Improved levan production by novel Calidifontibacillus erzurumensis LEV207 using one variable at a time approach

Improved levan production by novel Calidifontibacillus erzurumensis LEV207 using one variable at a time approach | 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 Improved levan production by novel Calidifontibacillus erzurumensis LEV207 using one variable at a time approach Omkar Prakash Palkar, Keerthi Rayasam, Vidyullatha Peddireddy, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4834548/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2024 Read the published version in International Microbiology → Version 1 posted 12 You are reading this latest preprint version Abstract Levan, an exopolysaccharide, has applications in the medical and food sectors. Under natural conditions, levan is produced at a low concentration by microorganisms. This current study focuses on optimizing levan production by a one-variable-at-a-time approach from a novel marine bacterium, Calidifontibacillus erzurumensis LEV207. The growth and levansucrase production by C. erzurumensis LEV207 were studied to optimize the media components and conditions. Sucrose and yeast extract proved to be a good levansucrase inducer. The pH of 6.0 and temperature of 30°C, along with MgSO 4 as salt, increased the enzyme activity. Sucrose and yeast extract at the concentration of 50% and 1%, respectively, produced the maximum enzyme activity of 427.4 µmol/mL/min after 48 hrs of incubation. The optimization process increased the levan (EPSLEV207) production 7-fold with a final yield of 65.58 g/L. To confirm the structure and nature of EPSLEV207, NMR and XRD were performed, and the functional groups present in EPSLEV207 were determined using FTIR. Chemical hydrolysis of levan confirmed fructose as the monomer, making EPSLEV207 a homopolysaccharide. EPSLEV207 exhibited 49% and 22% radical scavenging activity against DPPH and ABTS radicals, respectively, and promoted the growth of probiotic strain. EPSLEV207 showed an 88% water solubility index and 78% water retention capacity. The optimization process has increased the yield of levan up to 7-fold, highlighting the potential of C. erzurumensis LEV207 to be a viable option for industrial applications. Exopolysaccharide C. erzurumensis levan optimization purification antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Marine environments show diversity in biological and chemical molecules owing to their extensive ecological diversity. These environments harbour vast unexplored biomolecules and microorganisms with unique abilities. Marine microorganisms possess distinct physiological and metabolic adaptations that enable them to thrive in extreme environments, producing novel metabolites. Marine exopolysaccharides (EPS) are one of the metabolites of significant importance in industries, pharmaceuticals, and the medical sector (Concórdio-Reis et al. 2023 ; Sahana and Rekha 2019 ; El Halmouch et al. 2023 ). Microorganisms secrete EPS to protect against harsh environments. EPS originating from marine microorganisms has wide applications as a texturizer, stabilizer, thickening agent, and flocculating agent in wastewater treatment (Hafez, Abdallah, and Moustafa 2024 ). Exopolysaccharides with fructose as repeating monomers are termed “Fructooligosaccharides” (FOS). Bacterial levansucrase (EC number − 2.4.1.10) is responsible for the synthesis oflevan-type FOS, a fructan characterized by β-(2,6) glycosidic bonds (Belghith et al. 2012 ). Studies report the involvement of levan against biological stress in bacteria by modulating cell osmotic pressure and protection against low temperature, salinity, and drought (Versluys et al. 2018 ; Xu et al. 2021 ). Levan has wide applications in the food industry as a gelling agent, emulsifying agent, stabilizer, water-binding agent, and viscosifier. Levan also serves as a drug carrier for antitumor agents and is known for its antioxidant and anti-inflammatory activities (Phengnoi et al. 2022 ). Levan is known for its strong adhesive property, facilitating the formation of films. Its low viscosity and compatibility with surfactants and salts set levan apart from other types of EPS. Levan demonstrates high stability in acidic and alkaline conditions, as well as under high temperatures. Levan is characterized by high water retention and decent biocompatibility and is non-toxic in nature (De Siqueira et al. 2020 ). Levan is being utilized for the preparation of fibrous scaffolds with applications in tissue engineering (Avsar et al. 2018 ). Levan derived from Halomonas species exhibited proliferation of human keratinocyte and fibroblast cells when tested with levan-based skin regenerating cosmeceutical formulations (Erginer et al. 2023 ). Various studies have been conducted to isolate levan-producing bacteria, which include strains of Halomonas , Zymomonas , Microbacterium , and Bacillus species (Erdal Altıntaş et al. 2023b ; Silbir et al. 2014 ; Xu et al. 2023 ; Mehta, Shukla, and Saraf 2024 ). Marine EPS holds significant applications but currently constitutes a minor segment of the polymer market. This is primarily attributed to their low productivity and the associated high production costs (Concórdio-Reis et al. 2023 ). The search for cost-effective, high-fructan-yielding bacteria is imperative, as only a limited number of strains have shown remarkable production rates. In addition to lactic acid bacteria, diverse strains within the Bacillus species are well known for their ability to synthesize levan-type EPS with distinct physicochemical properties and molecular weights (Mummaleti et al. 2022 ; Thomas et al. 2024 ). Studies have been conducted on EPS biosynthesis by bacteria, with a particular focus on optimizing the process and components of the production, studying metabolic pathways responsible for production, and identifying their potential applications through characterization (Mehta, Shukla, and Saraf 2024 ). Factors such as temperature and pH, alongside nutritional components like higher carbon and lower nitrogen content, have a notable effect on microbial EPS production (Freitas, Torres, and Reis 2017 ). Certain salts, such as phosphate and iron (Fe2+), have been reported to enhance EPS production (Idogawa et al. 2014 ; Belghith et al. 2012 ). Free radicals produced as an outcome of many metabolic reactions pose a significant threat to living organisms and can lead to the onset of various degenerative diseases and disorders. To regulate the oxidative damage caused by free radicals, both natural and synthetic antioxidants are being employed. Yet the concern over the side effects of synthetic antioxidants has shifted the research towards natural alternatives. EPS, in particular, is considered to be an effective antioxidant (Khalid et al. 2022 ; Liu et al. 2009 ). The objective of the present study was to optimize the production of levan by Calidifontibacillus erzurumensis LEV207, a bacterium isolated from the Bay of Bengal coast, Visakhapatnam, Andhra Pradesh, India. A systematic optimization of the medium components was done using a one-variable-at-a-time approach. The purified levan (EPSLEV207) was characterized by Nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and Fourier-transform infrared (F-TIR) spectroscopy. The antioxidant activity of purified levan was evaluated. Further, the potential of levan as a carbon source to support the growth of the probiotic strain Lactobacillus rhamnosus MTCC 1423 was studied. The physicochemical properties, such as water solubility index and water retention capacity, were also investigated. To our knowledge, this is the first documented report of levan production by Calidifontibacillus species. Materials and methods Screening and identification of exopolysaccharide-producing strain Samples were obtained from the Bay of Bengal coast, Visakhapatnam, Andhra Pradesh, India. Isolates were grown in a basal medium consisting of 10% sucrose, 0.5% yeast extract, pH 7.0, and incubated at 37°C for 24 hrs in an orbital shaking incubator (150 rpm). Pure culture was obtained on 10% sucrose, 0.5% yeast extract, 2% agar-agar, and pH 7.0 medium plates. A colony with mucoidal consistency, indicating the production of EPS, was selected for 16S rRNA sequencing to identify the bacterial strain. The partial 16S rRNA sequence has indicated a 99% similarity with Calidifontibacillus erzurumensis P2. This partial sequence has been submitted to GenBank and was assigned an accession number, PP947779. The isolate was named Calidifontibacillus erzurumensis LEV207 and was preserved at -80°C in 60% (v/v) glycerol (Thakham et al. 2020 ). Optimization of medium composition and production condition To maximize the yield of levan by Calidifontibacillus erzurumensis LEV207, optimization of medium composition and conditions was done by using one variable at a time approach. Medium components and conditions such as carbon source, nitrogen source, initial pH, incubation temperature, salts and metal ions, concentration of carbon source, and concentration of nitrogen source were systematically studied. 5% inoculum of C. erzurumensis LEV207 was constant for every parameter under study. Analytical methods For all the optimization parameters, analytical methods were used to monitor microbial growth, enzyme activity, and thin-layer chromatography (TLC) to detect levan production. For every parameter, following incubation for 24 hrs, 5 mL of samples were withdrawn and centrifuged for 10 mins at 10,000 rpm. The resultant supernatant was utilized as a crude enzyme for calculating enzyme activity, while the pellet (cell mass) was washed twice with 0.85% saline and used for quantifying bacterial growth (OD at 600nm). To calculate enzyme activity, 0.1 mL of supernatant was aspirated into 0.9 mL of 20% sucrose dissolved in 20 mM acetate buffer (pH 5.6) and incubated at 37°C for 10 mins. To halt the enzymatic reaction, the mixture was placed at 90°C for 5 mins, followed by determining reducing sugars by the DNSA method (Saqib and Whitney 2011 ). Levansucrase activity was calculated as the amount of enzyme responsible for the release of 1µmol of glucose per minute. To detect the production of levan, TLC was performed by loading 2µl of the supernatant on silica gel G-60 and placed in a water: acetic acid: chloroform (1:7:6) solvent system. After completion of the solvent run, TLC plates were placed in a hot air oven at 115°C for 10 mins to evaporate the solvent. TLC plates were sprayed with a mixture of sulphuric acid: ethanol (1:9) and kept at 115°C in a hot air oven until spots were visualized (Thakham et al. 2020 ). Carbon and nitrogen source At the initial stages, the carbon source was maintained at 10% concentration to reduce the viscosity of the medium. Sucrose, maltose, glucose, fructose, and galactose at 10% concentration were studied as carbon sources. Yeast extract − 0.5% and pH − 7.0 were constant in each medium. These mediums were incubated in an orbital shaking incubator (150 rpm) at 37°C. Sucrose as a carbon source proved to have good enzyme activity and hence was used as the carbon source for further tests. Peptone, tryptone, and yeast extract at 0.5% concentration were studied as nitrogen sources, while sucrose − 10% and pH – 7.0 were constant in each medium. Yeast extract as a nitrogen source exhibited good enzyme activity and was used as a nitrogen source for subsequent tests. pH and temperature The impact of pH on optimal levan production was investigated by formulating the medium with pH in the range of 4.0–10.0. 10% sucrose and 0.5% yeast extract were maintained in each medium. pH 6.0 exhibited the highest enzyme activity and was utilized for further experiments. To study the impact of temperature on levan production, media were incubated at 30, 37, 42, and 50°C. Medium composition, sucrose 10%, yeast extract – 0.5%, pH – 6.0 was kept the same. An incubation temperature of 30°C was confirmed to have good enzyme activity. Metal ions and salts To study the impact of metal ions and salts on enzyme production, mediums were formulated by including compounds like FeSO 4 , MgSO 4, KCl, NaCl, CaCl 2, and CuSO 4 at 50 and 100 mM concentrations. Medium composition, sucrose 10%, yeast extract – 0.5%, pH – 6.0 was kept the same, and 30°C was the incubation temperature. MgSO 4 at 50 mM concentration showed the highest enzyme activity. Sucrose and yeast extract concentration To check the effect of carbon concentration, mediums were formulated with 10, 20, 30, 40, and 50% concentrations of sucrose; other optimized parameters were yeast extract 0.5%, pH 6.0, MgSO 4 − 50 mM, and incubation temperature 30°C. The highest enzyme activity was observed at 50% sucrose concentration. To check the impact of nitrogen concentration, media were formulated with 0.5, 1, 2, and 5% yeast extract; other parameters were sucrose – 50%, pH- 6.0, MgSO 4 − 50 mM, and incubation temperature 30°C. The incubation duration required to attain the highest enzyme activity was considered the optimal duration of incubation. All the optimization tests were performed in triplicates. Isolation and purification of levan 100 mL of each unoptimized and optimized media were inoculated with 5% C. erzurumensis LEV207 inoculum, following which the flasks were incubated at 30°C for 48 hrs (150 rpm). Subsequently, the fermented broths were centrifugated at 7000 rpm for 20 min at 4°C to remove the microbial cells. The levan in the supernatants was precipitated by mixing with cold absolute ethanol (1:2.5) (v/v), followed by incubation at -20°C for 24 hrs. To separate the precipitated levan, the liquid was centrifuged at 10000 rpm for 20 mins at 4°C. The precipitated levan was dissolved in hot water and deproteinized by using Sevag reagent consisting of n-butanol and chloroform (1:5) (v/v). The appearance of a whitish layer in between the chloroform and water layer indicates protein separation. The upper layer was separated, and the deproteinization process was repeated 2–3 times. Subsequently, the supernatant was transferred to dialysis bag tubing (12 kDa cutoff), and dialysis was carried on for five days at 4°C against distilled water. Distilled water was changed every 24 hrs. Upon completion of dialysis, samples were lyophilized. Lyophilized dry mass was used to determine the yield of levan (Thakham et al. 2020 ). Characterization of Levan Nuclear Magnetic Resonance (NMR) spectroscopy NMR spectroscopy was utilized to analyze the structure of EPSLEV207. For NMR analysis, D 2 O was used as a solvent to dissolve EPSLEV207. 1 H and 13 C NMR spectra were analyzed by running the sample at 400 MHz (Bruker, AVANCE) (Thakham et al. 2020 ). Fourier Transform Infrared (FTIR) spectroscopy To determine the presence of functional groups in EPSLEV207 structure FTIR analysis was carried out. ATR-FTIR spectrophotometer (Bruker, ALPHA-II), was utilized to record FTIR spectra from the wavelength 4000 to 500 cm − 1 (Mehta, Shukla, and Saraf 2024 ). X-ray Diffraction (XRD) analysis An XRD study was done on EPSLEV207 to determine its nature. For this analysis, Bruker D8 Advance X-ray diffractometer was utilized. The instrument employs CuKα radiation and operates in scan mode over a range of 5° to 60° at various 2θ angles (Mehta, Shukla, and Saraf 2024 ). Determination of monosaccharides of levan by chemical hydrolysis Lyophilized EPSLEV207 was chemically hydrolyzed to determine the monosaccharide composition. Levan (10 mg/mL) was treated with 0.25% oxalic acid and incubated in a boiling water bath for a time period of 10, 20, and 30 mins. The resultant hydrolyzed samples were analyzed by performing TLC, as explained in the section on analytical methods (Belghith et al. 2012 ). Biological activity of EPSLEV207 Antioxidant activity of EPSLEV207 DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity The antioxidant activity against DPPH radicals was evaluated by slightly modifying the protocol explained by Srinivash et al and Zhu et al (Srinivash et al. 2023 ; Zhu et al. 2022 ). EPSLEV207 was dissolved in water at concentrations 1, 2, 3, 4, and 5 mg/mL and mixed with an equal volume of DPPH solution (0.1 mM) prepared in methanol. Ascorbic acid at similar concentrations was used as standard, and all the tubes were incubated for 30 minutes under dark conditions. Methanol was used as the blank. The following formula was utilized to determine the DPPH radical scavenging activity by recording OD at 517 nm: DPPH radical scavenging activity (%) = ( \(\:\frac{\text{A}\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) ) × 100 Where, A sample denotes the optical density of the test sample, and A control denotes the optical density of the control. ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity The antioxidant activity against ABTS radicals was quantified by performing the protocol explained by Zhu et al (Zhu et al. 2022 ). In short, the solutions of potassium persulfate (2.45mM) and ABTS (7mM) were mixed in equal volumes and placed in the dark for 16 hrs, followed by dilution of the mixture to 0.70 ± 0.2 OD at 734nm by PBS (pH 7.4). EPSLEV207 was dissolved in water at different concentrations (1, 2, 3, 4, and 5 mg/mL). Aliquots of 400 µl from each concentration were aspirated in 3 mL of ABTS radicals, followed by incubation for 6 mins in a dark place. The following formula was utilized to determine the ABTS radical scavenging activity by recording OD at 734 nm: ABTS radical scavenging activity (%) = ( \(\:\frac{\text{A}\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) ) × 100 Where, A sample denotes the optical density of the test sample and A control denotes the optical density of the control. Effect of levan on the growth of probiotic strain EPSLEV207 was evaluated for its utilization as a carbon source by the probiotic bacterium Lactobacillus rhamnosus MTCC 1423 by slightly modifying the protocol explained by Wang et al (Wang et al. 2022 ). A basal MRS medium (carbon-free) was prepared and EPSLEV207 and glucose were added as carbon sources at 1% concentration, respectively. The mediums were sterilized, and L. rhamnosus MTCC 1423 cells (1 × 10 6 CFU/mL) were inoculated into the medium and incubated at 37°C for 48 hrs. Every 24 hrs, samples were removed, serially diluted, spread on MRS agas plates, and incubated for 48 hrs. Utilization of glucose and EPSLEV207 was calculated by counting the cells on the MRS plates, and the number of cells was expressed in log CFU/mL. Physicochemical properties of levan - Water solubility index (WSI) and Water retention capacity (WRC) EPSLEV207 was evaluated for its WSI by suspending 30 mg of EPS in 1 mL of distilled water and placing the tubes at 35°C for 1 hr under continuous agitation (Thomas et al. 2024 ). The EPS solution was centrifuged for 20 mins at 5000 rpm, the resultant supernatant was transferred to a pre-weighed tube, and the solubilized EPS was precipitated by mixing with ethanol in a ratio of 1:3. The tubes were kept at 80°C to dry the precipitate, and the difference in the tube weight was recorded. WSI was calculated by using the following formula: WSI (%) = \(\:\frac{dry\:weight\:of\:EPS\:in\:the\:supernatant}{initial\:weight\:of\:EPS\:\left(30\:mg\right)}\) × 100 EPSLEV207 was evaluated for its WRC by modifying the protocol explained by Thomas et al (Thomas et al. 2024 ). In short, 20 mg of EPSLEV207 was taken in a pre-weighed tube and suspended in 1 mL of distilled water, followed by centrifugation at 13,000 rpm for 30 mins. The resultant supernatant was discarded, and the tube with wet EPSLEV207 was weighed. The difference in the weight of the tube before and after was calculated to get the weight of EPS after water absorption. WRC was calculated by using the following formula: WRC (%) = \(\:\frac{weight\:of\:EPS\:after\:absorption}{initial\:weight\:of\:EPS\:\left(20\:mg\right)}\) × 100 Result Strain cultivation To check the production of levan, C. erzurumensis LEV207 was streaked on agar plates with and without sucrose. Colonies with slimy consistency were observed on the sucrose agar plate, which indicates the production of EPS. Normal colonies were observed on the agar plate without sucrose. Optimization of medium components Impact of carbon source on levansucrase activity The impact of five carbon sources on C. erzurumensis LEV207 growth and levansucrase production was studied. The data revealed the growth of C. erzurumensis LEV207 in the medium supplemented with glucose, sucrose, galactose, and maltose (Fig. 1a). Fructose showed very low bacterial growth. Levansucrase production (enzyme activity − 163.3 µmol/mL/min) and bacterial growth were highest in the sucrose medium (Fig. 1b). Sucrose was utilized both for bacterial growth and levansucrase production. Sucrose, as a carbon source, has proven to affect levansucrase production in Gluconobacter albidus TMW 2.1191 (Jakob et al. 2020 ). Bacterial growth was good in glucose, galactose, and maltose, but the enzyme production was lower compared with sucrose medium. Hence, sucrose was considered the best carbon source for further investigations. Sucrose acts as an inducer for the production of levansucrase in Bacillus spp and Klebsiella strain L1 (Belghith et al. 2012 ; Desai and Patel 2019 ). Impact of nitrogen source on levansucrase activity Nitrogen sources in the medium can impact the growth of bacterial cells and levansucrase production. In the current study, three types of nitrogen sources, i.e. yeast extract, peptone, and tryptone, were studied for their impact on cell growth and enzyme production. The medium supplemented with yeast extract showed the highest cell growth (Fig. 1c) as well as levansucrase production (enzyme activity − 163.6 µmol/mL/min), followed by peptone and tryptone (Fig. 1d). Yeast extract was observed to have higher levansucrase production than other nitrogen sources (Desai and Patel 2019 ; Belghith et al. 2012 ). Yeast extract was confirmed as the most effective nitrogen source. Impact of pH on levansucrase activity The role of pH in the production of levansucrase and cell growth was studied in the present work. Mediums with initial pH ranging from 4.0 to 10.0 were used in the study. The findings reveal optimum levansucrase production (enzyme activity − 202 µmol/mL/min) at pH 6.0. A decrease in enzyme production was observed from pH 5.0 to 4.0, and pH 7.0 to 10.0 (Fig. 1f). Maximum levansucrase production was in the range of 5.0 to 6.5 (Desai and Patel 2019 ). Bacterial growth was observed highest at pH 8.0, which is suitable for cell mass growth, but the enzyme production was reduced significantly at this pH (Fig. 1e), which might be due to the difference in optimal pH for cell growth and enzyme production. Focusing on optimizing the levan production, pH 6.0 was considered to be the ideal. Impact of temperature on levansucrase activity To study the impact of temperature on levansucrase production, the incubation temperature of 30, 37, 42, and 50°C was studied. In the present study, the highest levansucrase production was at 30°C (enzyme activity – 216.13 µmol/mL/min), followed by 37°C (200.76 µmol/mL/min) and 42°C (195.23 µmol/mL/min), respectively Levansucrase production at the temperature 50°C was lowest in our observation (139.43 µmol/mL/min) (Fig. 1h). In contrast, cell growth was highest at 37°C, followed by 42 and 50°C, whereas it was least at 30°C, respectively (Fig. 1g). Comparable with pH, optimum temperature also differed for cell growth and levansucrase production. 30°C was reported to be the optimum temperature for the production of levan by Suhomyces kilbournensis (González-Torres et al. 2024 ). In our present study, 30°C was considered to be optimal for the production of levansucrase. Impact of metal ions and salts on levansucrase activity FeSO 4 , MgSO 4 , KCl, NaCl, CaCl 2 , and CuSO 4 at 50 and 100mM concentrations were used to study the influence of metal ions and salts on levansucrase production. C. erzurumensis LEV207 showed specificity for growth and levansucrase production under the influence of metal ions. The highest bacterial growth was observed in KCl (50mM), followed by MgSO 4, NaCl, FeSO 4 , CuSO 4, and CaCl 2 . No growth was observed in KCl (100 mM) (Fig. 2a). Levansucrase production was highest in MgSO 4 (50 mM) (enzyme activity – 221.3 µmol/mL/min), followed by NaCl (100 mM) (202.6 µmol/mL/min), and KCl (50mM) (197.13 µmol/mL/min) (Fig. 2b). Metal ions such as FeSO 4, CaCl 2, and CuSO 4 completely inhibited the production of the enzyme. Zhang et al. ( 2021 ) reported that metal ions have the potential to enhance enzyme levansucrase activity (Zhang et al. 2021 ). MgSO 4 (50 mM) proved to be the best salt that increased levansucrase production. Impact of concentration of sucrose on levansucrase activity To evaluate the impact of the concentration of sucrose on bacterial growth and levansucrase activity, media were formulated with 10, 20, 30, 40, and 50% sucrose and incubated for 72 hrs. The highest cell growth was observed at 10% sucrose and showed a gradual decline as the concentration increased (Fig. 2c). The cells tend to adjust with the increasing osmotic pressure created by increasing sucrose concentration, which leads to lower cell growth. But in the case of levansucrase production, the opposite pattern was observed. As the sucrose concentration increased, levansucrase production increased significantly (Fig. 2d). The highest levansucrase production (enzyme activity – 410.4 µmol/mL/min) was observed at 50% sucrose after 48 hrs of incubation. Chen and Liu ( 1996 ) suggested that a significant portion of sucrose is utilized by cells for growth when supplemented with sucrose below 10% concentration, while higher sucrose concentrations result in enhanced enzyme induction with inhibition of cell growth (Chen and Liu 1996 ). Impact of concentration of yeast extract on levansucrase activity To study the impact of the concentration of yeast extract on bacterial growth and levansucrase activity, mediums were prepared with 0.5, 1, 2, and 5% yeast extract and incubated for 72 hrs. The highest cell growth was observed at 1% yeast extract concentration, with a further decline as the yeast extract concentration increased (Fig. 2e). Levansucrase activity was highest at 1% concentration after 48 hrs of incubation (enzyme activity 427.4 µmol/mL/min) (Fig. 2f). Chen and Liu ( 1996 ) stated that the enzyme activity increases with the increase in the yeast extract concentration (Chen and Liu 1996 ). Isolation and purification of levan Both unoptimized and optimized mediums were sterilized and inoculated with 5% inoculum of C. erzurumensis LEV207, then incubated at 30°C for 48 hrs. Cells were separated by centrifugation, and precipitation of levan was done by adding chilled ethanol, followed by deproteinization. The deproteinized samples were dialyzed and lyophilized. The dry weight of lyophilized levan was utilized to calculate the yield of levan from the unoptimized and optimized medium. The unoptimized medium yielded 9.27 g/L of levan, while the optimized medium and conditions yielded 65.58 g/L of levan. The yield has been increased 7-fold by optimizing the medium components and conditions. Characterization of Levan NMR NMR spectroscopy was performed to study the structural aspect of EPSLEV207. The 1 H spectra reveal the chemical shift in the proton signals, providing insight into the structural composition of monomer, in this case, fructose. The signals present in EPSLEV207 showed a chemical shift of 3.487 ppm (H6b), 3.617 ppm (H1b), 3.688 ppm (H1a), 3.841 ppm (H6a), 3.882 ppm (H5), 4.028 ppm (H4), and 4.111 ppm (H3) (Fig. 3a). These chemical shifts resemble the levan produced by Bacillus siamensis , and Bacillus megaterium GJT321 (Thakham et al. 2020 ; Yu et al. 2016 ). The 13 C spectra reveal the carbon involved in the formation of monosaccharides. The 13 C spectra for EPSLEV207 showed the presence of six signals at 59.89 (C1), 63.38 ppm (C6), 75.19 ppm (C4), 76.28 ppm (C3), 80.28 ppm (C5), and 104.10 ppm (C2) (Fig. 3b). The observed signals confirmed the presence of fructose as a monosaccharide in the EPSLEV207 due to the presence of anomeric carbon at 104.10 ppm (C2), methylene group at 59.89 (C1), and 63.38 ppm (C6), furanose ring present at 80.28 ppm (C5), and peaks at 76.28 (C3), and 75.19 ppm (C4) representing oxymethinic group. Similar results were reported by Thakham et al. ( 2020 ) (Thakham et al. 2020 ). F-TIR The functional groups present in the EPSLEV207 structure were analyzed using F-TIR spectroscopy. As illustrated in Fig. 3c, FTIR spectra for EPSLEV207 indicated the presence of a strong peak at 3290 cm-1, representing an O-H bond. Two peaks were detected at 2937 and 2884 cm − 1 , representing the presence of C-H bond. A peak was present at 1645 cm − 1 , indicating the existence of bound water. A peak was detected at 1419 cm − 1 , representing the bending of the methylene group of the C-H bond. A peak around 1123 cm-1 represents the C-O-H bond. A strong peak was detected at 1009 cm − 1 , representing the C-O-C, a glycosidic linkage specifically present in the pyranose or furanose ring structure. Two peaks at 922 and 808 cm − 1 confirm the presence of furanose units. Overall, the FTIR spectra of EPSLEV207 confirm the presence of polysaccharides and resemble the spectra represented by Yu et al. ( 2016 ) and Thakham et al. ( 2020 ) (Yu et al. 2016 ; Thakham et al. 2020 ). XRD The nature of EPSLEV207 was determined by XRD. The X-ray diffractogram of EPSLEV207 showed a broad peak pattern around 15–20° 2θ with the highest intensity (4646 ppm) peak at 17.61° 2θ (Fig. 3d). The broad nature of the X-ray diffractogram pattern indicates the amorphous nature of levan (Mendonça et al. 2021 ; Kekez et al. 2016 ). The amorphous property of levan indicates a lack of crystal structure but provides a high solubility rate, a desirable property in medical and pharmaceutical fields. Sadhana et al. ( 2024 ) prepared a levan-derived nanoparticle of dolutegravir, an anti-HIV drug, and reported an enhancement in the solubility of the drug due to the amorphous nature of levan (Sadhana et al. 2024 ). Chemical hydrolysis and monosaccharide detection To determine the monosaccharide composition of EPSLEV207, oxalic acid was used as a hydrolyzing agent. Samples were treated for 10, 20, and 30 minutes and were maintained in a boiling water bath (Belghith et al. 2012 ). The hydrolyzed samples were used for loading on a TLC plate along with sucrose, fructose, and glucose as standards. The TLC plate was analyzed, and a single spot concurrent to fructose was observed (Fig. 4a), indicating that EPSLEV207 is a homopolysaccharide linked by fructose as a monomer. Biological activity Antioxidant activity – DPPH radical scavenging activity The in vitro antioxidant activity of EPSLEV207 was studied by DPPH radical scavenging assay. The evaluation was conducted for concentrations 0 to 5mg/mL, respectively, for which the DPPH radical scavenging activity was found to be in the range of 40.92–49.15% (Fig. 4b). These results indicate that the EPSLEV207 had a noticeable radical scavenging ability. Furthermore, the DPPH radical scavenging activity for EPSLEV207 was greater than EPS derived from Lactobacillus plantarum YW32 (DPPH < 30%) (Wang et al. 2015 ). The property of EPSLEV207 to scavenge the DPPH radicals can be attributed to their ability to donate electrons (Bouallegue et al. 2020 ). ABTS radical scavenging activity The invitro antioxidant activity of EPSLEV207 was studied by ABTS radicals. The evaluation was conducted for concentrations 0 to 5mg/mL, and the ABTS radical scavenging activity was found to be in the range of 17.59–22% (Fig. 4c). Our results for EPSLEV207 align with the ABTS radical scavenging activity of EPS derived from Pediococcus pentosaceus M41 at the concentration of 5mg/mL (Ayyash et al. 2020 ). Effect of levan on the growth of probiotic strain EPSLEV207 was studied for its effect on the growth of L. rhamnosus MTCC 1423, a probiotic strain. Glucose and EPSLEV207 were used as a carbon source in basal MRS broth and inoculated with L. rhamnosus. As illustrated in Fig. 4d, after 24 hrs of incubation, cells reached 8.95 log CFU/mL in glucose-supplemented medium and 8.49 log CFU/mL in EPSLEV207 supplemented medium. After 48 hrs of incubation, cells reached 7.11 log CFU/m L glucose medium and 8.38 log CFU/mL in EPSLEV207 medium. Glucose is a monomeric form of carbohydrate and is easily utilized by the cells at the initial growth phase, while EPSLEV207 is a polymeric form and was not utilized predominantly, leading to lesser cell growth. After 48 hrs, the glucose in the medium was completely utilized, and cells attained the death phase. While in the EPSLEV207 medium, cells were more viable than those in the glucose medium. In general, Lactic acid bacteria might possess substrate-specific fermentation metabolism, which enables them to utilize prebiotic compounds selectively (Wang et al. 2022 ). Physicochemical properties - WSI and WRC The WSI and WRC of EPSLEV207 were 88.88 ± 1.57% and 78.33 ± 2.35%, respectively. The WSI of EPSLEV207 was higher than the levan derived from Bacillus velezensis VTX20, but the WRC was lower compared to the levan from the same organism (WSI − 81.9% and WRC − 100.2%) (Vu et al. 2021 ). The WSI and WRC were much lower than the levan derived from Bacillus subtilis PR-C1 (WSI − 90.2% and WRC – 205.6%) (Thomas et al. 2024 ). Previous reports suggest the involvement of various factors, such as monosaccharide composition, chain length, and linkage pattern, in determining the WSI and WRC of EPS (Thomas et al. 2024 ). Discussion In the present work, we isolated a novel strain of Calidifontibacillus erzurumensis LEV207 from a marine sample and investigated its ability to produce levan, an exopolysaccharide composed of repeating fructose units. Given the wide range of applications for levan in the food and pharmaceutical fields, optimizing the yield is essential. We aimed to exploit the levan-producing capability of C. erzurumensis LEV207 by enhancing its yield through the optimization of the media composition and conditions. To study the effect of different parameters, cell mass, and levansucrase activity were considered critical factors for defining an increase in levan production. Bacterial cells produce levan as a defense mechanism against environmental stress. We explored the enzyme production rate of C. erzurumensis LEV207 under various conditions and optimized the production of levan. Media components such as carbon source, nitrogen source, pH, and presence of salts and metal ions significantly impact levansucrase production. The concentration of sucrose as a carbon source notably influences cell growth and enzyme production due to the osmotic pressure it creates. This effect was previously reported in the case of levan production by Zymomonas mobilis strain 113S (Vigants, Zikmanis, and Bekers 1996 ). When microorganisms are subjected to elevated sucrose levels, they may encounter osmotic stress, modulating their metabolic pathways to sustain cellular homeostasis (Barros and Celligoi 2006 ). Similarly, the concentration of yeast extract can also induce osmotic pressure and affect cell growth and enzyme activity. Determining the optimal concentration is critical; insufficient levels may not induce adequate osmotic stress to trigger levansucrase synthesis, whereas excessively high concentrations can inhibit cell growth. This inhibition was due to the yeast extract exceeding 1%. Excess yeast extract concentration inhibited cell growth and levansucrase production in C. erzurumensis LEV207 (Han et al. 2021 ). The initial pH of the media and temperature have different influences on cell growth and levansucrase activity. The change in pH can alter the structure and activity of enzymes. Different microorganisms have different optimal pH and temperatures for cell growth and proliferation. The variations in optimal pH and temperature for cell growth and levansucrase can be attributed to the difference in optimum pH and temperature for levansucrase induction and synthesis (Queiroz Santos, Del Bianchi, and Garcia-Cruz 2014 ). The stress of surviving at lower temperatures might have induced the production of levansucrase at 30°C in C. erzurumensis LEV207 (Han et al. 2021 ). Additionally, the presence of salts and metal ions can impact the production of levansucrase, as certain enzymes require specific salts to achieve optimal activity. Salts aid in stabilization of enzyme structure and regulation of substrate binding (Aguiar-Oliveira and Maugeri 2013 ). The purified levan (EPSLEV207) was evaluated for its antioxidant activity against DPPH and ABTS radicals, showing radical scavenging activity of 49.15 and 22%, respectively. The antioxidant properties of levan have been reported in various bacterial species, including Pseudomonas fluorescens strain ES, Leuconostoc mesenteroides S81, Lactobacillus plantarum KX041, etc (Korany et al. 2021 ; Taylan, Yilmaz, and Dertli 2019 ; Wang et al. 2017 ). Gut microbiota and its homeostasis is one of the major research areas that deals with human health. Prebiotic compounds are substrates that are selectively utilized by human gut microbiota and confer health benefits. Fructooligosaccharides, galactooligosaccharides, inulin, and lactulose are a few examples of polysaccharides that meet the necessary criteria for being termed prebiotics (Pohlentz et al. 2022 ). To investigate the prebiotic activity of EPSLEV207 as the sole carbon source, L. rhamnosus MTCC 1423 was grown in a modified MRS medium. Our findings indicate that L. rhamnosus exhibited better survivability in EPSLEV207-containing medium compared to glucose-containing medium. The WSI and WRC of EPSLEV207 were 88.88 ± 1.57% and 78.33 ± 2.35%, respectively. The solubility of levan is crucial for its application as a stabilizer, thickener, and emulsifier. The ability of levan to hold moisture helps in promoting effective healing and skin hydration. The water solubility and water retention capacity influence the ability of levan to interact with other compounds and have been used in the formation of hydrogels and drug delivery systems (Erdal Altıntaş and Aytar Çelik 2023a ). C. erzurumensis LEV207, isolated from the marine environment, demonstrates notable osmotolerance and the ability to endure extreme salinity and temperature. These qualities of C. erzurumensis LEV207 were utilized in the current study to optimize the yield of levan. Conclusion The present work has shown the potential of a novel marine bacterium, Calidifontibacillus erzurumensis LEV207, in the production of levan. Focusing on increasing the yield of levan, medium components and conditions were optimized. Optimization by one variable at a time approach has proven effective as the yield has increased 7-fold. NMR spectra confirmed the levan structure, FTIR spectra indicated the presence of glycosidic bonds, and XRD confirmed the amorphous nature of EPSLEV207. EPSLEV207 has good antioxidant activity, water solubility, and water retention capacity. It has also been effective as a carbon source in the growth of probiotic strain L. rhamnosus MTCC 1423. The utilization of high-levan-producing Calidifontibacillus erzurumensis LEV207 in industrial applications is promising. Furthermore, the application of recombinant DNA technology and cloning might improve the expression of levansucrase and extend the scale of applications. Declarations Acknowledgments We would like to thank the funding agencies for their in-time support. Author contribution POP and KVC designed the experiments. POP and KR performed experiments. POP and KVC analyzed and interpreted data and wrote the manuscript. KVC and the VP supervised the manuscript. Funding declaration This work was supported by National Fellowship for Other Backward Classes (UGC JRF NFOBC-No.F.40-2/2019) – Junior Research Fellowship (JRF) to POP and National Backward Classes Finance and Development Corporation (No. NBCFDC/E-70291) – JRF to KR. Data availability The data that support the findings of this study can be made available by authors upon request. Competing interests The authors declare no competing interest. Ethical approval This study does not involve animal or human subjects, and thus no ethical approval is required. Consent of publications We hereby confirm that all co-authors of this study have reviewed the final version of the manuscript and have provided their full consent for the submission and publication of the paper in International Microbiology . References Aguiar-Oliveira E, Maugeri F (2013) Effects of the addition of substrate and salts in both the fructosyltransferase immobilization and its catalytic properties. 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Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2024 Read the published version in International Microbiology → Version 1 posted Editorial decision: Revision requested 27 Aug, 2024 Reviews received at journal 20 Aug, 2024 Reviews received at journal 17 Aug, 2024 Reviews received at journal 14 Aug, 2024 Reviewers agreed at journal 12 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviewers invited by journal 09 Aug, 2024 Editor assigned by journal 09 Aug, 2024 Submission checks completed at journal 09 Aug, 2024 First submitted to journal 31 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. 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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-4834548","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345596222,"identity":"dbf98e69-98b0-4195-b858-3476bd9ec0af","order_by":0,"name":"Omkar Prakash Palkar","email":"","orcid":"","institution":"GITAM (Deemed to be University)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Omkar","middleName":"Prakash","lastName":"Palkar","suffix":""},{"id":345596225,"identity":"6da4c0a0-526d-4747-be00-1dc390a46e19","order_by":1,"name":"Keerthi Rayasam","email":"","orcid":"","institution":"GITAM (Deemed to be University)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Keerthi","middleName":"","lastName":"Rayasam","suffix":""},{"id":345596226,"identity":"9cc9e245-0fe5-4e7c-b90d-e81a437611db","order_by":2,"name":"Vidyullatha Peddireddy","email":"","orcid":"","institution":"Central University of Haryana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vidyullatha","middleName":"","lastName":"Peddireddy","suffix":""},{"id":345596227,"identity":"770d04c6-e032-44d8-abe0-8bbfda25900d","order_by":3,"name":"Viswanatha Chaitanya Kolluru","email":"data:image/png;base64,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","orcid":"","institution":"GITAM (Deemed to be University)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Viswanatha","middleName":"Chaitanya","lastName":"Kolluru","suffix":""}],"badges":[],"createdAt":"2024-07-31 10:10:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4834548/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4834548/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10123-024-00597-5","type":"published","date":"2024-09-26T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63948835,"identity":"f5390be9-07dc-48d1-910b-e9ca0905548e","added_by":"auto","created_at":"2024-09-04 06:36:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":569339,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Impact of carbon sources (Glucose, fructose, sucrose, galactose, and maltose) on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24 hrs of incubation. (b) Impact of carbon sources (Glucose, fructose, sucrose, galactose, and maltose) on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24 hrs. (c) Impact of nitrogen sources (yeast extract, peptone, tryptone) on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24 hrs of incubation. (d) Impact of nitrogen sources (yeast extract, peptone, tryptone) on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24 hrs. (e) Impact of pH (4.0,5.0,6.0,7.0,8.0,9.0,10.0) on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24 hrs of incubation. (f) Impact of pH (4.0,5.0,6.0,7.0,8.0,9.0,10.0) on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24 hrs. (g) Impact of temperature (30,37,42,50°C) on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24 hrs of incubation. (h) Impact of temperature (30,37,42,50°C) on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24 hrs.\u003c/p\u003e","description":"","filename":"OnlineFig1..png","url":"https://assets-eu.researchsquare.com/files/rs-4834548/v1/8b9049d7324d1ecf361d19b3.png"},{"id":63948834,"identity":"fad0b4af-7a2f-4cf7-ba19-86f90eecf1a1","added_by":"auto","created_at":"2024-09-04 06:36:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":573857,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of salts and metal ions (FeSO\u003csub\u003e4\u003c/sub\u003e, MgSO\u003csub\u003e4, \u003c/sub\u003eKCl, NaCl, CaCl\u003csub\u003e2,\u003c/sub\u003e and CuSO\u003csub\u003e4\u003c/sub\u003e) at 50 and 100mM concentration on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24 hrs of incubation. (b) Effect of salts and metal ions (FeSO\u003csub\u003e4\u003c/sub\u003e, MgSO\u003csub\u003e4, \u003c/sub\u003eKCl, NaCl, CaCl\u003csub\u003e2,\u003c/sub\u003e and CuSO\u003csub\u003e4\u003c/sub\u003e) at 50 and 100mM concentration on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24 hrs. (c) Effect of sucrose concentration (10,20,30,40,50%) on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24, 48 and 72 hrs of incubation. (d) Effect of sucrose concentration (10,20,30,40,50%) on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24, 48 and 72 hrs of incubation. (e) Effect of yeast extract concentration (0.5, 1, 2, 5%) on \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 growth after 24, 48 and 72 hrs of incubation. (f) Effect of yeast extract concentration (0.5, 1, 2, 5%) on levansucrase production by \u003cem\u003eC. erzurumensis \u003c/em\u003eLEV207 after 24, 48 and 72 hrs of incubation.\u003c/p\u003e","description":"","filename":"OnlineFig2..png","url":"https://assets-eu.researchsquare.com/files/rs-4834548/v1/d94995c72cf66b4b9304a0f5.png"},{"id":63948838,"identity":"88913292-29d6-474c-b49d-e6ceed255a72","added_by":"auto","created_at":"2024-09-04 06:36:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":332736,"visible":true,"origin":"","legend":"\u003cp\u003e(a) \u003csup\u003e1\u003c/sup\u003eH spectra of EPSLEV207 determined by NMR. (b) \u003csup\u003e13\u003c/sup\u003eC spectra of EPSLEV207 determined by NMR. (c) FTIR spectra of EPSLEV207 (d) X-ray diffractogram of EPSLEV207\u003c/p\u003e","description":"","filename":"OnlineFig3..png","url":"https://assets-eu.researchsquare.com/files/rs-4834548/v1/ab0acdc69df3db3b97eb67af.png"},{"id":63948836,"identity":"82cb6124-b288-4d9c-b273-169bd398730d","added_by":"auto","created_at":"2024-09-04 06:36:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":386646,"visible":true,"origin":"","legend":"\u003cp\u003e(a) TLC plate for confirming the presence of fructose as the monomer in levan EPSLEV207. From left to right, standards – sucrose, glucose, fructose, unhydrolyzed EPSLEV207, reaction mixture after 10 mins of hydrolysis, reaction mixture after 20 mins of hydrolysis and reaction mixture after 30 mins of hydrolysis. (b) Antioxidant activity of EPSLEV207 against DPPH radicals. (c) Antioxidant activity of EPSLEV207 against ABTS radicals. (d) Effect of glucose and EPSLEV207 on the growth of probiotic strain L. rhamnosus MTCC 1423 after 24 and 48 hrs. Cell numbers are expressed in log CFU/mL.\u003c/p\u003e","description":"","filename":"OnlineFig4..png","url":"https://assets-eu.researchsquare.com/files/rs-4834548/v1/c0a5d06919e147f867df9acd.png"},{"id":65627789,"identity":"d707cab6-2308-4446-bf62-1355eb780aa6","added_by":"auto","created_at":"2024-09-30 16:16:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3893543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4834548/v1/f8ccc559-2167-4726-b88e-ce62ecd941f6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improved levan production by novel Calidifontibacillus erzurumensis LEV207 using one variable at a time approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMarine environments show diversity in biological and chemical molecules owing to their extensive ecological diversity. These environments harbour vast unexplored biomolecules and microorganisms with unique abilities. Marine microorganisms possess distinct physiological and metabolic adaptations that enable them to thrive in extreme environments, producing novel metabolites. Marine exopolysaccharides (EPS) are one of the metabolites of significant importance in industries, pharmaceuticals, and the medical sector (Conc\u0026oacute;rdio-Reis et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sahana and Rekha \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; El Halmouch et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microorganisms secrete EPS to protect against harsh environments. EPS originating from marine microorganisms has wide applications as a texturizer, stabilizer, thickening agent, and flocculating agent in wastewater treatment (Hafez, Abdallah, and Moustafa \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExopolysaccharides with fructose as repeating monomers are termed \u0026ldquo;Fructooligosaccharides\u0026rdquo; (FOS). Bacterial levansucrase (EC number \u0026minus;\u0026thinsp;2.4.1.10) is responsible for the synthesis oflevan-type FOS, a fructan characterized by β-(2,6) glycosidic bonds (Belghith et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Studies report the involvement of levan against biological stress in bacteria by modulating cell osmotic pressure and protection against low temperature, salinity, and drought (Versluys et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Levan has wide applications in the food industry as a gelling agent, emulsifying agent, stabilizer, water-binding agent, and viscosifier. Levan also serves as a drug carrier for antitumor agents and is known for its antioxidant and anti-inflammatory activities (Phengnoi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Levan is known for its strong adhesive property, facilitating the formation of films. Its low viscosity and compatibility with surfactants and salts set levan apart from other types of EPS. Levan demonstrates high stability in acidic and alkaline conditions, as well as under high temperatures. Levan is characterized by high water retention and decent biocompatibility and is non-toxic in nature (De Siqueira et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Levan is being utilized for the preparation of fibrous scaffolds with applications in tissue engineering (Avsar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Levan derived from \u003cem\u003eHalomonas\u003c/em\u003e species exhibited proliferation of human keratinocyte and fibroblast cells when tested with levan-based skin regenerating cosmeceutical formulations (Erginer et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVarious studies have been conducted to isolate levan-producing bacteria, which include strains of \u003cem\u003eHalomonas\u003c/em\u003e, \u003cem\u003eZymomonas\u003c/em\u003e, \u003cem\u003eMicrobacterium\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e species (Erdal Altıntaş et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Silbir et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mehta, Shukla, and Saraf \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Marine EPS holds significant applications but currently constitutes a minor segment of the polymer market. This is primarily attributed to their low productivity and the associated high production costs (Conc\u0026oacute;rdio-Reis et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The search for cost-effective, high-fructan-yielding bacteria is imperative, as only a limited number of strains have shown remarkable production rates. In addition to lactic acid bacteria, diverse strains within the \u003cem\u003eBacillus\u003c/em\u003e species are well known for their ability to synthesize levan-type EPS with distinct physicochemical properties and molecular weights (Mummaleti et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Thomas et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStudies have been conducted on EPS biosynthesis by bacteria, with a particular focus on optimizing the process and components of the production, studying metabolic pathways responsible for production, and identifying their potential applications through characterization (Mehta, Shukla, and Saraf \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Factors such as temperature and pH, alongside nutritional components like higher carbon and lower nitrogen content, have a notable effect on microbial EPS production (Freitas, Torres, and Reis \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Certain salts, such as phosphate and iron (Fe2+), have been reported to enhance EPS production (Idogawa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Belghith et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFree radicals produced as an outcome of many metabolic reactions pose a significant threat to living organisms and can lead to the onset of various degenerative diseases and disorders. To regulate the oxidative damage caused by free radicals, both natural and synthetic antioxidants are being employed. Yet the concern over the side effects of synthetic antioxidants has shifted the research towards natural alternatives. EPS, in particular, is considered to be an effective antioxidant (Khalid et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe objective of the present study was to optimize the production of levan by \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207, a bacterium isolated from the Bay of Bengal coast, Visakhapatnam, Andhra Pradesh, India. A systematic optimization of the medium components was done using a one-variable-at-a-time approach. The purified levan (EPSLEV207) was characterized by Nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and Fourier-transform infrared (F-TIR) spectroscopy. The antioxidant activity of purified levan was evaluated. Further, the potential of levan as a carbon source to support the growth of the probiotic strain \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e MTCC 1423 was studied. The physicochemical properties, such as water solubility index and water retention capacity, were also investigated. To our knowledge, this is the first documented report of levan production by \u003cem\u003eCalidifontibacillus\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening and identification of exopolysaccharide-producing strain\u003c/h2\u003e \u003cp\u003eSamples were obtained from the Bay of Bengal coast, Visakhapatnam, Andhra Pradesh, India. Isolates were grown in a basal medium consisting of 10% sucrose, 0.5% yeast extract, pH 7.0, and incubated at 37°C for 24 hrs in an orbital shaking incubator (150 rpm). Pure culture was obtained on 10% sucrose, 0.5% yeast extract, 2% agar-agar, and pH 7.0 medium plates. A colony with mucoidal consistency, indicating the production of EPS, was selected for 16S rRNA sequencing to identify the bacterial strain. The partial 16S rRNA sequence has indicated a 99% similarity with \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e P2. This partial sequence has been submitted to GenBank and was assigned an accession number, PP947779. The isolate was named \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207 and was preserved at -80°C in 60% (v/v) glycerol (Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of medium composition and production condition\u003c/h2\u003e \u003cp\u003eTo maximize the yield of levan by \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207, optimization of medium composition and conditions was done by using one variable at a time approach. Medium components and conditions such as carbon source, nitrogen source, initial pH, incubation temperature, salts and metal ions, concentration of carbon source, and concentration of nitrogen source were systematically studied. 5% inoculum of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 was constant for every parameter under study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical methods\u003c/h2\u003e \u003cp\u003eFor all the optimization parameters, analytical methods were used to monitor microbial growth, enzyme activity, and thin-layer chromatography (TLC) to detect levan production. For every parameter, following incubation for 24 hrs, 5 mL of samples were withdrawn and centrifuged for 10 mins at 10,000 rpm. The resultant supernatant was utilized as a crude enzyme for calculating enzyme activity, while the pellet (cell mass) was washed twice with 0.85% saline and used for quantifying bacterial growth (OD at 600nm). To calculate enzyme activity, 0.1 mL of supernatant was aspirated into 0.9 mL of 20% sucrose dissolved in 20 mM acetate buffer (pH 5.6) and incubated at 37°C for 10 mins. To halt the enzymatic reaction, the mixture was placed at 90°C for 5 mins, followed by determining reducing sugars by the DNSA method (Saqib and Whitney \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Levansucrase activity was calculated as the amount of enzyme responsible for the release of 1µmol of glucose per minute. To detect the production of levan, TLC was performed by loading 2µl of the supernatant on silica gel G-60 and placed in a water: acetic acid: chloroform (1:7:6) solvent system. After completion of the solvent run, TLC plates were placed in a hot air oven at 115°C for 10 mins to evaporate the solvent. TLC plates were sprayed with a mixture of sulphuric acid: ethanol (1:9) and kept at 115°C in a hot air oven until spots were visualized (Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCarbon and nitrogen source\u003c/h2\u003e \u003cp\u003eAt the initial stages, the carbon source was maintained at 10% concentration to reduce the viscosity of the medium. Sucrose, maltose, glucose, fructose, and galactose at 10% concentration were studied as carbon sources. Yeast extract − 0.5% and pH − 7.0 were constant in each medium. These mediums were incubated in an orbital shaking incubator (150 rpm) at 37°C. Sucrose as a carbon source proved to have good enzyme activity and hence was used as the carbon source for further tests. Peptone, tryptone, and yeast extract at 0.5% concentration were studied as nitrogen sources, while sucrose − 10% and pH – 7.0 were constant in each medium. Yeast extract as a nitrogen source exhibited good enzyme activity and was used as a nitrogen source for subsequent tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003epH and temperature\u003c/h2\u003e \u003cp\u003eThe impact of pH on optimal levan production was investigated by formulating the medium with pH in the range of 4.0–10.0. 10% sucrose and 0.5% yeast extract were maintained in each medium. pH 6.0 exhibited the highest enzyme activity and was utilized for further experiments. To study the impact of temperature on levan production, media were incubated at 30, 37, 42, and 50°C. Medium composition, sucrose 10%, yeast extract – 0.5%, pH – 6.0 was kept the same. An incubation temperature of 30°C was confirmed to have good enzyme activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMetal ions and salts\u003c/h2\u003e \u003cp\u003eTo study the impact of metal ions and salts on enzyme production, mediums were formulated by including compounds like FeSO\u003csub\u003e4\u003c/sub\u003e, MgSO\u003csub\u003e4,\u003c/sub\u003e KCl, NaCl, CaCl\u003csub\u003e2,\u003c/sub\u003e and CuSO\u003csub\u003e4\u003c/sub\u003e at 50 and 100 mM concentrations. Medium composition, sucrose 10%, yeast extract – 0.5%, pH – 6.0 was kept the same, and 30°C was the incubation temperature. MgSO\u003csub\u003e4\u003c/sub\u003e at 50 mM concentration showed the highest enzyme activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSucrose and yeast extract concentration\u003c/h2\u003e \u003cp\u003eTo check the effect of carbon concentration, mediums were formulated with 10, 20, 30, 40, and 50% concentrations of sucrose; other optimized parameters were yeast extract 0.5%, pH 6.0, MgSO\u003csub\u003e4\u003c/sub\u003e − 50 mM, and incubation temperature 30°C. The highest enzyme activity was observed at 50% sucrose concentration. To check the impact of nitrogen concentration, media were formulated with 0.5, 1, 2, and 5% yeast extract; other parameters were sucrose – 50%, pH- 6.0, MgSO\u003csub\u003e4\u003c/sub\u003e − 50 mM, and incubation temperature 30°C. The incubation duration required to attain the highest enzyme activity was considered the optimal duration of incubation. All the optimization tests were performed in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and purification of levan\u003c/h2\u003e \u003cp\u003e100 mL of each unoptimized and optimized media were inoculated with 5% \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 inoculum, following which the flasks were incubated at 30°C for 48 hrs (150 rpm). Subsequently, the fermented broths were centrifugated at 7000 rpm for 20 min at 4°C to remove the microbial cells. The levan in the supernatants was precipitated by mixing with cold absolute ethanol (1:2.5) (v/v), followed by incubation at -20°C for 24 hrs. To separate the precipitated levan, the liquid was centrifuged at 10000 rpm for 20 mins at 4°C. The precipitated levan was dissolved in hot water and deproteinized by using Sevag reagent consisting of n-butanol and chloroform (1:5) (v/v). The appearance of a whitish layer in between the chloroform and water layer indicates protein separation. The upper layer was separated, and the deproteinization process was repeated 2–3 times. Subsequently, the supernatant was transferred to dialysis bag tubing (12 kDa cutoff), and dialysis was carried on for five days at 4°C against distilled water. Distilled water was changed every 24 hrs. Upon completion of dialysis, samples were lyophilized. Lyophilized dry mass was used to determine the yield of levan (Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of Levan\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eNuclear Magnetic Resonance (NMR) spectroscopy\u003c/h2\u003e \u003cp\u003eNMR spectroscopy was utilized to analyze the structure of EPSLEV207. For NMR analysis, D\u003csub\u003e2\u003c/sub\u003eO was used as a solvent to dissolve EPSLEV207. \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were analyzed by running the sample at 400 MHz (Bruker, AVANCE) (Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared (FTIR) spectroscopy\u003c/h2\u003e \u003cp\u003eTo determine the presence of functional groups in EPSLEV207 structure FTIR analysis was carried out. ATR-FTIR spectrophotometer (Bruker, ALPHA-II), was utilized to record FTIR spectra from the wavelength 4000 to 500 cm\u003csup\u003e− 1\u003c/sup\u003e (Mehta, Shukla, and Saraf \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eX-ray Diffraction (XRD) analysis\u003c/h2\u003e \u003cp\u003eAn XRD study was done on EPSLEV207 to determine its nature. For this analysis, Bruker D8 Advance X-ray diffractometer was utilized. The instrument employs CuKα radiation and operates in scan mode over a range of 5° to 60° at various 2θ angles (Mehta, Shukla, and Saraf \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of monosaccharides of levan by chemical hydrolysis\u003c/h2\u003e \u003cp\u003eLyophilized EPSLEV207 was chemically hydrolyzed to determine the monosaccharide composition. Levan (10 mg/mL) was treated with 0.25% oxalic acid and incubated in a boiling water bath for a time period of 10, 20, and 30 mins. The resultant hydrolyzed samples were analyzed by performing TLC, as explained in the section on analytical methods (Belghith et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBiological activity of EPSLEV207\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eAntioxidant activity of EPSLEV207\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section4\"\u003e \u003ch2\u003eDPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity against DPPH radicals was evaluated by slightly modifying the protocol explained by Srinivash et al and Zhu et al (Srinivash et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). EPSLEV207 was dissolved in water at concentrations 1, 2, 3, 4, and 5 mg/mL and mixed with an equal volume of DPPH solution (0.1 mM) prepared in methanol. Ascorbic acid at similar concentrations was used as standard, and all the tubes were incubated for 30 minutes under dark conditions. Methanol was used as the blank. The following formula was utilized to determine the DPPH radical scavenging activity by recording OD at 517 nm:\u003c/p\u003e \u003cp\u003eDPPH radical scavenging activity (%) = ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{A}\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e ) × 100\u003c/p\u003e \u003cp\u003eWhere, A\u003csub\u003esample\u003c/sub\u003e denotes the optical density of the test sample, and A\u003csub\u003econtrol\u003c/sub\u003e denotes the optical density of the control.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity against ABTS radicals was quantified by performing the protocol explained by Zhu et al (Zhu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In short, the solutions of potassium persulfate (2.45mM) and ABTS (7mM) were mixed in equal volumes and placed in the dark for 16 hrs, followed by dilution of the mixture to 0.70 ± 0.2 OD at 734nm by PBS (pH 7.4). EPSLEV207 was dissolved in water at different concentrations (1, 2, 3, 4, and 5 mg/mL). Aliquots of 400 µl from each concentration were aspirated in 3 mL of ABTS radicals, followed by incubation for 6 mins in a dark place. The following formula was utilized to determine the ABTS radical scavenging activity by recording OD at 734 nm:\u003c/p\u003e \u003cp\u003eABTS radical scavenging activity (%) = ( \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{A}\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e ) × 100\u003c/p\u003e \u003cp\u003eWhere, A\u003csub\u003esample\u003c/sub\u003e denotes the optical density of the test sample and A\u003csub\u003econtrol\u003c/sub\u003e denotes the optical density of the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffect of levan on the growth of probiotic strain\u003c/h2\u003e \u003cp\u003eEPSLEV207 was evaluated for its utilization as a carbon source by the probiotic bacterium \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e MTCC 1423 by slightly modifying the protocol explained by Wang et al (Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A basal MRS medium (carbon-free) was prepared and EPSLEV207 and glucose were added as carbon sources at 1% concentration, respectively. The mediums were sterilized, and \u003cem\u003eL. rhamnosus\u003c/em\u003e MTCC 1423 cells (1 × 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) were inoculated into the medium and incubated at 37°C for 48 hrs. Every 24 hrs, samples were removed, serially diluted, spread on MRS agas plates, and incubated for 48 hrs. Utilization of glucose and EPSLEV207 was calculated by counting the cells on the MRS plates, and the number of cells was expressed in log CFU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical properties of levan - Water solubility index (WSI) and Water retention capacity (WRC)\u003c/h2\u003e \u003cp\u003eEPSLEV207 was evaluated for its WSI by suspending 30 mg of EPS in 1 mL of distilled water and placing the tubes at 35°C for 1 hr under continuous agitation (Thomas et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The EPS solution was centrifuged for 20 mins at 5000 rpm, the resultant supernatant was transferred to a pre-weighed tube, and the solubilized EPS was precipitated by mixing with ethanol in a ratio of 1:3. The tubes were kept at 80°C to dry the precipitate, and the difference in the tube weight was recorded. WSI was calculated by using the following formula:\u003c/p\u003e \u003cp\u003eWSI (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{dry\\:weight\\:of\\:EPS\\:in\\:the\\:supernatant}{initial\\:weight\\:of\\:EPS\\:\\left(30\\:mg\\right)}\\)\u003c/span\u003e\u003c/span\u003e × 100\u003c/p\u003e \u003cp\u003eEPSLEV207 was evaluated for its WRC by modifying the protocol explained by Thomas et al (Thomas et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In short, 20 mg of EPSLEV207 was taken in a pre-weighed tube and suspended in 1 mL of distilled water, followed by centrifugation at 13,000 rpm for 30 mins. The resultant supernatant was discarded, and the tube with wet EPSLEV207 was weighed. The difference in the weight of the tube before and after was calculated to get the weight of EPS after water absorption. WRC was calculated by using the following formula:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eWRC (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{weight\\:of\\:EPS\\:after\\:absorption}{initial\\:weight\\:of\\:EPS\\:\\left(20\\:mg\\right)}\\)\u003c/span\u003e\u003c/span\u003e × 100\u003c/h2\u003e \u003cdiv id=\"Sec23\" type=\"Results\" class=\"Section3\"\u003e \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \n\n \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003cdiv id=\"Sec35\" class=\"Section4\"\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e\n\n \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003ch2\u003eStrain cultivation\u003c/h2\u003e\u003cp\u003eTo check the production of levan, \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 was streaked on agar plates with and without sucrose. Colonies with slimy consistency were observed on the sucrose agar plate, which indicates the production of EPS. Normal colonies were observed on the agar plate without sucrose.\u003c/p\u003e\u003ch2\u003eOptimization of medium components\u003c/h2\u003e\u003ch2\u003eImpact of carbon source on levansucrase activity\u003c/h2\u003e\u003cp\u003eThe impact of five carbon sources on \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 growth and levansucrase production was studied. The data revealed the growth of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 in the medium supplemented with glucose, sucrose, galactose, and maltose (Fig.\u0026nbsp;1a). Fructose showed very low bacterial growth. Levansucrase production (enzyme activity − 163.3 µmol/mL/min) and bacterial growth were highest in the sucrose medium (Fig.\u0026nbsp;1b). Sucrose was utilized both for bacterial growth and levansucrase production. Sucrose, as a carbon source, has proven to affect levansucrase production in \u003cem\u003eGluconobacter albidus\u003c/em\u003e TMW 2.1191 (Jakob et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Bacterial growth was good in glucose, galactose, and maltose, but the enzyme production was lower compared with sucrose medium. Hence, sucrose was considered the best carbon source for further investigations. Sucrose acts as an inducer for the production of levansucrase in \u003cem\u003eBacillus spp\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e strain L1 (Belghith et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Desai and Patel \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eImpact of nitrogen source on levansucrase activity\u003c/h2\u003e\u003cp\u003eNitrogen sources in the medium can impact the growth of bacterial cells and levansucrase production. In the current study, three types of nitrogen sources, i.e. yeast extract, peptone, and tryptone, were studied for their impact on cell growth and enzyme production. The medium supplemented with yeast extract showed the highest cell growth (Fig.\u0026nbsp;1c) as well as levansucrase production (enzyme activity − 163.6 µmol/mL/min), followed by peptone and tryptone (Fig.\u0026nbsp;1d). Yeast extract was observed to have higher levansucrase production than other nitrogen sources (Desai and Patel \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Belghith et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Yeast extract was confirmed as the most effective nitrogen source.\u003c/p\u003e\u003ch2\u003eImpact of pH on levansucrase activity\u003c/h2\u003e\u003cp\u003eThe role of pH in the production of levansucrase and cell growth was studied in the present work. Mediums with initial pH ranging from 4.0 to 10.0 were used in the study. The findings reveal optimum levansucrase production (enzyme activity − 202 µmol/mL/min) at pH 6.0. A decrease in enzyme production was observed from pH 5.0 to 4.0, and pH 7.0 to 10.0 (Fig.\u0026nbsp;1f). Maximum levansucrase production was in the range of 5.0 to 6.5 (Desai and Patel \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Bacterial growth was observed highest at pH 8.0, which is suitable for cell mass growth, but the enzyme production was reduced significantly at this pH (Fig.\u0026nbsp;1e), which might be due to the difference in optimal pH for cell growth and enzyme production. Focusing on optimizing the levan production, pH 6.0 was considered to be the ideal.\u003c/p\u003e\u003ch2\u003eImpact of temperature on levansucrase activity\u003c/h2\u003e\u003cp\u003eTo study the impact of temperature on levansucrase production, the incubation temperature of 30, 37, 42, and 50°C was studied. In the present study, the highest levansucrase production was at 30°C (enzyme activity – 216.13 µmol/mL/min), followed by 37°C (200.76 µmol/mL/min) and 42°C (195.23 µmol/mL/min), respectively Levansucrase production at the temperature 50°C was lowest in our observation (139.43 µmol/mL/min) (Fig.\u0026nbsp;1h). In contrast, cell growth was highest at 37°C, followed by 42 and 50°C, whereas it was least at 30°C, respectively (Fig.\u0026nbsp;1g). Comparable with pH, optimum temperature also differed for cell growth and levansucrase production. 30°C was reported to be the optimum temperature for the production of levan by \u003cem\u003eSuhomyces kilbournensis\u003c/em\u003e (González-Torres et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In our present study, 30°C was considered to be optimal for the production of levansucrase.\u003c/p\u003e\u003ch3\u003eImpact of metal ions and salts on levansucrase activity\u003c/h3\u003e\u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e, MgSO\u003csub\u003e4\u003c/sub\u003e, KCl, NaCl, CaCl\u003csub\u003e2\u003c/sub\u003e, and CuSO\u003csub\u003e4\u003c/sub\u003e at 50 and 100mM concentrations were used to study the influence of metal ions and salts on levansucrase production. \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 showed specificity for growth and levansucrase production under the influence of metal ions. The highest bacterial growth was observed in KCl (50mM), followed by MgSO\u003csub\u003e4,\u003c/sub\u003e NaCl, FeSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4,\u003c/sub\u003e and CaCl\u003csub\u003e2\u003c/sub\u003e. No growth was observed in KCl (100 mM) (Fig.\u0026nbsp;2a). Levansucrase production was highest in MgSO\u003csub\u003e4\u003c/sub\u003e (50 mM) (enzyme activity – 221.3 µmol/mL/min), followed by NaCl (100 mM) (202.6 µmol/mL/min), and KCl (50mM) (197.13 µmol/mL/min) (Fig.\u0026nbsp;2b). Metal ions such as FeSO\u003csub\u003e4,\u003c/sub\u003e CaCl\u003csub\u003e2,\u003c/sub\u003e and CuSO\u003csub\u003e4\u003c/sub\u003e completely inhibited the production of the enzyme. Zhang et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that metal ions have the potential to enhance enzyme levansucrase activity (Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MgSO\u003csub\u003e4\u003c/sub\u003e (50 mM) proved to be the best salt that increased levansucrase production.\u003c/p\u003e\u003ch2\u003eImpact of concentration of sucrose on levansucrase activity\u003c/h2\u003e\u003cp\u003eTo evaluate the impact of the concentration of sucrose on bacterial growth and levansucrase activity, media were formulated with 10, 20, 30, 40, and 50% sucrose and incubated for 72 hrs. The highest cell growth was observed at 10% sucrose and showed a gradual decline as the concentration increased (Fig.\u0026nbsp;2c). The cells tend to adjust with the increasing osmotic pressure created by increasing sucrose concentration, which leads to lower cell growth. But in the case of levansucrase production, the opposite pattern was observed. As the sucrose concentration increased, levansucrase production increased significantly (Fig.\u0026nbsp;2d). The highest levansucrase production (enzyme activity – 410.4 µmol/mL/min) was observed at 50% sucrose after 48 hrs of incubation. Chen and Liu (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) suggested that a significant portion of sucrose is utilized by cells for growth when supplemented with sucrose below 10% concentration, while higher sucrose concentrations result in enhanced enzyme induction with inhibition of cell growth (Chen and Liu \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eImpact of concentration of yeast extract on levansucrase activity\u003c/h2\u003e\u003cp\u003eTo study the impact of the concentration of yeast extract on bacterial growth and levansucrase activity, mediums were prepared with 0.5, 1, 2, and 5% yeast extract and incubated for 72 hrs. The highest cell growth was observed at 1% yeast extract concentration, with a further decline as the yeast extract concentration increased (Fig.\u0026nbsp;2e). Levansucrase activity was highest at 1% concentration after 48 hrs of incubation (enzyme activity 427.4 µmol/mL/min) (Fig.\u0026nbsp;2f). Chen and Liu (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) stated that the enzyme activity increases with the increase in the yeast extract concentration (Chen and Liu \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eIsolation and purification of levan\u003c/h2\u003e\u003cp\u003eBoth unoptimized and optimized mediums were sterilized and inoculated with 5% inoculum of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207, then incubated at 30°C for 48 hrs. Cells were separated by centrifugation, and precipitation of levan was done by adding chilled ethanol, followed by deproteinization. The deproteinized samples were dialyzed and lyophilized. The dry weight of lyophilized levan was utilized to calculate the yield of levan from the unoptimized and optimized medium. The unoptimized medium yielded 9.27 g/L of levan, while the optimized medium and conditions yielded 65.58 g/L of levan. The yield has been increased 7-fold by optimizing the medium components and conditions.\u003c/p\u003e\u003ch2\u003eCharacterization of Levan\u003c/h2\u003e\u003ch2\u003eNMR\u003c/h2\u003e\u003cp\u003eNMR spectroscopy was performed to study the structural aspect of EPSLEV207. The \u003csup\u003e1\u003c/sup\u003eH spectra reveal the chemical shift in the proton signals, providing insight into the structural composition of monomer, in this case, fructose. The signals present in EPSLEV207 showed a chemical shift of 3.487 ppm (H6b), 3.617 ppm (H1b), 3.688 ppm (H1a), 3.841 ppm (H6a), 3.882 ppm (H5), 4.028 ppm (H4), and 4.111 ppm (H3) (Fig.\u0026nbsp;3a). These chemical shifts resemble the levan produced by \u003cem\u003eBacillus siamensis\u003c/em\u003e, and \u003cem\u003eBacillus megaterium\u003c/em\u003e GJT321 (Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e13\u003c/sup\u003eC spectra reveal the carbon involved in the formation of monosaccharides. The \u003csup\u003e13\u003c/sup\u003eC spectra for EPSLEV207 showed the presence of six signals at 59.89 (C1), 63.38 ppm (C6), 75.19 ppm (C4), 76.28 ppm (C3), 80.28 ppm (C5), and 104.10 ppm (C2) (Fig.\u0026nbsp;3b). The observed signals confirmed the presence of fructose as a monosaccharide in the EPSLEV207 due to the presence of anomeric carbon at 104.10 ppm (C2), methylene group at 59.89 (C1), and 63.38 ppm (C6), furanose ring present at 80.28 ppm (C5), and peaks at 76.28 (C3), and 75.19 ppm (C4) representing oxymethinic group. Similar results were reported by Thakham et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003ch3\u003eF-TIR\u003c/h3\u003e\u003cp\u003eThe functional groups present in the EPSLEV207 structure were analyzed using F-TIR spectroscopy. As illustrated in Fig.\u0026nbsp;3c, FTIR spectra for EPSLEV207 indicated the presence of a strong peak at 3290 cm-1, representing an O-H bond. Two peaks were detected at 2937 and 2884 cm\u003csup\u003e− 1\u003c/sup\u003e, representing the presence of C-H bond. A peak was present at 1645 cm\u003csup\u003e− 1\u003c/sup\u003e, indicating the existence of bound water. A peak was detected at 1419 cm\u003csup\u003e− 1\u003c/sup\u003e, representing the bending of the methylene group of the C-H bond. A peak around 1123 cm-1 represents the C-O-H bond. A strong peak was detected at 1009 cm\u003csup\u003e− 1\u003c/sup\u003e, representing the C-O-C, a glycosidic linkage specifically present in the pyranose or furanose ring structure. Two peaks at 922 and 808 cm\u003csup\u003e− 1\u003c/sup\u003e confirm the presence of furanose units. Overall, the FTIR spectra of EPSLEV207 confirm the presence of polysaccharides and resemble the spectra represented by Yu et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Thakham et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Yu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Thakham et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eXRD\u003c/h2\u003e\u003cp\u003eThe nature of EPSLEV207 was determined by XRD. The X-ray diffractogram of EPSLEV207 showed a broad peak pattern around 15–20° 2θ with the highest intensity (4646 ppm) peak at 17.61° 2θ (Fig.\u0026nbsp;3d). The broad nature of the X-ray diffractogram pattern indicates the amorphous nature of levan (Mendonça et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kekez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The amorphous property of levan indicates a lack of crystal structure but provides a high solubility rate, a desirable property in medical and pharmaceutical fields. Sadhana et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) prepared a levan-derived nanoparticle of dolutegravir, an anti-HIV drug, and reported an enhancement in the solubility of the drug due to the amorphous nature of levan (Sadhana et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eChemical hydrolysis and monosaccharide detection\u003c/h2\u003e\u003cp\u003eTo determine the monosaccharide composition of EPSLEV207, oxalic acid was used as a hydrolyzing agent. Samples were treated for 10, 20, and 30 minutes and were maintained in a boiling water bath (Belghith et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The hydrolyzed samples were used for loading on a TLC plate along with sucrose, fructose, and glucose as standards. The TLC plate was analyzed, and a single spot concurrent to fructose was observed (Fig.\u0026nbsp;4a), indicating that EPSLEV207 is a homopolysaccharide linked by fructose as a monomer.\u003c/p\u003e\u003ch2\u003eBiological activity\u003c/h2\u003e\u003ch2\u003eAntioxidant activity –\u003c/h2\u003e\u003cp\u003e \u003cb\u003eDPPH radical scavenging activity\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe in vitro antioxidant activity of EPSLEV207 was studied by DPPH radical scavenging assay. The evaluation was conducted for concentrations 0 to 5mg/mL, respectively, for which the DPPH radical scavenging activity was found to be in the range of 40.92–49.15% (Fig.\u0026nbsp;4b). These results indicate that the EPSLEV207 had a noticeable radical scavenging ability. Furthermore, the DPPH radical scavenging activity for EPSLEV207 was greater than EPS derived from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e YW32 (DPPH \u0026lt; 30%) (Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The property of EPSLEV207 to scavenge the DPPH radicals can be attributed to their ability to donate electrons (Bouallegue et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003eABTS radical scavenging activity\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe invitro antioxidant activity of EPSLEV207 was studied by ABTS radicals. The evaluation was conducted for concentrations 0 to 5mg/mL, and the ABTS radical scavenging activity was found to be in the range of 17.59–22% (Fig.\u0026nbsp;4c). Our results for EPSLEV207 align with the ABTS radical scavenging activity of EPS derived from \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e M41 at the concentration of 5mg/mL (Ayyash et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003eEffect of levan on the growth of probiotic strain\u003c/b\u003e \u003c/p\u003e\u003cp\u003eEPSLEV207 was studied for its effect on the growth of \u003cem\u003eL. rhamnosus\u003c/em\u003e MTCC 1423, a probiotic strain. Glucose and EPSLEV207 were used as a carbon source in basal MRS broth and inoculated with \u003cem\u003eL. rhamnosus.\u003c/em\u003e As illustrated in Fig.\u0026nbsp;4d, after 24 hrs of incubation, cells reached 8.95 log CFU/mL in glucose-supplemented medium and 8.49 log CFU/mL in EPSLEV207 supplemented medium. After 48 hrs of incubation, cells reached 7.11 log CFU/m L glucose medium and 8.38 log CFU/mL in EPSLEV207 medium. Glucose is a monomeric form of carbohydrate and is easily utilized by the cells at the initial growth phase, while EPSLEV207 is a polymeric form and was not utilized predominantly, leading to lesser cell growth. After 48 hrs, the glucose in the medium was completely utilized, and cells attained the death phase. While in the EPSLEV207 medium, cells were more viable than those in the glucose medium. In general, Lactic acid bacteria might possess substrate-specific fermentation metabolism, which enables them to utilize prebiotic compounds selectively (Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003ePhysicochemical properties - WSI and WRC\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe WSI and WRC of EPSLEV207 were 88.88 ± 1.57% and 78.33 ± 2.35%, respectively. The WSI of EPSLEV207 was higher than the levan derived from \u003cem\u003eBacillus velezensis\u003c/em\u003e VTX20, but the WRC was lower compared to the levan from the same organism (WSI − 81.9% and WRC − 100.2%) (Vu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The WSI and WRC were much lower than the levan derived from \u003cem\u003eBacillus subtilis\u003c/em\u003e PR-C1 (WSI − 90.2% and WRC – 205.6%) (Thomas et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Previous reports suggest the involvement of various factors, such as monosaccharide composition, chain length, and linkage pattern, in determining the WSI and WRC of EPS (Thomas et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present work, we isolated a novel strain of \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207 from a marine sample and investigated its ability to produce levan, an exopolysaccharide composed of repeating fructose units. Given the wide range of applications for levan in the food and pharmaceutical fields, optimizing the yield is essential. We aimed to exploit the levan-producing capability of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 by enhancing its yield through the optimization of the media composition and conditions. To study the effect of different parameters, cell mass, and levansucrase activity were considered critical factors for defining an increase in levan production. Bacterial cells produce levan as a defense mechanism against environmental stress. We explored the enzyme production rate of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 under various conditions and optimized the production of levan.\u003c/p\u003e \u003cp\u003eMedia components such as carbon source, nitrogen source, pH, and presence of salts and metal ions significantly impact levansucrase production. The concentration of sucrose as a carbon source notably influences cell growth and enzyme production due to the osmotic pressure it creates. This effect was previously reported in the case of levan production by \u003cem\u003eZymomonas mobilis\u003c/em\u003e strain 113S (Vigants, Zikmanis, and Bekers \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). When microorganisms are subjected to elevated sucrose levels, they may encounter osmotic stress, modulating their metabolic pathways to sustain cellular homeostasis (Barros and Celligoi \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Similarly, the concentration of yeast extract can also induce osmotic pressure and affect cell growth and enzyme activity. Determining the optimal concentration is critical; insufficient levels may not induce adequate osmotic stress to trigger levansucrase synthesis, whereas excessively high concentrations can inhibit cell growth. This inhibition was due to the yeast extract exceeding 1%. Excess yeast extract concentration inhibited cell growth and levansucrase production in \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe initial pH of the media and temperature have different influences on cell growth and levansucrase activity. The change in pH can alter the structure and activity of enzymes. Different microorganisms have different optimal pH and temperatures for cell growth and proliferation. The variations in optimal pH and temperature for cell growth and levansucrase can be attributed to the difference in optimum pH and temperature for levansucrase induction and synthesis (Queiroz Santos, Del Bianchi, and Garcia-Cruz \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The stress of surviving at lower temperatures might have induced the production of levansucrase at 30\u0026deg;C in \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, the presence of salts and metal ions can impact the production of levansucrase, as certain enzymes require specific salts to achieve optimal activity. Salts aid in stabilization of enzyme structure and regulation of substrate binding (Aguiar-Oliveira and Maugeri \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe purified levan (EPSLEV207) was evaluated for its antioxidant activity against DPPH and ABTS radicals, showing radical scavenging activity of 49.15 and 22%, respectively. The antioxidant properties of levan have been reported in various bacterial species, including \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e strain ES, \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e S81, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e KX041, etc (Korany et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Taylan, Yilmaz, and Dertli \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Gut microbiota and its homeostasis is one of the major research areas that deals with human health. Prebiotic compounds are substrates that are selectively utilized by human gut microbiota and confer health benefits. Fructooligosaccharides, galactooligosaccharides, inulin, and lactulose are a few examples of polysaccharides that meet the necessary criteria for being termed prebiotics (Pohlentz et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To investigate the prebiotic activity of EPSLEV207 as the sole carbon source, \u003cem\u003eL. rhamnosus\u003c/em\u003e MTCC 1423 was grown in a modified MRS medium. Our findings indicate that L. rhamnosus exhibited better survivability in EPSLEV207-containing medium compared to glucose-containing medium. The WSI and WRC of EPSLEV207 were 88.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57% and 78.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35%, respectively. The solubility of levan is crucial for its application as a stabilizer, thickener, and emulsifier. The ability of levan to hold moisture helps in promoting effective healing and skin hydration. The water solubility and water retention capacity influence the ability of levan to interact with other compounds and have been used in the formation of hydrogels and drug delivery systems (Erdal Altıntaş and Aytar \u0026Ccedil;elik \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207, isolated from the marine environment, demonstrates notable osmotolerance and the ability to endure extreme salinity and temperature. These qualities of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 were utilized in the current study to optimize the yield of levan.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present work has shown the potential of a novel marine bacterium, \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207, in the production of levan. Focusing on increasing the yield of levan, medium components and conditions were optimized. Optimization by one variable at a time approach has proven effective as the yield has increased 7-fold. NMR spectra confirmed the levan structure, FTIR spectra indicated the presence of glycosidic bonds, and XRD confirmed the amorphous nature of EPSLEV207. EPSLEV207 has good antioxidant activity, water solubility, and water retention capacity. It has also been effective as a carbon source in the growth of probiotic strain \u003cem\u003eL. rhamnosus\u003c/em\u003e MTCC 1423. The utilization of high-levan-producing \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207 in industrial applications is promising. Furthermore, the application of recombinant DNA technology and cloning might improve the expression of levansucrase and extend the scale of applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe would like to thank the funding agencies for their in-time support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003ePOP and KVC designed the experiments. POP and KR performed experiments. POP and KVC analyzed and interpreted data and wrote the manuscript. KVC and the VP supervised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u0026nbsp;\u003c/strong\u003eThis work was supported by National Fellowship for Other Backward Classes (UGC JRF NFOBC-No.F.40-2/2019) \u0026ndash; Junior Research Fellowship (JRF) to POP and National Backward Classes Finance and Development Corporation (No. NBCFDC/E-70291) \u0026ndash; JRF to KR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The data that support the findings of this study can be made available by authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This study does not involve animal or human subjects, and thus no ethical approval is required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent of publications\u003c/strong\u003e We hereby confirm that all co-authors of this study have reviewed the final version of the manuscript and have provided their full consent for the submission and publication of the paper in \u003cem\u003eInternational Microbiology\u003c/em\u003e.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAguiar-Oliveira E, Maugeri F (2013) Effects of the addition of substrate and salts in both the fructosyltransferase immobilization and its catalytic properties. 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Int J Biol Macromol 210: 504\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2022.04.203\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2022.04.203\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\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":"Exopolysaccharide, C. erzurumensis, levan, optimization, purification, antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-4834548/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4834548/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLevan, an exopolysaccharide, has applications in the medical and food sectors. Under natural conditions, levan is produced at a low concentration by microorganisms. This current study focuses on optimizing levan production by a one-variable-at-a-time approach from a novel marine bacterium, \u003cem\u003eCalidifontibacillus erzurumensis\u003c/em\u003e LEV207. The growth and levansucrase production by \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 were studied to optimize the media components and conditions. Sucrose and yeast extract proved to be a good levansucrase inducer. The pH of 6.0 and temperature of 30\u0026deg;C, along with MgSO\u003csub\u003e4\u003c/sub\u003e as salt, increased the enzyme activity. Sucrose and yeast extract at the concentration of 50% and 1%, respectively, produced the maximum enzyme activity of 427.4 \u0026micro;mol/mL/min after 48 hrs of incubation. The optimization process increased the levan (EPSLEV207) production 7-fold with a final yield of 65.58 g/L. To confirm the structure and nature of EPSLEV207, NMR and XRD were performed, and the functional groups present in EPSLEV207 were determined using FTIR. Chemical hydrolysis of levan confirmed fructose as the monomer, making EPSLEV207 a homopolysaccharide. EPSLEV207 exhibited 49% and 22% radical scavenging activity against DPPH and ABTS radicals, respectively, and promoted the growth of probiotic strain. EPSLEV207 showed an 88% water solubility index and 78% water retention capacity. The optimization process has increased the yield of levan up to 7-fold, highlighting the potential of \u003cem\u003eC. erzurumensis\u003c/em\u003e LEV207 to be a viable option for industrial applications.\u003c/p\u003e","manuscriptTitle":"Improved levan production by novel Calidifontibacillus erzurumensis LEV207 using one variable at a time approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 06:35:56","doi":"10.21203/rs.3.rs-4834548/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-27T08:01:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-20T08:34:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T22:49:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-14T20:19:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17725200060316011694704098191421955252","date":"2024-08-12T06:14:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336414161734241965778587347711051945880","date":"2024-08-11T07:01:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248420814653963851303313866516505948555","date":"2024-08-09T20:02:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62051207659957140473152812279879079552","date":"2024-08-09T14:48:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-09T14:43:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-09T14:39:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-09T04:30:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2024-07-31T10:09:32+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":"c53ef080-8fd1-4c4d-bf56-d0de781fe40b","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:09:17+00:00","versionOfRecord":{"articleIdentity":"rs-4834548","link":"https://doi.org/10.1007/s10123-024-00597-5","journal":{"identity":"international-microbiology","isVorOnly":false,"title":"International Microbiology"},"publishedOn":"2024-09-26 15:57:15","publishedOnDateReadable":"September 26th, 2024"},"versionCreatedAt":"2024-09-04 06:35:56","video":"","vorDoi":"10.1007/s10123-024-00597-5","vorDoiUrl":"https://doi.org/10.1007/s10123-024-00597-5","workflowStages":[]},"version":"v1","identity":"rs-4834548","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4834548","identity":"rs-4834548","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}