective against biofilm formed by \u003cem\u003eK. pneumoniae\u003c/em\u003e, even at low (100 \u0026micro;g/mL) concentration, while EPS 61 (1000 \u0026micro;g/mL) had the highest effect with the inhibition ratio of 14% against \u003cem\u003eP. aeruginosa\u003c/em\u003e PA01.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibiofilm activity of EPSs on A) \u003cem\u003eE. coli\u003c/em\u003e 3055, B) \u003cem\u003eS. aureus\u003c/em\u003e RN4220, C) \u003cem\u003eK. pneumoniae\u003c/em\u003e 5108, D) \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, E) \u003cem\u003eE. faecalis\u003c/em\u003e JH2-2. Values are means of three replicates with standard errors shown.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003eof EPS (\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eInhibition ratio of different bacterial antibiofilm (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e41\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e38\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEPS 261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThere are limited number of studies regarding EPS production by thermophilic bacteria which include \u003cem\u003eG. thermodenitrificans\u003c/em\u003e, \u003cem\u003eA. gonensis, A. pushchinoensis, P. toebii, A. pallidus, Brevibacillus thermoruber\u003c/em\u003e and \u003cem\u003eA. kestanboliensis\u003c/em\u003e species (Nicolaus et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Arena et al. 2009; Minana-Galbis et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Radchenkova et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yasar Yildiz et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; A.I. Ahmed, 2018; Panosyan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Genc et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Karadayi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To the best of our knowledge, strains belonging to \u003cem\u003eA. suryakundensis, A. flavithermus, P.thermoglucosidasius\u003c/em\u003e species are the first to examine for their EPS production and characterization in our report. EPSs producing thermophilic bacilli, crude EPS, and protein amounts produced by themselves are summarized in (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThermophilic Gram positive bacilli with EPS production levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolation source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrude EPS yield (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein amount (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e HBB-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoil, Ortak\u0026ccedil;ı, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e419.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus gonensis\u003c/em\u003e HBB-61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermal pool, Alang\u0026uuml;ll\u0026uuml;, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus suryakundensis\u003c/em\u003e HBB-74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoil, Alang\u0026uuml;ll\u0026uuml;, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e269.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus flavithermus\u003c/em\u003e HBB-76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoil, Alang\u0026uuml;ll\u0026uuml;, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e186.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParageobacillus toebii\u003c/em\u003e HBB-78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermal mud, Alang\u0026uuml;ll\u0026uuml;, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e224.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAeribacillus pallidus\u003c/em\u003e HBB-89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermal mud, Davutlar, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e187.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParageobacillus thermoglucosidasius\u003c/em\u003e HBB-106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermal mud, G\u0026uuml;m\u0026uuml;şk\u0026ouml;y, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e352.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus kestanbolensis\u003c/em\u003e HBB-134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHot spring, Alang\u0026uuml;ll\u0026uuml;, Aydın\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e141.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e HBB-261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSediment, Yenice, Denizli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e357.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus\u003c/em\u003e sp. strain WSUCF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompost facility, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Wang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e ArzA-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArzakan geothermal spring, Armenia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Panosyan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus toebii\u003c/em\u003e ArzA-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArzakan geothermal spring, Armenia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Panosyan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBrevibacillus thermoruber\u003c/em\u003e 423\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGradechnista hot spring, Bulgaria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Yasar Yildiz et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus\u003c/em\u003e sp. R4-33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRadioactive radon hot spring, China\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Zhao et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAeribacillus pallidus\u003c/em\u003e 418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRupi hot spring, Bulgaria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Radchenkova et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus\u003c/em\u003e sp. 4004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydrothermal vent, Italy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Poli et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus\u003c/em\u003e (\u003cem\u003eParageobacillus\u003c/em\u003e) \u003cem\u003ethermantarcticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrater of Mount Melbourne, Antarctica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Aliyu et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus stearothermophilus\u003c/em\u003e 1A60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydrothermal vent, Italy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Gugliandolo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGeobacillus thermodenitrifcans\u003c/em\u003e B3-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydrothermal vent, Italy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Arena et al. 2009)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnoxybacillus tepidamans\u003c/em\u003e V264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVelingrad hot spring, Bulgaria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e111.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Kambourova et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Coorevits et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAnoxybacillus\u003c/em\u003e sp. R4-33, which is isolated from radioactive radon hot spring was the most EPS productive (1083 mg/L) thermophilic bacilli so far (Zhao et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eGeobacillus\u003c/em\u003e sp. WSUCF1 and \u003cem\u003eG. thermantarcticus\u003c/em\u003e were also reported as thermophiles with high EPS yields of 404 mg/L and 400 mg/L, respectively. \u003cem\u003eGeobacillus\u003c/em\u003e sp. TS3-9, (87 mg/L), \u003cem\u003eG\u003c/em\u003e. \u003cem\u003etepidanmans\u003c/em\u003e V264 (111.4 mg/L), \u003cem\u003eG. thermodenitrifcans\u003c/em\u003e ArzA-6 (76 mg/L), \u003cem\u003eG. toebii\u003c/em\u003e ArzA-8 (80 mg/L), \u003cem\u003eAeribacillus pallidus\u003c/em\u003e (53 mg/L), \u003cem\u003eGeobacillus toebii\u003c/em\u003e (50 mg/L) and \u003cem\u003eAnoxybacillus kestanbolensis\u003c/em\u003e (25.3 mg/L) showed productivity to a lesser extent (Radchenkova et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Panosyan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). EPS yields of our thermophilic isolates ranged between 117\u0026ndash;419 mg/L which means that their production capacities are comparable with those of other thermophiles reported, previously.\u003c/p\u003e \u003cp\u003eHu et al. obtained 4 fractions by ion exchange chromatography (DEAE-52) (EPS 1, EPS 2, EPS 3, EPS 4) with the yields % of 21.2-25.0-18.8-20.0%, respectively. These four fractions were then purified by gel filtration chromatography (Sephadex G-100). It was found to have 88.0-90.1-89.1-87.9 percent purification, respectively (Hu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Schiano Moriello et al. purified EPS of 4004 coded thermophilic bacteria belonging to the genus \u003cem\u003eGeobacillus\u003c/em\u003e, gel filtration chromatography (Sephadex G-50; 2.5 x 50cm) and ion exchange chromatography (Sepharose DEAE CL-6B; 1.5 x 40cm) were applied, respectively. Total EPS purification percentage were 80% and 70%, respectively (Schiano Moriello et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). It was observed that our study demonstrated higher purification yields than that of above-mentioned studies.\u003c/p\u003e \u003cp\u003eIt has been reported that the allowable level of hemolysis for biomaterials is 5%. The percentages of hemolysis for our EPSs at the highest concentration (5000\u0026micro;g/mL) were in the range of 0-0.3% for EPS 61, 76, 78, 106, 134, 261, while EPS 20 and EPS 89 showed hemolysis potential of 2.5% and 3.9%, respectively. Among the EPSs purified from nine thermophilic bacteria, only EPS 74 was evaluated as cytotoxic due to its high (11.1%) hemolytic percentage. It was observed that eight of the EPSs obtained from nine different bacteria used in our study were not cytotoxic.\u003c/p\u003e \u003cp\u003eAbinaya et al. reported that EPS obtained from \u003cem\u003eBacillus licheniformis\u003c/em\u003e Dahb1 had a cytotoxicity of 1.2% at a concentration of 5000\u0026micro;g/mL (Abinaya et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Genc et al., isolated EPS from \u003cem\u003eA. pushchinoensis\u003c/em\u003e G11 and revealed that EPS had a dose-independent cytotoxic effect on A-549, Caco-2 and HT-29 cell lines (Genc et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In another study, Arena et al. tested the cytotoxic effect of EPS from \u003cem\u003eG. thermodenitrificans\u003c/em\u003e B3-72 on human peripheral blood mononuclear normal cells and observed dose-dependent inhibition on healthy cells (Arena et al. 2009). As a result of cytotoxicity assay, Wang et al., showed no significant effect on HEK-293 cell viability when they tested EPS-1 and EPS-2 from \u003cem\u003eGeobacillus\u003c/em\u003e sp. WSUCF1 even at high concentrations (2 mg/mL of EPS-1 and 3 mg/mL of EPS-2) (Wang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the antibacterial activity tests of EPSs produced by lactic bacteria were reported many times in the literature, works on those produced by thermophiles are very rare. Tuşar et al., tested the antibacterial effects of EPSs by thermophilic \u003cem\u003eBacillus zhangzhounesis\u003c/em\u003e 2CA and \u003cem\u003eBacillus licheniformis\u003c/em\u003e 2CS strains against pathogenic \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e. EPS produced by \u003cem\u003eB. licheniformis\u003c/em\u003e 2CS showed the highest antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e (with 16 mm zone diameter) when grown in M3 medium (0.2% yeast extract\u0026thinsp;+\u0026thinsp;1% sucrose) (Tuşar et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, Gen\u0026ccedil; examined the antimicrobial activity of EPS by \u003cem\u003eA. pushchinoensis\u003c/em\u003e G11 against \u003cem\u003eSalmonella enteritidis\u003c/em\u003e ATCC13076, \u003cem\u003eAeromonas hydrophila\u003c/em\u003e ATCC 35654, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, \u003cem\u003eBacillus subtilis\u003c/em\u003e ATCC 6633, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853, \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e ATCC 13883 and \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 10231 with final densities of 0.01, 0.25, 1, 1.25 and 2.5 g/L of freeze-dried sample. According to the test results, authors stated that antibacterial and antifungal activities were not detected (Genc et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the studies by mesophilic EPSs, Rani et al., tested EPS from \u003cem\u003eLactobacillus gasseri\u003c/em\u003e FR4, and they reported antibacterial activity against \u003cem\u003eE. faecalis\u003c/em\u003e at a concentration of 10 mg/mL similarly to our results (Rani et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the study conducted by Ghalem, EPS obtained from the yogurt starter bacteria mixture exhibited antimicrobial activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e with an inhibitory zone of 13 mm and 9 mm, respectively (Ghalem, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The EPSs formed by \u003cem\u003eL. plantarum\u003c/em\u003e 47FE, and \u003cem\u003eL. pentosus\u003c/em\u003e 68FE showed inhibitory effect against representatives of both Gram-positive and Gram-negative bacteria and \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. typhimurium\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e were found to have the highest sensitivities in their study (Saif and Sakr, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe mechanism that enables exopolysaccharides to show antimicrobial activity is thought to be the functional groups of EPS (Abdalla et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is known that negatively charged EPSs which contain sulfate groups interact better with Gram-positive bacteria that have higher positive charge on their cell walls (Saif and Sakr, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This result may be due to the negative charge of thermophilic EPSs, and the sulfate groups they may contain affect bacterial cell surface communication.\u003c/p\u003e \u003cp\u003eThe DPPH radical scavenging activity of EPSs has been investigated in many studies and generally positive results have been demonstrated. It was reported that EPS of the thermophilic bacterium \u003cem\u003eGeobacillus\u003c/em\u003e sp. showed high antioxidant activity at a concentration higher than 8 mg/mL (Wang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The EPS produced by a thermo-halophilic bacteria \u003cem\u003eHalomonas nitroreducens\u003c/em\u003e strain WB1 had antioxidant properties at the concentration of 5.0 mg/ml. The EPS and ascorbic acid showed 83.3% and 90.2% of DPPH radical scavenging activity, respectively (Chikkanna et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In our study, EPS (76 and 134) obtained from thermophilic bacteria were found to have 86% and 91% activity at 1 mg/mL concentration, respectively. Additionally, at higher concentration the activity increased. EPS (76, 106, 134 and 261) showed 90-92-99-90% DPPH radical scavenging activity at 4 mg/mL concentration, respectively.\u003c/p\u003e \u003cp\u003eIt has been reported that the use of a compost fermented with thermophiles as feed prevents lipid peroxidation in the liver in rats. Under these conditions, antioxidants were not decreased in the livers of rats fed compost extract (Miyamoto et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). EPSs of \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e, EPS 1 and EPS 2 showed 41.45% and 50.43% lipid peroxidation inhibition activity at 4 mg/mL concentration, respectively (Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eL. paracasei\u003c/em\u003e subsp. \u003cem\u003eparacasei\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e EPS have been reported to have linoleic acid peroxidation inhibiting activities of 43.48%-27.57%, respectively, at 10 mg/mL (Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a previously published study, antioxidant activity was determined in \u003cem\u003eL. acidophilus\u003c/em\u003e EPS (2 mg/mL) by reducing power analysis at an absorbance value of 1.047 (Amiri et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the study conducted with EPS (5 mg/mL) of \u003cem\u003eL. rhamnosus\u003c/em\u003e, it was observed that the absorbance value of the reducing power was between the highest (0.2\u0026ndash;0.3). In addition, it has been reported that the fact that the reducing power antioxidant activity is higher than the others is due to the excess sulfate content in the structure of EPS and the low molecular weight (Hu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It was determined that \u003cem\u003eL. plantarum\u003c/em\u003e EPS has a reducing power of 1.38 at a concentration of 2 mg/mL (Dilna et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In our study, EPS (76 and 134) absorbance values of 0.578\u0026ndash;1.176 were measured at 1 mg/mL concentration, respectively. Concentration-dependent antioxidant activity increased. EPS (76, 106, 134, 261) at 5 mg/mL concentration and absorbance values of 2.128-1.026-4.603-0.621, respectively, and reducing power and antioxidant activity were determined.\u003c/p\u003e \u003cp\u003eDilna et al. found 40% inhibition of alpha-amylase for \u003cem\u003eL. plantarum\u003c/em\u003e RJF4 EPS (0.8 mg/mL) and 98% of acarbose at the same concentration (Dilna et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Jiang et al. studied α-amylase inhibition of EPS (2 mg/mL) of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e. As a result, inhibition of α-amylase was determined as 37.4% (Jiang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Xu et al. studied α-amylase inhibition of two EPSs of \u003cem\u003eBacillus licheniformis\u003c/em\u003e, BL-P1 and BL-P2 at 150 \u0026micro;g/mL. According to the findings, EPS BL-P1 and BL-P2 showed 67.24% and 75.63% inhibition levels, respectively, while positive control, acarbose, had an inhibition percentage of around 90% at the same concentration (Xu et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It can be concluded that alpha-amylase inhibition levels exhibited by our EPSs were relatively lower than those reported by other authors.\u003c/p\u003e \u003cp\u003e \u003cem\u003eL. delbrueckii bulgaricus\u003c/em\u003e EPS tested for prebiotic activity determination and the index was found to be between 7.9 and 10.1 (Hussein et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Lee et al. investigated the prebiotic activity of \u003cem\u003eL. paracasei\u003c/em\u003e EPS at a concentration of 20 mg/mL and determined that the prebiotic index was between 15\u0026ndash;25 (Lee et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). EPS produced by \u003cem\u003eEnterobacter\u003c/em\u003e sp. ACD2 was reported as non-active because the prebiotic index was less than 1 when it was tested at concentration of 15 mg/mL (Almutairi and Helal, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In our study, higher prebiotic activity results were obtained compared to the literature.\u003c/p\u003e \u003cp\u003eIt was reported by Al-Nahas et al. that EPS (2 mg/tube) from \u003cem\u003ePseudoalteromonas\u003c/em\u003e sp. AM exhibited a fibrinolytic activity score of +\u0026thinsp;3 (Al-Nahas et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Almutairi and Helal studied the fibrinolytic activity of EPS (2 mg/mL) of \u003cem\u003eEnterobacter\u003c/em\u003e sp. ACD2. Hemoclar (2 mg/mL) was used as standard. Activity results were determined as 100% and 75% lysis, respectively (Almutairi and Helal, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Saif and Sakr investigated the fibrinolytic activity of EPSs of \u003cem\u003eL. plantarum\u003c/em\u003e 47FE, and \u003cem\u003eL. pentosus\u003c/em\u003e 68FE in their study. They reported that both EPSs (10 mg/mL) showed\u0026thinsp;+\u0026thinsp;5 fibrinolytic activity (Saif and Sakr, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It can be concluded that the blood clot lytic activity of thermophilic EPSs is low.\u003c/p\u003e \u003cp\u003eIn the study conducted by Gen\u0026ccedil; et al, EPS of \u003cem\u003eAnoxybacillus pushchinoensis\u003c/em\u003e showed antibiofilm activity at a concentration of 2000 \u0026micro;g/mL. It inhibited biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae, B. subtilis, C. albicans\u003c/em\u003e and \u003cem\u003eS. enteritidis\u003c/em\u003e with inhibition rates between 4.29\u0026ndash;10.46% (Genc et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Zammuto et al., investigated the antibiofilm and antiadhesive effects of the EPS B3-15, produced by a thermophilic strain of \u003cem\u003eBacillus licheniformis\u003c/em\u003e (B3-15), on different surfaces such as, a polyvinyl-chloride medical device, polystyrene microplates and human epithelial nasal cells. The EPS was effective on bacterial adhesion of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 29213 at a concentration of 300 \u0026micro;g/mL but had no activity on mature biofilms. EPS B3-15 also reduced the adhesion of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e (five logs-scale and one log, respectively) on human nasal epithelial cells (Zammuto et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A novel thermophilic EPS, named as EPS1-T14 produced by \u003cem\u003eB. licheniformis\u003c/em\u003e strain T14 was evaluated for its effects on biofilm formation by \u003cem\u003eStaphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e clinical strains. The EPS1-T14 was found to active on biofilms formed by all the target pathogenic bacteria without antibacterial effects and it showed a dose-dependent inhibitory effect depend on the strain tested (Span\u0026ograve; et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Since only EPS261 was found to be effective against \u003cem\u003eE. faecalis\u003c/em\u003e JH2-2 during antibacterial activity assays, antibiofilm ability of other exopolysaccharides could not be caused by growth inhibition. It is thought that the antibiofilm activity occurs by the EPS preventing the attachment of pathogenic bacteria and thus the formation of biofilm. EPS inhibits the initial attachment and association of bacteria. It does this by reducing cell-cell surface communication and weakening cell surface modifications (Kim and Kim, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Thus it can be proposed that EPSs, except for produced by HBB 261, might interfere with the steps of biofilm formation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, exopolysaccharides produced by thermophilic strains belonging to species of \u003cem\u003eA. suryakundensis, A. flavithermus, P. thermoglucosidasius\u003c/em\u003e have been isolated and characterized for the first time in this report. Beside of this, a lot of biological activity tests were performed for EPSs, which have been rarely studied for thermophilic ones. Nine thermophilic EPSs were highly purified by gel filtration and ion exchange chromatography. According to our results, most remarkable biological characteristics of EPSs are prebiotic and antioxidant activities and their lack of cytotoxicity. EPSs produced by nine thermophilic isolates were also found to have antimicrobial, anti-diabetic and fibrinolytic properties. Therefore, EPSs of thermophilic origin from our study have a high potential for use in health and food fields. Future investigations on chemical structures of EPSs will reveal new insights into thermophilic exopolysaccharides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Aydın Adnan Menderes University (T\u0026uuml;rkiye) Research fund, by the project FEF-20026. Mehmet AYTAR received funding from Higher Education Council of T\u0026uuml;rkiye (YOK) as a participant of the 100/2000 PhD scholarship.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.A., wrote the first draft, prepared Figures 2-7 and Tables 1-5.G. B. wrote the main manucsript text, prepared Table 6. D.A.U. prepared Figure 1, edited language.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe sequence data obtained in the study were deposited in GenBank, under accession numbers OR896922.1.- OR897028.1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmed AIS, Ibrahim AI, Agha MK (2018) \u003cem\u003eBrevibacillus\u003c/em\u003e spp. in Agroecology: The beneficial impacts in biocontrol of plant pathogens and soil bioremediation. 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Appl Biochem Biotechnol 172; 2732\u0026ndash;2746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12010-013-0680-6\u003c/span\u003e\u003cspan address=\"10.1007/s12010-013-0680-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Thermophilic Bacteria, Exopolysaccharide, Purification, Biological Activity, Characterization","lastPublishedDoi":"10.21203/rs.3.rs-4679730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4679730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA total of one hundred seven thermophilic bacteria were screened for their ability to produce exopolysaccharides. Nine isolates belonging to \u003cem\u003eGeobacillus\u003c/em\u003e, \u003cem\u003eParageobacillus\u003c/em\u003e, \u003cem\u003eAeribacillus\u003c/em\u003e and \u003cem\u003eAnoxybacillus\u003c/em\u003e genera with highest EPS quantities were chosen and purified EPSs used for biological activity studies. EPS yields of selected thermophilic bacteria ranged between 117\u0026ndash;419 mg/L. Among the tested EPSs, 61, 106 and 261 showed antibacterial effect against \u003cem\u003eE. faecalis\u003c/em\u003e JH2-2 at a concentration of 15 mg/mL. EPS samples had significant antioxidant capacity, especially EPS 134, with highest DPPH radical scavenging activity of 100% at a concentration of 5 mg/mL and strongest reducing power. EPS 20, showed highest lipid peroxidation inhibition effect at a rate of 31%. EPSs displayed weak alpha amylase inhibition activity when compared with standart acarbose. The prebiotic indices of EPSs 20, 61, 76, 89, 134 and 261 were found to be higher than that of inulin, a representative prebiotic carbohydrate for all tested lactic acid bacteria in the study. All examined EPSs inhibited the biofilms formed by various bacteria depending on the test strain. Results indicated that thermophilic EPSs had remarkable antioxidant, prebiotic, and antibiofilm activities. Therefore, EPSs characterized in this study may have technological applications in health and food fields.\u003c/p\u003e","manuscriptTitle":"Production and Biological Activities of Exopolysaccharides Synthesized by Thermophilic Bacilli Isolated from Hot Springs in Türkiye","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 17:24:35","doi":"10.21203/rs.3.rs-4679730/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-06T09:07:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-03T09:32:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-02T17:34:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192509074530679282969819463722395960049","date":"2024-07-31T10:02:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152640922363946078780033445780370302344","date":"2024-07-15T14:45:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-15T11:59:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-05T15:13:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-04T13:38:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2024-07-03T10:34:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f8d55e31-dcc7-4715-bfa8-9fe2ecae4e0b","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:09:03+00:00","versionOfRecord":{"articleIdentity":"rs-4679730","link":"https://doi.org/10.1007/s10123-024-00588-6","journal":{"identity":"international-microbiology","isVorOnly":false,"title":"International Microbiology"},"publishedOn":"2024-09-09 15:57:06","publishedOnDateReadable":"September 9th, 2024"},"versionCreatedAt":"2024-07-29 17:24:35","video":"","vorDoi":"10.1007/s10123-024-00588-6","vorDoiUrl":"https://doi.org/10.1007/s10123-024-00588-6","workflowStages":[]},"version":"v1","identity":"rs-4679730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4679730","identity":"rs-4679730","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}