Introduction

Microbial polysaccharides are secreted by the cell as capsular polysaccharides or exopolysaccharides. Exopolysaccharides are high molecular weight natural polymers that are produced by plant, algae, fungi, and bacteria (Dilna et al. 2015; Staudt et al. 2004). Exopolysaccharides (EPS) are large polymers of glycosides linked by glycosidic bonds. Their molecular weights are high and range from 100 to 2000 kDa. The monomeric ratio and additive degree provide them with electrical charge (cationic, neutral, anionic), chain conformation (straight, branched) that gives them their hydrophilic or hydrophobic nature, and their unique physicochemical and functional characteristics (López-Ortega et al. 2021). EPSs are polysaccharides consisting of long-chain, branched, repeating sugars or sugar derivatives. They are classified into two groups depending on the composition of the repeating units and the biosynthesis pathway: homopolysaccharides (HoPS) and heteropolysaccharides (HePS) (Mende et al. 2016).

Although many bacteria can produce exopolysaccharides, bacteria isolated from extreme environments have become more promising because of their adaptation to harsh conditions. In exopolysaccharide production, thermophilic bacteria provide various advantages such as shorter fermentation time, better mass transfer and reduced viscosity of the fermentation broth. It has been reported in the literature that Bacillus and Geobacillus genera are good EPS producers (Panosyan et al. 2018; Radchenkova et al. 2013; Wang et al. 2021). Examples of EPS production by thermophilic bacteria in the literature are as follows, Parageobacillus thermantarcticus ex Bacillus thermantarcticus, (Nicolaus et al. 2004), Geobacillus tepidamans (Wang et al. 2017), Geobacillus sp. TS3-9, (Wang et al. 2017), G. toebii (Panosyan et al. 2018; Radchenkova et al. 2013), G. thermodenitrificans (Panosyan et al. 2018), Aeribacillus pallidus, (Radchenkova et al. 2013), Anoxybacillus kestanbolensis, (Radchenkova et al. 2013), Brevibacillus thermoruber (Yıldız et al. 2014), A. puschinoensis (Genç et al. 2021), Geobacillus sp. WSUCF1 (Wang et al. 2021).

Exopolysaccharides have strong biological activities. EPSs show remarkable antimicrobial activity against both gram-positive and gram-negative bacterial pathogens. Its antagonistic activity against bacterial pathogens has been reported in many studies (Ayyash et al. 2020; Rani et al. 2018; Saha et al. 2004; Wu et al. 2010). EPSs can also have an antioxidant activity, and they significantly delay or inhibit oxidation at low concentrations, in foods and body. These compounds protect the body against the different types of oxidative damage caused by reactive oxygen species (Saha et al. 2004). Besides, EPSs can have an antidiabetic role by inhibiting alpha amylase enzyme (Dilna et al. 2015). Recently, the prebiotic effect has become even more important. The number of probiotic bacteria is increased by prebiotics. These are non-digestible food ingredients that are beneficial to the host by triggering the growth of bacterial species already present in the colon. Thus, prebiotics improve host health (Tsuda & Miyamoto 2010). Moreover, EPSs with fibrinolytic activity are important (Al-Nahas et al. 2011) due to their potential in medical for paralyzed patients. Finally, some of EPSs are known to prevent biofilm formation. Microbial biofilm studies have started to attract attention in recent years. Bacteria in the biofilm exhibit high resistance to antibiotics, disinfectants, and host immune system clearance. Biofilm is very important in the medical, environmental and industrial field (Stepanović et al. 2007).

In this study, the EPS production conditions (time, temperature, pH, NaCl concentration, sugar type and sugar concentration) of a thermophilic bacterium HBB 111 (identified as Geobacillus thermodenitrificans HBB 111) were optimized, and the obtained exopolysaccharide was purified by ion exchange chromatography and gel filtration chromatography. Characterization of the purified exopolysaccharide was carried out by TLC, FTIR–ATR, NMR, GC–MS, SEM, and SEM–EDX techniques and hemolytic activity, antimicrobial activity, antioxidant activity, alpha-amylase enzyme inhibition activity, prebiotic activity, fibrinolytic activity and antibiofilm activity properties were examined.

Materials and methods

Bacterial isolate and identification

A thermophilic bacterium, HBB-111 was previously isolated by Dr. Gamze Başbülbül (Başbülbül et al. 2009, 2012) and used for EPS production. Bacterial isolate was stored at −20 °C and grown on Tryptic Soy Agar (TSA) medium at 55 °C for regrowth. Genomic DNA sample of HBB-111 was isolated using easyDNA genomic DNA isolation kit (R-Tech, Turkey) according to manufacturer instructions. 20F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1390R (5′-GACGGGCGGTGTGTACAA-3′) primers were used to amplify16S rDNA gene by PCR. Amplicon was sequenced by a company (MEDSANTEK) and after alignment with the sequences from NCBI database closest phylogenetic relative of isolate was determined (http://www.ncbi.nlm.nih.gov).

Isolation and purification of EPS

For preincubation in TSB medium, thermophilic bacteria were incubated overnight at 55 °C in a 180 rpm shaking incubator for 24 h. The preincubated culture was inoculated in 5% volume of modified basal 2 medium (Wang et al. 2021). Thermophilic bacterium was incubated with 20 g/L sucrose modified basal medium 2 (B2) for 24 h at 55 °C. The effects of time, temperature, pH, NaCl concentration, sugar type and sugar concentration on the EPS production of the thermophilic isolate were examined and EPS production conditions were also optimized. Then, culture supernatant was taken and autoclaved, and then the volume (300 mL) was reduced to 10 mL by evaporation in an incubator at 70 °C for 3 days (Li et al. 2015; Patwal and Baranwal 2021; Radchenkova et al. 2013). Trichloroacetic acid (TCA) solution was added to the concentrated supernatant with a final concentration of 4% and incubated in a mixer at room temperature for 30 min. The culture was then centrifuged at 2500×g for 10 min. Pure alcohol, twice its volume, was added to the supernatant and kept at + 4 °C for 24 h for precipitation. It was then centrifuged at 2500×g for 10 min. The pellet was dissolved with distilled water and lyophilized and stored at −80 °C for analysis (Bajpai et al. 2016; Wang et al. 2015). While the amount of sugar in the produced raw EPS samples was determined by the phenol–sulfuric acid method (Masuko et al. 2005), the amount of protein was determined by the Bradford method (Bradford 1976).

EPS purification was first performed by ion exchange chromatography column (1,2 cm × 50 cm) with Diethylaminoethyl (DEAE)-Cellulose by using 1.0 mL of EPS (40 mg/mL). 25 mL of solutions were passed through the column as NaCl gradient (0.1/0.25/0.5/0.75/1 M) at flow rate 1.0 mL/min. Secondly, both size separation and desalting were performed by gel filtration chromatography column (1,2 cm × 30 cm) with Sephadex G-100 at flow rate 0.5 mL/min. The column was loaded with 15 mL EPS obtained from ion exchange chromatography. Following, 25 mL of distilled water was passed through the column and samples were collected in 5 mL fractions each.

Determination of the chemical structure of EPS

Thin layer chromatography was used to analyze the products formed as a result of the sulfuric acid hydrolysis of EPS (Dilna et al. 2015; Jouault et al. 2001). 5 µL of different sugars (2 mg/mL of maltose, galactose, fructose, glucose, sucrose) and hydrolyzed EPS sample at 2 mg/mL concentration were applied to the layer and air-dried. A 5:3:2 mixture of n-butanol: ethanol: water was prepared for the mobile phase and added to the thin layer chromatography tank, the dried layer was placed in the tank and the samples were carried out. After the run-up, the sheet was air-dried. For the dyeing process, 0.2% orcinol prepared in sulfuric acid: methanol 1:9 mixture was used. The color change was observed by spraying the dye solution and keeping it in an oven at 100 °C for 10 min.

FTIR-ATR spectroscopy is widely used for the preliminary determination of functional groups in the structure of EPSs. FTIR-ATR spectroscopy of thermophilic EPS was performed using the FTIR-ATR device (Perkin Elmer Spectrum2). For this purpose, the FTIR-ATR spectrum was taken by placing 5 mg of exopolysaccharide on the sample loading part and the obtained spectra were evaluated (Seveiri et al. 2020). NMR analysis was performed for the structural examination of the produced EPS by using the NMR device (Agilent Technologies Varian 400). In order to determine the monosaccharide content of the purified exopolysaccharide by GC–MS analysis, 10 mg of EPS sample was hydrolyzed with 2 M 2 mL trifluoroacetic acid (TFA) for 2 h at 100 °C. After hydrolysis, TFA was evaporated. Then, methanol was added to the sample and the sample was evaporated again (Amiri et al. 2019). Chromatograms were taken by injecting 1 µL of the hydrolysate into the GC–MS device (Shimadzu QP2010 ultra). The GS-MS operation conditions: carrier gas: helium; flow rate: 1.0 mL/min; temperature: 250 °C. SEM analysis of the surface morphology and structure of the purified EPS (10 mg) was examined with the SEM (Zeiss Sigma 300) device. For this purpose, the sample was coated with gold and given to the SEM–EDX device. Obtained SEM photographs and EDX analysis were evaluated.

Determination of biological activities of EPS

Hemolytic activity was first evaluated qualitatively on blood agar as described by Filik and Kubilay2020. Briefly, the medium was prepared by adding 5 mL of sheep blood sample to 100 mL of basal medium and poured into petri dishes. Then EPS solutions (0.025–0.50–1,0–2.5 and 5.0 mg/mL) were filled into the wells by the agar well method and incubated at 37 °C for 24 h. The hemolytic activity test was also quantitatively performed according to the method applied by Abinaya et al. 2018. For this purpose, EPS solutions were added to each test tube and mixed by gently inverting. PBS was used as blank and distilled water was used as control. After separating the supernatant, absorbance was measured at 540 nm, and hemolysis percentage (%) was calculated (Guezennec et al. 2012; Li et al. 2018).

The agar well diffusion method was used to examine the antimicrobial activity. Test microorganisms (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Proteus vulgaris) were incubated overnight at 37 °C on Nutrient agar (NA). Candida albicans was incubated overnight at 28 °C on Potato Dextrose Agar (PDA). The next day, suspensions at 0.5 McFarland turbidity were spread onto Müeller Hinton Agar (MHA) petri dishes. Gentamicin (10 µg) and flucanazole (25 µg) discs were used as control for bacteria and yeast, respectively. Exopolysaccharide (1,5,15 mg/mL) was added to the wells as 100 µL. Zones of inhibition around the wells were measured after 24 h of incubation (El Essawy et al. 2016).

Determination of DPPH radical scavenging activity was made according to Blois (1958) (Cao et al. 2020). BHT and ascorbic acid were used as standard antioxidants. The percent inhibition value was calculated from the formula % inhibition = (Acontrol−Asample)/Acontrol × 100 (Acontrol: Absorbance of control, Asample: Absorbance of sample/standard antioxidant), and the IC50 values were calculated. Determination of reducing power described by Lin et al. 2012was performed.

Alpha-amylase enzyme (from Aspergillus oryzae) inhibition activity assay was performed as described method by Dilna et al. 2015. Results are expressed as percent inhibition of α-amylase and calculated according to the formula, % inhibition = (Acontrol−Atest)/Acontrol × 100 (OD control; acarbose, OD test; EPS).

Prebiotic activity of EPS was determined by using Lactobacillus acidophilus 66, L. bulgaricus 118 and L. plantarum DSM 20174 bacteria and examining bacterial culture densities in liquid medium (El Essawy et al. 2016).

The fibrinolytic activity of EPS was investigated by modifying the USP 28-NF 23 pharmacopeia (2005) method. For this purpose, blood clot lysis was monitored using sheep blood and lysis levels were expressed at 5 diagnostic levels (+ 1, + 2, + 3, + 4, + 5) (Li et al. 2014; Saha et al. 2004).

The antibiofilm activity was performed as described method by Stepanović et al. 2007. Antibiofilm activity of EPS was tested on biofilm-forming bacteria Escherichia coli (3055), Klebsiella pneumoniae (5108), Pseudomonas aeruginosa (PAO1) and Staphylococcus aureus (RN4220). 96-well microplates were used to assess the biofilm formation of bacterial strains by spectrophotometrically at 595 nm (Li et al. 2014). The antibiofilm activity was expressed as percentage inhibition of biofilm formation.

Statistical analysis

Data were statistically analyzed to determine statistical differences in the effects of the tested concentrations by one-way analysis of variance (ANOVA) using SPSS 2317.0 software. p < 0.05 value was used to identify statistically significant differences. Experimental results were expressed as the means of three parallel experiments ± standard deviation (S.D.).

Results and discussion

BLAST result

As a result of BLAST analysis, the similarity rate of HBB 111 with other species in the database was determined. HBB 111 showed highest (94,35%) homology to Geobacillus thermodenitrificans strain ArzA-9/1 the isolate after alignment of 580 bp section of 16S rDNA gene. The sequence data was deposited in GenBank, under accession number OR896966.

Isolation and purification of EPS

A limited number of thermophilic bacteria have been reported to be EPS producers in the literature. For example, Panosyan et al. (2018) determined that only two of the 16 thermophilic bacilli isolated from the Arzakan (Armenia) geothermal springs were EPS producers. One of these species was reported to be 99.7% similar to G. thermodenitrificans subsp. calidus F84bT (ArzA-6), and the other was reported to be 98.9% similar to G. toebii NBRC 107807 T (ArzA-8) (Panosyan et al. 2018). Similarly, in our study, the EPS producer bacterium was the Geobacillus species.

The effects of time, temperature, pH, NaCl concentration, sugar type and sugar concentration on the EPS production conditions of the Geobacillus thermodenitrificans HBB111 isolate were examined, and the optimum EPS production conditions were selected as 24 h incubation, 55 °C, pH 8.0, NaCl-free medium and 2.4% lactose-containing medium (Fig. S1). The amount of EPS produced under these conditions was found to be 44.0 mg/L. The amount of protein in the isolated raw EPS sample was calculated as 1.9 mg/L. The obtained EPSs were lyophilized, dried and stored for use in chromatographic processes. Purification of EPS from the raw sample was carried out using ion exchange chromatography and then gel filtration chromatography, and the results are presented in Fig. 1. The obtained results showed that 0.1 M NaCl concentration was suitable for EPS purification and the presence of a single exopolysaccharide as a result of purification. The purification percentage of ion exchange chromatography was calculated as 39.4% and the purification percentage of gel filtration chromatography was calculated as 94.1%. The initial EPS concentration loaded on the columns was chosen as 40 mg/mL, and the amount of EPS obtained as a result of the chromatographic steps was found to be 14.84 mg/mL. As a result of the purification processes, the amount of protein in the exopolysaccharide sample was not determined. The purified EPS was named as EPS 111 and stored for characterization.

Fig. 1
figure 1

Purification of EPS 111 by ion exchange chromatography (A) and gel filtration chromatography (B).

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Characterization of EPS

Thin layer chromatography of EPS 111 was investigated comparatively with standard sugars of maltose, galactose, fructose, glucose, and sucrose, and shown in Fig. S2. From the thin layer chromatogram, it is seen that the exopolysaccharide contains glucose, fructose, galactose sugars because of sulfuric acid hydrolysis.

FTIR-ATR analysis was performed for the characterization of purified EPS 111 and is shown in Fig. 2A. In the FTIR analysis of EPS, the band at 3261 cm−1 is due to stretching vibrations of the O–H groups of sugars and the band at 2931 cm−1 is due to C–H stretching vibrations. The vibrations seen at 1626 and 1409 cm−1 belong to the asymmetric and symmetric stretching vibrations of the carboxyl (-COOH) groups. Peaks at 1103 and 1042 cm−1 are due to C–O–H stretching vibration and C–O–C stretching vibration of the pyranose ring, respectively (Hu et al. 2021). The peak at 830 cm−1 is due to the α-glycosidic bond. It is the fingerprint area between 800–1200 cm−1 and is useful in diagnosing different sugars (Amiri et al. 2019). In addition, Li et al. reported that the peak at 922 cm−1 originates from the β-pyranose ring of a hexose and the β-pyranose ring of the peak mannose units at 830 cm−1 (Li et al. 2014).

Fig. 2
figure 2

A FTIR-ATR spectrum, B Proton NMR analysis, C 13C NMR analysis of EPS 111.

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Proton NMR and 13C NMR analysis results of exopolysaccharides are shown in Fig. 2B and C, respectively. Two signals between 4.5 and 5.5 ppm as a result of proton NMR represent anomeric C. Both of these may belong to the α-pyranose ring. On 13C NMR, the signal seen at 103.49 ppm indicates β-anomeric C, the signal seen at 91.99 ppm indicates α-anomeric C, and the signal seen at 81.18 indicates furanosidic C shifts. In addition, the signals seen between 60 and80 ppm are due to shifts of C2-C6 carbons (Hu et al. 2020).

GC–MS analysis of EPS 111 was performed, and the obtained chromatogram is presented in Fig. 3. When the GC–MS chromatogram is examined, hydroxymethyl furfural (RT 9432 min) and levulinic acid (pentanoic acid, 4 oxo, RT 4746 min) peaks are seen at different retention times. It has been reported in the literature that hexose sugars (especially glucose) are hydrolyzed in TFA to form hydroxymethyl furfural and levulinic acid (Marzialetti et al. 2008). It is also known that pentose sugars form furfural by acid hydrolysis and may result from the breakdown of a furfural-5-methyl pentose sugar, which occurs in 3.66 min. The peak at 5,436 min in the chromatogram belongs to maltol. It is known that maltol is formed via dehydration by Maillard reaction as a result of heating reducing sugars with amino acids (Okur and Seydim2011; Yıldız et al. 2010).

Fig. 3
figure 3

GC-MS chromatogram of EPS 111.

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The surface structure and morphology of EPS 111 were examined by SEM device. For this purpose, photographs of the exopolysaccharide were taken at different magnifications (100X, 1.00KX, 5.00KX, 10.00KX) and are shown in Fig. 4A–D, respectively. The photographs obtained showed that the exopolysaccharide has an amorphous, rough, and layered dense polymeric surface. The obtained EDX result is presented in Fig. 4E, revealing that the exopolysaccharide is densely composed of C and O.

Fig. 4
figure 4

Scanning electron microscope (SEM) images A 100X, B 1.00KX, C 5.00KX, D 10.00KX, and E Energy dispersive X-ray spectroscopy (EDX) analysis of EPS 111

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Biological activities of purified EPS

Hemolytic activity test was performed to determine whether the EPS 111 were cytotoxic after purification. As a result of qualitative cytotoxicity method (Fig. 5A and B) no hemolytic activity was detected for EPS 111. Figure. 5C represents hemolytic potential of EPS 111 with 5 different concentrations (25–500-1000–2500-5000 µg/mL), while Fig. 5B represents positive control/S. pyogenes. Quantitative cytotoxicity tests of EPS 111 were evaluated according to its hemolytic potential on red blood cells, and results are given in Fig. 5C. It has been reported that the allowable level of hemolysis for biomaterials is 5%. EPS 111 showed 2,3% cytotoxicity at 5000 µg/mL concentration, so it was evaluated as not cytotoxic.

Fig. 5
figure 5

Qualitative determination of cytotoxicity of EPSs by hemolytic potential test (A: EPS 111, B: P.C./S. pyogenes).C Hemolysis percentage of EPS 111. Values are means of three replicates with standard error bars shown.

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The antimicrobial activity of EPS 111 against E. coli, E. faecalis, P. aeruginosa, S. aureus, P. vulgaris, C. albicans pathogens was investigated, and it was determined that EPS 111 demonstrated antimicrobial activity only against E. faecalis (Fig. 6). EPS 111 showed antimicrobial activity at the concentration of 15 mg/mL, with the zones of 21 mm.

Fig. 6
figure 6

Agar well diffusion test of EPS 111 on E. faecalis JH2-2 bacteria.

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To evaluate the antioxidant activity of EPS 111, DPPH radical scavenging activity and reducing power were carried out. The DPPH radical scavenging activity of the exopolysaccharide was demonstrated in Fig. 7A, and it was observed that the % inhibition rate increased with increasing EPS concentration and the IC50 value was calculated as 585 µg/mL. The reducing power of exopolysaccharides was also studied, and experimental results are given in Fig. 7B. In the reducing power analysis, the color change that occurs with the reduction of Fe3+ to Fe2+ is examined as an indicator of antioxidant activity. In the light of the results obtained, it was seen that the reducing power increased with the increase of EPS concentration.

Fig. 7
figure 7

A DPPH radical scavenging activity, and B Reduction Power of EPS 1

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The determination of α-amylase inhibition activity was performed to evaluate the potential of EPS 111 for use in the antidiabetic field. The α-amylase inhibition activity of exopolysaccharides was measured at the concentration of 800 µg/mL. While acarbose, used as a standard, showed 37% activity at 1000 µg/mL concentration, EPS 111 showed the inhibition activity 12% at 800 µg/mL. The prebiotic activities of EPS 111 were investigated using lactic acid bacteria and enteric bacteria. EPS was used at the concentration of 2 mg/mL. Prebiotic indexes against L. acidophilus 66, L. bulgaricus 118 and L. plantarum DSM 20174 were found to be 4.33, 4.70 and 5.36, respectively. Prebiotic indexes being greater than 1 showed that EPS 111 has prebiotic activity. The fibrinolytic activity of EPS 111 in the study was investigated to determine their capacity to dissolve blood clots and thus their potential as drugs. Evaluation of fibrinolytic activity results was made by considering the amount of clot fragmentation between (+ 1 and + 5) levels. The results are given in Table 1. Clot fragmentation activity pictures of EPS 111 demonstrated in Fig S3andS4. When the results obtained were examined, it was seen that EPS 111 had moderate (+ 3) fibrinolytic activity.

Table 1 Fibrinolytic activity results of EPS 111 (2 mg/3 mL), P.C. (positive control: Actilyse 50 mg), N.C (negative control)
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Antibiofilm activity of EPS 111 was tested against different bacteria, and the results are given in Fig. 8. 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). In this study, it was determined that the biofilm formation of E. faecalis was most inhibited. Inhibition rate of EPS 111 at 100 µg/mL concentration was 41%.

Fig. 8
figure 8

Antibiofilm activity of EPS 111 on A E. coli 3055. B K. pneumoniae 5108.C S. aureus RN4220. D P. aeruginosa PAO1, E E. faecalis JH2-2. Values are means of three replicates with standard error bars shown.

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Thermophilic bacteria generally exhibit a high growth rate. This leads to a shortening of their doubling times and offer advantage for metabolite production (Panikov et al. 2003). Among the thermophilic endospore forming bacteria, the highest EPSs production was observed for the Anoxybacillus sp. R4-33 strain (1083 mg/L)isolated from a radon hot spring in China (Zhao et al. 2014), followed by B. thermoruber strain 423 (863 mg/L) isolated from Gradechnista hot spring in Bulgaria (Yildiz et al. 2014).

The structural analysis revealed that heteropolysaccharide composition of EPS111 was similar to those from other thermophilic bacteria. (Table 2). Moreover, Panosyan et al. 2018, stated that, thermophilic EPSs are heteropolysaccharides which composed of glucose, mannose and galactose generally as we detected for EPS111.

Table 2 EPS production yields, chemical compositions and biological activities of EPSs from thermophilic bacilli
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The biological activities of thermophilic EPSs have been studied less than those excreted by mesophilic ones. Despite of the relatively low yield of EPS111, as far as we know, this is the most detailed report regarding an EPS producing thermophile in terms of biological activities. As can be seen from Table 2, the antioxidant, antimicrobial and antitumor activities of some thermophilic EPSs have been reported in the literature. However, in our study, a total of seven biological activities of EPS111 were examined and detected. Purified EPS 111 had low cytotoxicity (2.3%) and exhibited high antioxidant capacity and remarkable antidiabetic, prebiotic and fibrinolytic activities.

Conclusions

In recent years, studies on thermophilic bacteria producing EPS have been increasing and the biological activities of the produced exopolysaccharides have been demonstrated and characterized. Thermophilic EPSs are promising in these areas due to their biological and therapeutic potential, as well as their use as functional food ingredients. In this study, optimization of EPS production by Geobacillus thermodenitrificans HBB 111 was done and EPS 111 was purified using ion exchange and gel filtration chromatography techniques. Purified EPS was characterized, and its biological potential was examined by different methods such as hemolytic activity, antimicrobial activity, antioxidant activity, antidiabetic activity, prebiotic activity, fibrinolytic activity and antibiofilm activity. It was observed that the purified EPS in this study had multiple biological activities, unlike those in the literature. The use of natural source EPSs instead of synthetic or compounds with predicted toxicity is valuable in terms of biotechnology and biomedicine, and the EPS111 purified in this study has the potential to be used in this context.