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.