IntroductionExopolysaccharides (EPS) are long chains of sugar units such as glucose, galactose, and rhamnose found in various bacteria1. Polysaccharides may be embedded in the cells of diverse bacterial species including storage polysaccharides for example glycogen2. Structural polysaccharides that comprise cell walls as well as exocellular polysaccharides leading to the formation of glycocalyces. These exocellular polysaccharides include capsular polysaccharides and EPS2. Some of these EPS have been shown to possess valuable properties such as biodegradability, non-toxicity, biocompatibility, cell adhesion, environmental protection, and energy storage3. Heteropolysaccharides like hyaluronic acid and homopolysaccharides such as dextran are examples of EPS that exhibit monosaccharide composition-based classification4. This is because their unique rheological properties make them highly useful in industries such as food, cosmetics, pharmaceuticals and textiles4. Other applications include using these polymers as emulsifiers and stabilizers among other functions such as moisture retention4. EPS has potential to wound healing, environmental applications such as heavy metal tolerance and metal biosorption5,6.Simple sugars such as glucose and sucrose commonly serve as the primary carbon and energy sources, whereas amino acids or ammonium salts serve as nitrogen sources. They are required for microbial growth and production of biomolecules7. The ratio of carbon and nitrogen (C:N) available in environmental conditions has an important role in EPS production; low nitrogen levels enhance EPS production8. There are predominant routes to the EPS biosynthesis in bacterial species, the Wzx/Wzy-dependent, ABC transporters-dependent, synthase-dependent, and extracellular synthesis mediated by sucrase protein9,10.EPS in nature can be produced through different fermentation techniques such as Solid state fermentation (SSF) and submerged fermentation (SmF), which are utilized to produce exopolysaccharides and other beneficial substances under controlled environments. The SmF was selected over SSF due to its superior control over critical process parameters such as pH, temperature, aeration, and agitation, which are essential for consistent and enhanced EPS production1,4. SmF also facilitates easier downstream processing, uniform nutrient distribution, and scalability, making it more suitable for industrial applications and large-scale bioprocess optimization89. Fermentation leverages the metabolic pathways of microorganisms to convert complex substrates into value-added secondary metabolites, such as exopolysaccharides (EPS). In microbial systems, the type and yield of bioactive metabolites are species-specific and significantly influenced by environmental parameters such as nutrient availability, pH, temperature, and oxygen levels. These conditions modulate the biosynthesis of industrially valuable compounds, including antibiotics, pigment enzymes, and antioxidants11. Some of the bacterial strains that secrete EPS were reported, such as Lactobacillus plantarum1Bacillus cereus12Lacticaseibacillus paracasei CIDCA 8339, CIDCA 83,123 and CIDCA 83,12413, Leuconostoc pseudomesenteroids 56 and Weissella cibaria 21 and 6414, and Alkalibacillus sp.w15Bacillus haynesi CamB616, and Streptococcus thermophilus17.EPS has different functionalities like antioxidant activity, antitumor effects, immunomodulatory properties, and acid hydrolysis properties. Several parameters affect the antioxidant potential of bacterial exopolysaccharides (EPS), such as fermentation conditions, the bacterial growth and fermentation kinetics, the type of monosaccharides, glycosidic linkage patterns, degree of branching18,19. Acid hydrolysis is commonly employed to analyse the monosaccharide composition of EPS by cleaving glycosidic linkages to release individual sugar units. Variations in monosaccharide compositions contribute to functional differences among EPS types. For instance, glucose-rich EPS have demonstrated strong scavenging activity against superoxide and DPPH radicals19. Similar to their function in modulating monosaccharide proportion in structure heteropolysaccharide, bacterial fermentation also plays a significant role in determining the structure and corresponding radical DPPH scavenging/Hydroxyl radical scavenging for EPS, such as having a high mannose to galactose ratio has potent hydroxyl radical scavenging and DPPH reduction abilities20. The antioxidant properties of EPS, composed of glucose, galactose, and rhamnose as the main monosaccharide constituents, have also been reported. Bacterial fermentation conditions, such as pH, govern EPS structure and hence affect antioxidant properties. This is made possible through regulation of the proportion of monosaccharides in the EPS that occurs as a result of varying pH leading to differences in the level of antioxidant activity exhibited by such polysaccharides21. These parameters indicate that different bacterial EPS could be used for various applications having antioxidant ability as indicated22.The ability of exopolysaccharides (EPS) to fight against microbes is influenced by their features. EPS consists of groups, like carbonyl phosphate and hydroxyl groups that interact with bacterial cell membranes to exhibit antimicrobial effects23. Research indicates that EPS made up of glucose and rhamnose shows properties against pathogens such as Staphylococcus aureus, Bacillus subtilis, and Bacillus pertussis24. Furthermore, EPS produced by bacteria with glucose mannose, galactose, fucose, and uronic acid fractions have displayed effects against various pathogens. The composition of EPS including the types of monosaccharides and the presence of groups plays a crucial role in determining their antimicrobial activity which make them a promising options for antimicrobial applications22.Exopolysaccharides (EPS) from a variety of bacterial species have been the subject of much investigation, but little is known about novel or underreported microbial strains that can produce EPS with strong reducing and antioxidant capabilities. Furthermore, psychrotolerant or lesser-known genera like Sporocarcina are substantially ignored in the majority of the research currently in publication, which concentrates on well-studied genera like Lactobacillus1Streptococcus16 and Bacillus12. Additionally, there aren’t many thorough investigations that include purification, structural characterization, optimum fermentation conditions, and assessment of bioactivities including antioxidant capacity in a single workflow for EPS made from these novel strains. Despite growing interest in mesophilic bacteria as novel EPS producers, Sporosarcina psychrophila, a psychrotolerant, Gram-positive, spore-forming bacterium, remains underexplored. Originally isolated from cold environments, S. psychrophila is known for its unique enzymatic and metabolic adaptations, which may facilitate EPS biosynthesis under low-temperature or nutrient-stress conditions. For cryoprotection, food preservation, and cosmetics, its psychrotolerance facilitates the creation of EPS with improved solubility, stability, and function at low temperatures. Rare sugars or new connections may be present in these EPS, providing special structural, rheological, or bioactive qualities that are advantageous in cold-chain applications. While Sporosarcina species have been previously noted for urease activity and bioremediation potential25 there is a lack of comprehensive studies on their capacity for EPS production. To date, there is no detailed report evaluating the EPS yield, structural properties, or antioxidant activity of EPS from S. psychrophila. This gap presents an opportunity to explore novel EPS with therapeutic potential from psychrotolerant sources.In this work, the submerged fermentation process for producing EPS from Sporosarcina psychrophila MTCC-2908 was optimized. To assess the pharmacological potential of the crude EPS, it was purified, characterized, and its antioxidant and reducing activities were examined. To the best of our knowledge, this is the first research showing that S. psychrophila MTCC-2908 produces EPS with bioactive qualities, which adds to our understanding of the possible uses of psychrotolerant bacterial EPS.Materials and methodsMaterialsAll chemicals, reagents, and media components used in this study were of analytical grade and procured from Loba Chemie, Sigma-Aldrich, and HiMedia,Bacterial strain and preservationThe bacterial strain Sporocarcina psychrophila MTCC-2908 was procured from the cell culture facility of the Institute of Microbial Technology (IMTECH), Chandigarh-India, in a lyophilized form. The Glycerol stock of the strain was prepared and preserved at − 80 °C for further use.Preparation of inoculumThe bacterial strain was inoculated into nutrient broth (NB) media and retrieved the active nature of the culture. The strain was further sub-cultured into 100 mL NB and stored at 4 °C for maintenance. During the course of inoculum preparations, the growth profile of the Sporocarcina psychrophila MTCC-2908 was constructed as shown in Fig. 1. The mid-exponential culture of 10 h was used for inoculation.Fig. 1Growth curve of Sporocarcina psychrophila MTCC-2908 in nutrient broth.Full size imageSubmerged fermentation for EPS productionA 100 mL fermentation media consisting of (% w/v): Glucose, 20; NH4Cl, 6; K2HPO4, 1.1; MgSO4·7H2O, 0.3; MgSO4·H2O, 0.3; CaCl2·2H2O, 0.2; FeCl3, 0.2, and initial pH 4.0 was prepared. The fermentation media was inoculated with 7.5 mL of inoculum and kept in an incubator at 180 rpm and 32 °C 48–120 h.Initial screening of process parameters using Plackett- Burman design (PBD)A statistical experimental model was created to screen the influential parameters among 10 parameters, for EPS production using PBD with JMP software (Trial Version) as shown in Table S1. The experimental design matrix of PBD with real and coded levels is shown in Table 1. The study was carried out in a 500 mL flask with 100 mL of fermentation media.Table 1 The orthogonal design matrix with real and coded levels of PBD with experimental and predicted EPS yield.Full size tableOptimization of the fermentation media using central composite designFive significant parameters were screened from the Plackett-Burman design (Glucose, NH4Cl, K2HPO4, MgSO4·7H2O, MnSO4 2H2O) and they are further optimized by central composite design (CCD) to find the optimal conditions. The design matrix was generated using JMP software to elucidate the interaction effect of the above five significant parameters for optimal EPS production. An experiment design of 32 different trials with five different levels was formulated as depicted in Tables S2 and 2.Table 2 The experimental and predicted values of central composite design matrix EPS production.Full size tableExtraction and purification of EPSA 50 mL fermentation broth was centrifuged at 10,000 rpm, and 4 °C for 15 min. Next, the chilled absolute ethanol was added to the supernatant in the ratio of 2:1 for the precipitation of the exopolysaccharides. Then, the mixture was kept overnight at 4 °C and later again it was centrifuged at 10,000 rpm at 4 °C for 15 min to collect the EPS in the form of pellets. The dry weights of the pellets were noted to estimate the amount of crude EPS yield. The EPS extracted was further purified by dissolving in distilled water and centrifuged at 10,000 rpm at 4 ℃ for 15 min20,21,24,26. Finally, the purified EPS was lyophilized for further characterization and application studies.Characterization of the EPS produced by Sporosarcina psychrophila MTCC − 2908The purified EPS was characterized using different thermoanalytical techniques, including SEM, AFM, XRD, TGA, and FTIR, offer insights into the structural, morphological, and thermal stability of EPS. Collectively, these techniques provide compressive chemical characterization, including identification of monomeric residues, glycosidic linkages, and branching pattern25,26.Fourier transform infrared spectroscopy (FTIR)FTIR spectroscopy is used to detect functional groups and molecular bonds based on characteristic absorption frequencies. This was acquired in transmittance mode with a Shimadzu spectrophotometer The EPS sample was transferred into the container and 50 scans were performed in 4 cm− 1 resolution.ThermoGravimetric analysis (TGA)TGA was used to measure the mass over time as its temperature changes. TGA of the exopolysaccharide was performed using Hitachi STA7200 Thermal Analysis System. The EPS sample of 1.0 mg was heated in a linear range rate of 20 °C over a temperature range of 40–730 °C under nitrogen flow of 200 mL, and the corresponding weight loss was monitored and recorded.Atomic force microscopy (AFMS)AFMS is a technique used for the imaging of the surface of the samples. The atomic force microscopy of the EPS was carried out as described elsewhere17. Briefly, the glass slides were treated with a blend of 15 mL HCl and 5mL HNO3 for 30 min. Meanwhile, the EPS sample was subjected to a mixture of 20 mL H2SO4 and 5 mL H2O2 for 30 min. Next, the glass slides were rinsed with double distilled water, and stored for further use. Then, a fresh EPS solution was prepared by dissolution in double distilled water. Approximately 10 µL of EPS sample was deposited onto the surface of glass slide and allowed to air dry at room temperature. Finally, the AFMS images were captured using the Innova SPM Atomic Force Microscope.X-ray diffraction (XRD)To determine the physical characteristics of the purified EPS, XRD was recorded using a powder diffractometer. Scanning was carried out at a range of 2\(\:\theta\:\) angles (2–70 °C). The crystallinity index was found with the ratio between the area under the crystalline peaks and the total area of the amorphous and crystalline peaks as shown below:27$$CI\, \% = \frac{{\sum {Area\,Under\,Crystalline\,peaks} }}{{\sum {Area\,Under\,Crystalline\,peaks} \, + \, \sum {Area\,under\,Amorphous\,peaks} }} \times 100$$Scanning electron microscopy–energy-dispersive X-ray analysisScanning electron microscopy of lyophilized EPS samples was carried out using Zeiss EVO 18 Special edition Scanning Electron Microscope at 15 KeV accelerating voltage and images at magnifications of 3000×, 5000× and 10,000× were recorded. For SEM analysis of EPS was made conductive by gold-coating, using quorum gold sputtering unit. The elemental mapping of the EPS was performed by energy-dispersive X-ray spectroscopy for analysis of carbon/oxygen/nitrogen/phosphorus and sulphur composition. The X-ray spectrum of the elements was obtained at an accelerating voltage of 15 KeV28.Total antioxidant capacity (TAC) of EPSThe TAC test measures how a sample removes free radicals, and thereby assess the antioxidant capacity of biological samples. TAC was prepared by dissolving 4 mM Ammonium molybdate, 28 mM Sodium sulphate, and 45 mL of sulfuric acid in 250 mL of water. Different amounts of EPS ranging from (300–1200 µg) was dissolved in 1mL of the TAC mix and kept for incubation at room temperature for 1 h and then absorbance was measured at 695 nm using a spectrophotometer. Ascorbic acid was used as standard (10–40 µg)16.Reducing power of EPSThe Reducing power of the EPS was assessed using a ferric reducing antioxidant power (FRAP) assay. This assay reflects the electron-donating ability of the EPS, which is crucial for neutralizing free radicals and preventing oxidative damage in biological and food systems21,29. Different concentrations of EPS (300–500 µg) dissolved in 1 mL of distilled water were combined with 2.5 mL of 1% potassium ferricyanide. This mixture was then subjected to incubation at 50 °C for 20 min. Subsequently, 2.5 mL of 10% trichloroacetic acid was introduced and the resulting mixture was centrifuged at 3000 rpm for 10 min. To the 2.5 mL supernatant, 2.5 mL of distilled water and 0.5 mL of ferric chloride (0.1%) were added. Then, incubated for 10 min, and later the absorbance was measured at 700 nm using a spectrophotometer with ascorbic acid as standard. A higher absorbance value indicates greater reducing power29,30.Result and discussionInitial screening of fermentation media using Plackett-Burman designIn the experimental PBD, the EPS yield was ranged from 0 to 7.33 g/L, which indicates the significance of parameters selected for the optimization of production of EPS (Table 1). This widespread distinction too imitates the importance of fermentation media optimization for accomplishing higher yield. The PBD showed 6-fold increase in EPS yield when compared to unoptimized condition (Table 1; Run order 12). This result is in good agreement with the results reported elsewhere31,32,33. The first order mathematical model of PBD based on the regression analysis with coded values is depicted below:$$\begin{aligned} Y & = 2.619 + 0.017\,{\text{ X}}_{1} + 0.013\,{\text{ X}}_{2} + 0.227\,{\text{ X}}_{3} + 1.524\,{\text{ X}}_{4} + 8.757\,{\text{ X}}_{5} + 6.01\,{\text{ X}}_{6} \\ & \quad + 6.356\,{\text{ X}}_{7} {-}0.165\,{\text{X}}_{8} + 0.006\,{\text{X}}_{9} + 0.0003\,{\text{X}}_{{10}} \\ \end{aligned}$$(1)where Y represents EPS yield (g/L); X1, Glucose; X2, NH4Cl; X3, K2HPO4; X4, MgSO4·7H2O; X5, MnSO4·H2O; X6, CaCl2·2H2O; X7, FeCl3; X8, Inoculum size; X9, Agitation Speed; and X10, Fermentation time.The model (Eq. 1) exhibited good predictability with a coefficient of determination (R2) of 0.83, indicating that 83% of the variation in EPS yield could be explained by the linear combination of the variables. The minimal residuals observed between experimental and Predicted values further validated model adequacy (Fig. 2). The prediction profiler generated by JMP software screened five significant parameters such as Glucose, NH4Cl, K2HPO4, MgSO4·7H2O, and MnSO4·H2O (Fig. 3). Further, these significant parameters were optimized with central composite design to find the optimal values for an enhanced EPS yields.Fig. 2A normal plot between actual and predicted values of EPS yield of PBD.Full size imageFig. 3A prediction profiler generated by PBD for the EPS production.Full size imageCentral composite design (CCD)The significant parameters such as glucose concentration, NH4Cl, K2 HPO4, MgSO4·7H2O, and MnSO4·H2O, were further optimized for the production of EPS using CCD at 5 levels and 6 central points with 33 different experimental runs by Sporosarcina psychrophila MTCC–2908. The CCD matrix with coded and uncoded values of above-mentioned parameters with EPS yield are shown in Tables S2 and 2. These data showed that the EPS yield increased to 16.2 g/L (Run 28) to 3.9 g/L (Run 5) through submerged fermentation. The results of the experimental design matrix were fitted with a polynomial equation as a function of five parameters with coded values and are shown in Eq. 2 for EPS production.$$\begin{aligned} Y & = - 12.696 + 0.081\,{\text{ X}}_{1} + 0.543\,{\text{ X}}_{2} + 4.625\,{\text{X}}_{3} + 4.166\,{\text{X}}_{4} + 15.25\,{\text{X}}_{5} - 0.002\,\left( {{\text{X}}_{1} } \right)^{2} + 0.088\,{\text{X}}_{1} \,{\text{X}}_{2} + 2.071\,\left( {{\text{X}}_{2} } \right)^{2} + 0.042\,{\text{X}}_{1} \,{\text{X}}_{3} \\ & \quad - 1.05\,{\text{X}}_{2} \,{\text{X}}_{3} + 15.383\,\left( {{\text{X}}_{3} } \right)^{2} - 0.136\,{\text{X}}_{1} \,{\text{X}}_{4} - 4.125\,{\text{ X}}_{2} \,{\text{X}}_{4} - 5.375\,{\text{X}}_{3} \,{\text{X}}_{4} + 9.008\,\left( {{\text{X}}_{4} } \right)^{2} + 0.08\,{\text{ X}}_{1} \,{\text{X}}_{5} + 0.7\,{\text{X}}_{2} \,{\text{X}}_{5} \\ & \quad - 9.937\,{\text{X}}_{3} \,{\text{X}}_{5} - 3.5\,{\text{X}}_{4} \,{\text{X}}_{5} + 13.196\,\left( {{\text{X}}_{5} } \right)^{2} \\ \end{aligned}$$(2)where Y, EPS yield of Sporosarcina psychrophila MTCC–2908 (g/L); X1, Glucose concentration (% w/v); X2, NH4Cl (% w/v); X3, K2HPO4 (% w/v); X4, MgSO4·7H2O (% w/v); X5, MnSO4·H2O (% w/v).The results of the statistical analysis of variance (ANOVA) obtained in this study for the production of EPS are shown in Table 3. The results are in good agreement with the general facts of higher F value (F = 26.052, p