Production and partial characterization of exopolysaccharide from a newly isolated halophilic strain Halomonas sp. DT-Z4

Production and partial characterization of exopolysaccharide from a newly isolated halophilic strain Halomonas sp. 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DT-Z4 Fengqian Yang, Fangyan Wang, Longzhan Gan, Chengyang Wang, Chunbo Dong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6584835/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Halomonas sp. DT-Z4, a moderately halophilic strain isolated from saline and alkali soil in Qinghai Province, was used to characterize the structure and function of a novel extracellular polysaccharide (EPS-Z4). By optimizing the fermentation conditions (carbon source: sucrose 9% w/v; Nitrogen source: peptone 12 g/L; pH 9.0; 25°C), EPS production reached 3.09 g/L, indicating its adaptability to extreme salinity environment. Structural characterization showed that EPS-Z4 is a fructose-dominated heteropolysaccharide (97.6%) with small amounts of glucose (1.4%), glucuronic acid (0.5%) and amino sugar (0.1%). Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used to confirm its α-(1→6) linkage and high thermal stability (Td = 275.9°C). Scanning electron microscopy (SEM) showed that it had a dense lamellar network morphology and a zeta potential of -21.4 mV, indicating that it had moderate colloidal stability. Functional analysis showed that EPS-Z4 had excellent water solubility (WSI = 85.79%) and oil retention (OHC = 321%), which were better than most traditional microbial polysaccharides. EPS-Z4 could be used as a stabilizer for high-temperature food processing and as a biological agent for saline-alkali soil remediation. Exopolysaccharides Halophilic bacterium Preliminary optimization Structural characterization Halomonas sp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Microbial Exopolysaccharides (EPS) are high molecular weight polymers secreted by microorganisms, including bacteria and fungi. Owing to their distinctive rheological properties, biocompatibility, and functional versatility, EPS has demonstrated significant potential for applications in the food industry, biomedicine, and environmental remediation [1; 2; 3; 4]. In recent years, the exploration of microbial resources from extreme environments has advanced research on EPS derived from halophilic bacteria to a new level. These microorganisms produce polysaccharides that are rich in sulfate groups and uronic acids, resulting in EPS characterized by high thermal stability, salt tolerance, and exceptional emulsification capabilities [ 5 ]. Such properties confer enhanced biological activity and functionality. For instance, their remarkable emulsifying characteristics, metal chelation ability, as well as antioxidant activities position them as ideal alternatives to traditional industrial polysaccharides such as xanthan gum. Although studies have successfully isolated EPS-producing strains, such as Kocuria rosea ZJUQH [ 6 ] and Halorubrum sp. TBZ112 [ 7 ], from extreme environments like saline lakes and saline-alkali soils, significant knowledge gaps remain in the understanding of EPS biosynthetic networks among strains derived from high-salt ecosystems. For instance, while Halomonas sulphidaeis has been demonstrated to alleviate salt stress through EPS secretion [ 8 ], the structure-activity relationship between the proportion of fructose units in its linear branching structure and the emulsifying properties of Halomonas sulphidaeis remains unclear. Furthermore, traditional lactic acid bacteria such as Leuconostoc mesenteroides BD170 can produce up to 32 g/L of EPS under low-salt conditions [ 9 ]; however, their adaptation to high-salt industrial fermentation environments is challenging due to their reliance on sucrose substrates and limited salt tolerance (1% NaCl). Therefore, developing new halophilic bacteria with high yield potential and exceptional environmental adaptability is crucial for overcoming the current technical bottleneck. In this study, we investigated the microbiome of high-saline-alkali soil in Qinghai Province and successfully isolated a halophilic strain, designated DT-Z4, which exhibited significant EPS production. This strain was identified as Halomonas sp. through 16S rDNA sequencing. Through single-factor optimization experiments, we achieved an EPS production level of 3.09 g/L under the following conditions: carbon source (sucrose at 9% w/v), nitrogen source (peptone at 12 g/L), pH 9.0, and a temperature of 25°C. The structural characterization of EPS-Z4 revealed that fructose constituted the main chain (97.6%), with trace amounts of glucose, uronic acid, and amino sugars contributing to its unique linear branching configuration. The surface morphology of EPS-Z4 was examined using scanning electron microscopy (SEM), revealing a dense layered network structure. Functional analyses demonstrated that this polysaccharide possesses excellent thermal stability (Td = 275.9°C), high water solubility index (WSI = 85.79%), and remarkable oil holding capacity (OHC = 321%). These properties suggest its potential applications in high-temperature food processing and saline-alkali soil remediation. This study represents the first comprehensive analysis of the structure-function relationship for fructosyl EPS derived from Halomonas, providing theoretical support for targeted engineering and large-scale production efforts involving EPS from this genus while expanding our understanding of microbial resources in extreme environments. Materials and Methods Isolation and screening of EPS-producing halophilic bacteria A gradient dilution coating method was employed to isolate strains from saline-alkali soil samples collected in Qinghai Province. A 1 g soil suspension was diluted in 9 mL of sterile saline containing 5% NaCl, followed by gradient dilutions ranging from 10⁻¹ to 10⁻ 8 . Subsequently, 100 µL of each diluted solution was evenly spread on solid medium composed of K 2 HPO 4 (0.5 g/L), MgSO 4 ·7H 2 O (0.15 g/L), peptone (1.5 g/L), yeast extract (2.5 g/L), sucrose (20 g/L), agar (10 g/L), NaCl (80 g/L) at a pH of 8.0 ± 0.2. The viscous and mucoid colonies, characterized by irregular edges and a wet metallic luster, were evaluated using a double-blind method. The target strains were inoculated into liquid fermentation medium and incubated at 37°C with shaking at 200 rpm for a duration of 48 hours, after which the content of EPS was quantitatively measured. The strains were preserved in a glycerol suspension at -80°C for future analysis. For EPS quantification, the absorbance value was determined at an optical density of 490 nm using the modified phenol-sulfuric acid method [ 10 ]; quantification was achieved through comparison with a glucose standard curve. Each experimental group included three biological replicates to ensure statistical validity. Morphological and molecular characteristics Samples of strain DT-Z4 in logarithmic growth phase were washed with isotonic PBS buffer (pH 7.4), fixed in 2.5% (v/v) glutaraldehyde solution at 4 ° C for 12 h, and then dehydrated in 30%, 50%, 70%, 80%, 90%, and 100% gradient ethanol (10 min per stage). After critical point drying, the cells were fixed on a copper mesh and coated with gold film. Imaging was performed using a field emission scanning electron microscope (Thermo Scientific Apreo 2C) equipped with a T2 secondary electron detector operating at 10 kV and representative images were captured at 20,000× and 100,000× magnification to resolve surface structure. The Coico staining method was employed for Gram stain identification, while the morphological characteristics of the cells were systematically characterized according to the Bergey's Manual of Systemaic Bacteriology (9th edition, 2004). The DNA template of strain DT-Z4 was prepared utilizing the Chelex-100 boiling method [ 11 ]. Universal primers 27F (5 '-AGA GTT TGA TCM TGG CTC AG-3') and 1492R (5 '-CTA CGG CTA CCT TGT TAC GA-3') [ 12 ] were used to amplify the 16S rDNA gene. The purified products were cloned into pUCm-T vector and subjected to Sanger bidirectional sequencing. The nearly complete sequences obtained were compared against multiple databases via BLAST on the EzBioCloud server ( https://www.ezbiocloud.net/ ) [ 13 ] to identify highly homologous sequences. A phylogenetic tree was constructed based on default parameters in MEGA version 7.0 software [ 14 ]; specifically, the Neighbor-Joining method was applied to calculate genetic distances, while bootstrap analysis was conducted to assess topology reliability. Optimization of the culture conditions for EPS production Based on the basic fermentation medium described above (pH 8.0, temperature 30°C, rotation speed 200 rpm, inoculum volume 5%), a single-factor optimization strategy was employed to systematically investigate the composition of the medium and fermentation parameters for EPS production by the strain. Following activation in LB medium for 24 hours, the strains were inoculated into the fermentation medium as seed solutions. The following variables were examined sequentially: first, carbon sources (sucrose, fructose, lactose, maltose, mannitol, sorbitol; at a concentration of 20 g/L) and nitrogen sources (yeast extract, peptone, tryptone, beef extract; at a concentration of 5 g/L). After identifying the optimal carbon and nitrogen sources, concentration gradients were established (carbon source: 1–10 g/L; nitrogen source: 0–20 g/L). Building upon this optimized medium composition, we evaluated the effects of various fermentation parameters on EPS synthesis including fermentation time (ranging from 24 to 120 hours with intervals of 24 hours), inoculum size (from 1–13%, with increments of 2%), agitation speed (50–250 rpm with increments of 50 rpm), pH levels (from pH 5.0 to pH 10.0 with increments of pH 1.0), and temperature settings (ranging from 20°C to 40°C with increments of 5°C). Throughout each parameter test conducted during this study,the other variables were maintained at their baseline values. Crude extraction and purification of EPS Based on Kim's methodological framework and the characteristics of halophilic bacteria, the fermentation broth of strain DT-Z4 was centrifuged at 7000 rpm for 10 minutes at 4°C to remove cellular debris. The supernatant was then mixed with absolute ethanol in a volume ratio of 1:3 (v/v) and thoroughly stirred before being allowed to stand at 4°C for 12 hours to facilitate polysaccharide precipitation [ 15 ]. The resultant precipitate was collected via centrifugation at 7000 rpm for 20 minutes and subsequently redissolved in ultrapure water to yield a crude EPS solution. Deproteinization was performed using a modified Sevage method: the crude extract was combined with chloroform-n-butanol (4:1, v/v) in a volume ratio of 2:1, vortexed for 20 minutes, and centrifuged at 7000 rpm for 15 minutes to separate denatured proteins from the aqueous phase into the organic phase. This procedure was repeated until no white flocculent material remained at the interface. The deproteinized solution was transferred into an dialysis bag with a molecular weight cut-off of 8–14 kDa and dialyzed continuously against ultra-pure water for a duration of 72 hours at 4°C [ 16 ]. Finally, the product obtained after freeze-drying under vacuum conditions for 48 hours resulted in an off-white powder designated as EPS-Z4, which is intended for further characterization. Ultraviolet–visible and fourier transform infrared spectroscopy The purity of EPS-Z4 was assessed using UV-visible spectrophotometry. Specifically, a 10 mg sample of the polysaccharide EPS-Z4 was dissolved in 10 mL of ultrapure water. The resulting aqueous solution was subjected to analysis with a UV-visible spectrophotometer (U-3900H, Hitachi, Japan). Scanning was conducted across wavelengths ranging from 200 to 700 nm at intervals of 0.5 nm. Peaks corresponding to protein and nucleic acid absorption were identified at wavelengths of 260 nm and 280 nm [ 17 ]. To evaluate the functional groups and glycosidic bonds present within EPS-Z4, the freeze-dried powder was mixed with potassium bromide and compressed into pellets using a hydraulic press. An FTIR spectrophotometer (Nicolet iS10, Thermo Scientific, USA) was utilized to record spectra over the range of 4000 to 500 cm − 1 , achieving a resolution of 4 cm − 1 [ 17 ]. Data acquisition and processing were performed using OMNIC spectral software. Monosaccharide composition analysis The monosaccharide composition of EPS-Z4 was determined by high-performance anion-exchange chromatography (HPAEC) with some modifications as outlined by Gan et al [ 18 ]. Five mg of purified EPS-Z4 was hydrolyzed with 2 mL of 3 M trifluoroacetic acid (TFA) for 2 h at 121°C in a sealed glass ampoule. The acid hydrolysate was aspirated into a tube and dried under a stream of nitrogen (N2) atmosphere, and finally the residue was dissolved into ultrapure water. The released monomers were detected using an ion chromatography system (ICS5000, Thermo Fisher). Standard monosaccharides (rhamnose, arabinose, fucose, fructose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, mannuronic acid, gururonic acid, ribose, galactosamine hydrochloride, glucosamine hydrochloride and xylose) were used as references for the identification and quantification of the corresponding peaks. Zeta potential, particle size, and scanning electron microscopy examination The EPS-Z4 aqueous solution was filtered through a 0.22 µm membrane and subsequently dissolved in ultrapure water to achieve a final concentration of 1 mg/mL. The particle size distribution and zeta potential of EPS-Z4 were assessed using a Zetasizer Nano ZSP (Malvern, UK) at a temperature of 25°C [ 19 ]. The morphology of EPS-Z4 was examined using scanning electron microscopy (SEM; JSM-7500F, JEOL, Japan). Dried samples were affixed to copper stubs and coated with a conductive gold layer approximately 10 nm thick for SEM characterization. Observations were conducted at magnifications of 1000× and 10000× under an accelerating voltage of 7 kV [ 20 ]. Determination of thermal stability To investigate the thermal properties of the obtained EPS-Z4, a 3 mg sample of EPS-Z4 was subjected to thermal stability testing in an Al 2 O 3 crucible. The experiments were conducted using a thermogravimetric analyzer (TG209F1, Netzsch, Germany) [ 21 ] and a differential scanning calorimeter (DSC214, Shanghai). The samples underwent TGA analysis with nitrogen as the carrier gas and were heated from 35°C to 800°C at a linear heating rate of 10°C/min. Concurrently, DSC measurements were performed within the temperature range of 20°C to 400°C to ensure consistency with other parameters according to DTG guidelines [ 22 ]. Water solubility index (WSI) The water solubility index (WSI) of the samples was determined using modified method of Yang. A total of 50 mg of EPS-Z4 was dissolved in 0.5 mL of distilled water. The solution was vigorously agitated at room temperature for 2 hours using a vortex mixer to achieve a uniform suspension. The precipitate was collected by centrifugation at 8000 rpm for 20 minutes. Subsequently, the supernatant was decanted, freeze-dried, and the weight of the tube was recorded [ 23 ]. WSI was calculated according to Eq. (1). Water holding capacity The 50 mg sample was dissolved in water in 1 mL ultrapure solution and shaken in a vortex mixer for 1min. The solution was allowed to rest for 30 min, shaken every 10 min, centrifuged at 8000rpm for 20 min, the upper water was poured, weighed, and the holding power (WHC) of the polysaccharide was calculated using Eq. (2) [ 24 ]. Oil holding capacity One mL of soybean oil was added to a centrifuge tube of known weight containing 50 mg of sample and dispersed by vortexing for minutes using a vortex mixer. The plates were allowed to stand for 30 min at room temperature, shaken every 10 min, and centrifuged at 8000 rpm for 20 min [ 18 ]. The supernatant was aspirated, weighed, and the oil holding power (OHC) of the polysaccharide was calculated using Eq. (3). Results and Discussion Screening and identification of EPS-producing strain Strain DT-Z4 was isolated from saline soil in Qinghai Province. The exopolysaccharide (EPS) production of DT-Z4 was measured at 1.66 ± 0.14 g/L under conditions of 48 hours fermentation, pH 8.0, and 8% NaCl, indicating that it is a typical moderate halophile. Scanning electron microscopy (SEM) observations revealed that DT-Z4 consists of Gram-negative short bacilli with a length ranging from 1.5 to 1.8 µm and a diameter between 0.4 and 0.7 µm (Fig. 1 ); the colonies appeared as thick milky white, round or oval shapes with slightly convex edges. Phylogenetic analysis based on the complete sequence of the 16S rDNA (1545 bp, GenBank accession number OR690787) demonstrated that DT-Z4 shares a remarkable sequence similarity of 99.58% with Halomonas sulfidaeris ATCC BAA-803 T (Fig. 2 ), surpassing the bacterial species threshold of 98.7% [ 25 ]. The phylogenetic tree constructed using the neighbor-joining method (MEGA version 7.0, bootstrap 1000) indicated that DT-Z4 and Halomonas sulfidaeris ATCC BAA-803 T form an independent evolutionary branch with high confidence (99%), suggesting that DT-Z4 may represent a homologous species to Halomonas sulfidaeris . Notably, Halomonas sulphoides MV-19 [ 26 ], which was isolated from mud volcanoes, has also been shown to regulate soil osmotic potential through EPS secretion to mitigate salt stress effects on crops; this suggests potential applications for DT-Z4 in ecological restoration efforts aimed at saline soils. Although strain DT-Z4 exhibited lower EPS yield compared to the reported high EPS-producing strain Leuconostoc mesenteroides BD170 [ 27 ] which produced up to 32 g/L EPS, its stable glucose production capability under extreme salinity conditions demonstrates greater environmental adaptability. Optimization analysis of fermentation conditions Through systematic optimization of the type and concentration of carbon and nitrogen sources, as well as fermentation parameters, we identified the optimal culture conditions for EPS synthesis by the strain (Fig. 3 ). The carbon source screening experiment demonstrated that EPS production reached 1.83 g/L when sucrose was utilized as a substrate, significantly surpassing that observed with other carbon sources: fructose (0.40 g/L), lactose (0.51 g/L), mannitol (1.08 g/L), and sorbitol (0.73 g/L). This finding aligns with the metabolic characteristics of halophilic bacteria, which preferentially utilize disaccharide substrates [ 28 ]. Notably, EPS production was not detected in the experimental group using maltose as a carbon source (Fig. 3 a). This may be attributed to the strain's lack of α-1,4 glycosidase activity, rendering it incapable of effectively catabolizing maltose into usable monosaccharides. In addition, the sucrose gradient experiment showed that EPS production followed a bimodal curve with increasing concentration, reaching a peak of 2.91 g/L at 9% (w/v) (Fig. 3 b), and then decreased beyond the threshold (2.82 g/L at 10%), possibly related to the inhibition of sugar transporters activity by the hypertonic environment [ 29 ]. In nitrogen source optimization, organic nitrogen sources significantly promoted EPS synthesis, which may be due to the important role of vitamins and cofactors present in organic nitrogen sources in the induction of growth and EPS production[ 30 ], with the highest yield in the peptone group (2.29 g/L), which was higher than other nitrogen sources (yeast extract 1.70 g/L, tryptone 0.74 g/L, beef extract 0.63 g/L) (Fig. 3 c). When peptone concentration was increased to 12 g/L, EPS production reached 2.45 g/L (Fig. 3 d), but further increasing concentrations (16–20 g/L) resulted in a decrease in production, possibly related to the flow of carbon metabolism to TCA cycle rather than EPS synthesis caused by C/N imbalance. The dynamic study of fermentation parameters revealed that the synthesis of EPS exhibited typical growth-coupling characteristics. EPS production reached 2.76 g/L at a stirring speed of 200 rpm (Fig. 4 a), which was significantly higher than the yield observed at lower speeds (50 rpm: 1.97 g/L). This finding indicates that moderate levels of dissolved oxygen can enhance glycosyltransferase activity. The yield peaked at 2.59 g/L after 96 hours (Fig. 4 b) but subsequently decreased to 2.53 g/L after 120 hours, likely due to substrate depletion or the accumulation of metabolic by-products, consistent with the typical fermentation kinetics associated with moderately halophilic bacteria. The highest yield recorded was 3.09 g/L achieved with a 7% inoculum; however, an excessively high inoculum level (13%) led to increased competition for nutrients and resulted in a reduced yield of only 1.13 g/L (Fig. 4 c). Furthermore, temperature experiments indicated that an optimal condition was found at 25°C, yielding approximately 1.76 g/L (Fig. 4 d), aligning with the room temperature adaptability characteristic of most moderately halophilic bacteria. Additionally, it was observed that a neutral to weakly alkaline environment (pH range: 8.0–9.0) favored EPS synthesis, achieving a maximum production rate of 1.99 g/L at pH 9.0; conversely, an acidic culture environment (pH5.0: 0.44 g/L) significantly inhibited bacterial metabolism (Fig. 4 e). UV-Vis absorption spectroscopy and FTIR spectroscopy analysis The purified EPS-Z4 was successfully obtained by ethanol gradient precipitation, Sevage deproteinization, 8–14 kDa dialysis membrane purification and freeze-drying. Uv-vis spectrum analysis (200–400 nm) showed that EPS-Z4 had no significant absorption peak at 260 nm (nucleic acid characteristic absorption) and 280 nm (protein characteristic absorption) (Fig. 5 a) [ 31 ], indicating that its nucleic acid impurity content was negligible (A260/A280 < 0.5), which met the purity standard of food-grade microbial polysaccharides. This result is consistent with the evaluation system of the purity of biological macromolecules by the Lowry method [ 32 ]. Fourier transform infrared spectroscopy (FTIR) revealed the molecular backbone characteristics of EPS-Z4 (Fig. 5 c) : The broad absorption peak at 3370.9 cm⁻¹ was attributed to the stretching vibration of O-H bond of polysaccharide [ 33 ], while the strong peak at 1645.9 cm⁻¹ might be due to the H-O-H bending vibration of bound water or the C = O stretching of amide I band [ 34 ], suggesting that EPS-Z4 may have trace amount of bound water or protein residue. The characteristic peak at 1080 cm⁻¹ corresponded to the C-O-C stretching vibration of the galactopyranose ring [ 35 ], indicating that the monosaccharide unit might exist in the form of a pyranose ring. The absorption peaks at 850 cm⁻¹ and 935.6 cm⁻¹ locations indicated the C1-H out of plane bending vibration and C-O-C asymmetric stretching vibration of α-type pyarose ring, respectively [ 18 ], suggesting that the glycosidic linkage of the main chain might be α-(1→6). The weak peak (COO⁻ symmetric stretching vibration) at 1409.2 cm⁻¹ [ 36 ] combined with the possible C = O signal at 1645.9 cm⁻¹ [ 37 ] further supported that EPS-Z4 contains uronic acid components such as glucuronic acid, which is commonly found in microbial polysaccharides with immunomodulatory functions. The C-H stretching vibration at 2928.8 cm⁻¹ and the α-terminal carbon vibration at 764 cm⁻¹ [ 38 ] indicated that the molecule might have a complex branching structure. Compared with Halomonas EPS, EPS-Z4 showed significantly lower C-O-H bending vibration peak intensity at 1035.5 cm⁻¹ compared with Halomonas elongata EPS [ 39 ], suggesting differences in the distribution or connection mode of sugar ring substituents. A similar phenomenon has also been reported in Halomonas sulfidaeris [ 8 ], which may be related to the specificity of glycosyltransferases in the biosynthetic pathway. Nuclear magnetic resonance (NMR) is needed to further analyze the monosaccharide configuration and linkage order. The monosaccharide composition of the EPS By conducting a comparative analysis of HPLC retention times (Fig. 5 b), the composition of EPS-Z4 was characterized. The primary components identified were fructose (97.60%) and glucose (1.40%). Additionally, trace amounts of glucuronic acid (0.50%), mannose (0.10%), xylose (0.10%), galactose (0.10%), arabinose (0.10%), glucosamine hydrochloride (0.06%), and galactosamine hydrochloride (0.04%) were also present in the sample. This compositional profile indicates that EPS-Z4 is classified as a heteropolysaccharide with fructose as its predominant component. The fructose-dominant composition pattern observed in EPS-Z4 markedly differs from that reported for exopolysaccharides derived from lactic acid bacteria in the existing literature; for instance, exopolysaccharides hydrolyzed by Lactobacillus casei [ 40 ] are primarily composed of glucose (89.4%) and mannose (10.6%). Furthermore, the EPS of most Streptococcus thermophilus isolates was dominated by glucose (58–75%) and galactose (25–40%) [ 41 ]. It is noteworthy that N-acetylaminocarbohydrate derivatives detected within EPS-Z4, totaling 0.1%, exhibit compositional characteristics similar to those described for exopolysaccharides produced by Streptococcus thermophilus DGCC 7785 and other strains documented in the literature [ 41 ]. The presence of such functional groups may influence both the physicochemical properties, such as charge distribution and biological activities, including immunomodulatory or antioxidant capacities of polysaccharides. Compared to homopolysaccharides (HoPS), the intricate composition of EPS-Z4 aligns more closely with the classification of heteropolysaccharides (HePS). HePS are characterized by their inclusion of both neutral sugars, such as fructose and glucose, and acidic sugars like glucuronic acid within their structure [ 42 ]. This charge heterogeneity may influence the rheological properties and functional activities of EPS-Z4. Moreover, when compared to conventional heteropolysaccharides, the high fructose content (> 97%) observed in EPS-Z4 suggests that it may possess a unique glycosidic linkage or synthetic pathway. Particle size and zeta potential examination The charge characteristics of the solution system are a critical parameter for evaluating the stability of colloids, as they govern particle aggregation behavior through the dynamic balance between electric bilayer repulsive forces and van der Waals forces [ 43 ]. Systematic characterization of the colloidal properties of an aqueous solution of EPS-Z4 revealed that polysaccharide particles were uniformly distributed (Fig. 6 a), with a hydrodynamic diameter distribution ranging from 68.1 to 342 nm and a mean Z-average particle size of 172.9 nm (Fig. 6 b). Notably, EPS-Z4 exhibited a zeta potential of − 21.4 mV (Fig. 6 b), indicating its negatively charged nature, which suggests that the surface of the polysaccharide is rich in ionizable groups (e.g., carboxylate or phosphate groups) and possesses electron-donor capacity consistent with the anionic properties typically associated with microbial exopolysaccharides [ 44 ]. From the perspective of colloid stability, it is generally accepted that systems exhibit high stability when the absolute value of zeta potential exceeds 30 mV, while moderate or partial stability is observed when zeta potential falls within ± 10–30 mV range [ 45 ]. The measured value of -21.4 mV in this study indicates that the EPS-Z4 solution system falls into the medium stability category; its stability may be influenced by steric effects arising from physicochemical parameters such as pH and ionic strength, as well as by molecular weight distribution within the polysaccharide [46; 47]. Importantly, although EPS-Z4's absolute zeta potential did not reach levels indicative of high stability, its negative charge characteristics can still effectively inhibit rapid particle aggregation through electrostatic repulsion, thereby providing essential support for subsequent functional applications. Thermal stability of EPS-Z4 The thermal stability of polysaccharides is a crucial factor influencing their practical applications. As illustrated in Fig. 6 c and 6 d, the thermal properties of EPS-Z4 were systematically evaluated using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The TGA curve indicates that EPS-Z4 underwent three distinct stages of thermal weight loss during linear heating from 35°C to 800°C. In the first stage (35 to 72.3°C), a weight loss of 6.2% was primarily attributed to the removal of adsorbed and bound water from the samples, which aligns with the dehydration behavior observed in glucan derived from pseudomesenteroides Leuconostoc YF32, as reported by Yang et al., who noted a similar weight loss of 6.4% [ 48 ]. No significant weight loss was detected in the second stage (72.3 to 210.4°C), indicating that EPS-Z4 exhibits excellent structural stability within this temperature range; thus, it is advisable to maintain application temperatures below 210.4°C to prevent molecular chain degradation [ 49 ]. In the third stage (210.4 to 461.2°C), a substantial weight loss rate of 56.3% was observed, likely resulting from depolymerization of the polysaccharide backbone and cleavage of glycosidic bonds. The subsequent weight loss recorded between 461.2°C and 800°C (11.6%) can be associated with oxidative decomposition processes affecting organic residues [ 50 ]. The DTG curve further indicated that the degradation temperature (Td) of EPS-Z4 was 275.9°C, which is significantly higher than the heat sterilization temperature commonly employed in food processing (typically ≤ 121°C). This finding suggests its potential applicability as a stabilizer or thickener in hot-processed foods [ 51 ]. In contrast, Yang et al. reported that the Td of YF32 glucan reached as high as 307.62°C [ 48 ], while Guo et al. did not provide a clear report on the Td of Edwardsiella tarda exocellular polysaccharide ETW1 [ 50 ]. However, they noted that its antioxidant activity remained well-preserved at elevated temperatures. These results indicate that although EPS-Z4 exhibits lower thermal stability compared to other similar substances documented in the literature, it still fulfills industrial requirements effectively. DSC analysis further revealed the phase transition properties of EPS-Z4 (Fig. 6 d). The Tg of EPS-Z4 was 72.2°C, indicating that EPS-Z4 had an amorphous structure in the solid state. The Tm was 213.4°C, which was consistent with the stability results of the second stage in TGA. Compared with commercial polysaccharides, the Tm of EPS-Z4 was significantly higher than that of xanthan gum (153.4°C) and locust gum (109.11°C), and slightly lower than that of guar gum (490.1°C) [ 52 ], which highlights its potential as a heat resistant food additive. Scanning electron microscopy The microscopic morphology of EPS-Z4 was characterized using scanning electron microscopy (SEM) (Fig. 7 ), which revealed a distinctive surface topology for this polysaccharide. At a magnification of 1000 ×, EPS-Z4 exhibited a complex network composed of densely arranged irregular lamellae and interwoven dendrites. This hierarchical porous structure bears resemblance to the "multi-branched flat sheet structure" observed in the polysaccharide mhEPS produced by the moderately halophilic Gracilibacillus sp. SCU50, as reported by Gan et al [ 51 ]. Upon further magnification at 10,000 ×, the surface of EPS-Z4 transitioned to a smooth and dense sheet morphology, whose specular reflection characteristics starkly contrast with the porous scintillation surface features of Leuconostoc citreum NM105 exopolysaccharide studied by Yang et al [ 48 ]. These findings suggest that polysaccharides derived from different strains may exhibit morphological differentiation due to variations in molecular chain arrangement. Partial functional properties of the freeze-dried EPS-Z4 EPS-Z4 demonstrated remarkable water solubility, achieving a WSI of 85.79 ± 0.11%. This high solubility may be attributed to the synergistic effect of its linear (1→6)-α-D-glucan structure combined with short branched chains, which facilitate water permeation by reducing intermolecular hydrogen bond density. The WHC of EPS-Z4 was only 14.06 ± 4.62%, which was significantly lower than that of Leuconostoc lactis KC117496 (117 ± 7.5%) [ 53 ]. This relatively low WHC could be associated with its lower molecular weight and dense lamellar microstructure: smaller molecular sizes limit the network formation capability of molecular chains, while smooth surfaces decrease the adsorption sites for water molecules. This observation aligns with the proposition made by Gan et al. [ 51 ], suggesting that low molecular weight and reduced porosity work synergistically to diminish WHC. Furthermore, EPS exhibiting low water holding capacity can enhance brittleness, minimize breakage, and improve the processability of extruded products. The OHC was 321.00 ± 9.90%, which was three times higher than that of Bacillus licheniformis PASS26 (101.7%) [ 54 ], but significantly lower than that of mhEPS (1023.34%) [ 18 ]. Notably, the OHC/WHC ratio of EPS-Z4 was 22.8, which was much higher than that of mhEPS (68.7). This unique property makes EPS-Z4 especially suitable for composite food systems where both water transport and oil oxidation need to be controlled, such as coating of fried food. Conclusion A novel exopolysaccharide (EPS-Z4) was isolated from Halomonas sp. DT-Z4. Through the optimization of fermentation conditions, EPS production reached 3.09 g/L at 8% NaCl, pH 9.0, and a temperature of 25°C, demonstrating its potential for large-scale production in high-salinity environments. Structural characterization revealed that EPS-Z4 is a fructose-dominated heteropolysaccharide containing trace amounts of glucose, glucuronic acid, and amino sugars; these components collectively form a unique linear backbone with short branches. The main properties of EPS-Z4 include high thermal stability (Td = 275.9°C), excellent water solubility (WSI = 85.79%), significant oil-holding capacity (OHC = 321%), and moderate colloidal stability (zeta potential = -21.4 mV). The distinctive lamellar microstructure observed via scanning electron microscopy (SEM), along with its fructose-rich composition, sets EPS-Z4 apart from conventional polysaccharides derived from lactobacillus species that are primarily composed of glucose or galactose. The thermoelasticity and solubility characteristics of EPS-Z4 render it suitable for use as a stabilizer or thickener in hot processed foods. Additionally, its salt tolerance presents promising applications in the ecological restoration of saline soils. Declarations Acknowledgements Not applicable. Author Contributions Fengqian Yang: Data curation, Writing-original draft. Fangyan Wang: Conceptualization, Visualization. Longzhan Gan: Visualization, Formal analysis, Conceptualization, Methodology, Supervision, Writing-review and editing. Chengyang Wang: Data curation, Methodology, Formal analysis. Chunbo Dong: Formal analysis, Supervision, Resources. Xiao Zou: Methodology, Visualization, Writing - review and editing. All authors contributed to the article and approved the version as submitted. Funding This work was financially supported by National Natural Science Foundation of China (No. 32460033), Guizhou Provincial Basic Research Program (Natural Science) [No. QiankeheFoundation-ZK(2024) General 089], Guizhou Provincial Department of Education Youth Science and Technology Talent Development Project [No. Qianjiaoji (2024) 35] and Special Project of Natural Science Foundation of Guizhou University [No. Gui Da Te Gang He Zi(2023)16]. Data Availability The data that support the fndings of this study are available on request from the corresponding author. Ethical Approval This study has no ethical issue. Consent to Participate Not applicable. Consent to Publish Not applicable. Competing Interests The authors declare that they have no competing interests. References Banerjee, A., Sarkar, S., Govil, T., González-Faune, P., Cabrera-Barjas, G., Bandopadhyay, R., & Sani, R. K. (2021). Extremophilic Exopolysaccharides: Biotechnologies and Wastewater Remediation. Volume 12–2021 . 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International Journal Of Biological Macromolecules , 120 (Pt B), 1441–1450. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6584835","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453774717,"identity":"5a794d68-4ecd-49d4-bc1b-cc9cbab79a79","order_by":0,"name":"Fengqian Yang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Fengqian","middleName":"","lastName":"Yang","suffix":""},{"id":453774718,"identity":"1b6c864c-410c-41d6-b783-476c77e79ca0","order_by":1,"name":"Fangyan Wang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Fangyan","middleName":"","lastName":"Wang","suffix":""},{"id":453774719,"identity":"ddae2e4e-9869-4cea-b8a8-5d9a0c2d5760","order_by":2,"name":"Longzhan Gan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACPjBZAeFIEKWFDUyeIVkLYxtJWviPX/xcOK/W3uAA88HbPAx2eUTYcqZYeua248wGB9iSrXkYkosJa2HsSZDm3XaMzeAAj5k0D8OBxAaCWph5kn/zzjnGY3CA/xuRWtjYj0nzNtRIAG1hI1ILDw+bNc+xAwaSh9mMLecYJBPWws9//PFtnpo6e77jzQ9vvKmwI6yFgYHHAEgcZmBgBnEMCKsHAvYHQKKOKKWjYBSMglEwQgEA5QcyggJHR98AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0000-3618-5682","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Longzhan","middleName":"","lastName":"Gan","suffix":""},{"id":453774720,"identity":"4ad3959d-1afa-4e2a-97b5-4916a1063ef5","order_by":3,"name":"Chengyang Wang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Chengyang","middleName":"","lastName":"Wang","suffix":""},{"id":453774721,"identity":"00325351-f08b-4d4a-89b9-255d82c1a675","order_by":4,"name":"Chunbo Dong","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Chunbo","middleName":"","lastName":"Dong","suffix":""},{"id":453774722,"identity":"0c258dd9-cda0-418f-b5c5-fd48b31c3170","order_by":5,"name":"Xiao Zou","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Zou","suffix":""}],"badges":[],"createdAt":"2025-05-03 15:15:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6584835/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6584835/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82611894,"identity":"a8e239d1-4d66-43b8-9aee-ef6591ee13ba","added_by":"auto","created_at":"2025-05-13 10:54:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":257441,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of strain DT-Z4 morphology at 20000 × (a), 100000 × (b) magnification.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/28d4e52bf2c883415e513843.png"},{"id":82611855,"identity":"dcf88620-3f03-4fdc-b590-25c15d4d70cd","added_by":"auto","created_at":"2025-05-13 10:53:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111653,"visible":true,"origin":"","legend":"\u003cp\u003eDisplays a neighbour-joining tree of 16S rDNA sequences indicating the genetic relatedness between \u003cem\u003eHalomonas\u003c/em\u003e sp. DT-Z4 and other species. GenBank accession numbers are provided in parentheses alongside strain names. Bootstrap values from 1000 replications label the branching points, with only values ≥50% shown. The bar represents 0.01 substitutions per nucleotide position.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/17c717b50632c5d6885c4375.png"},{"id":82611878,"identity":"44ae04f5-786b-4e8d-8fca-3f78abe4d9a3","added_by":"auto","created_at":"2025-05-13 10:54:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64634,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of media components on the yield of EPS from \u003cem\u003eHalomonas\u003c/em\u003e sp. DT-Z4: (a) Carbon sources, (b) Sucrose concentration, (c) Nitrogen sources, (d) Peptone concentration.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/97a2375a18f4032cea14f6dc.png"},{"id":82612145,"identity":"9c4b4a29-cda3-4d67-902a-6bf36af4794e","added_by":"auto","created_at":"2025-05-13 11:02:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68773,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of fermentation conditions on the yield of EPS from the strain DT-Z4: (a) Rotation speed, (b) Fermentation time, (c) Inoculation volume, (d) Temperature, (e) pH concentration.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/4ece502ec04407c6e6caa06c.png"},{"id":82611891,"identity":"ccbc55b1-7c8d-4d59-90ca-3f516171ec3b","added_by":"auto","created_at":"2025-05-13 10:54:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43515,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis spectrum (a), HPLC chromatogram (b), and FT-IR spectrum (c) of EPS-Z4.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/ec50058e83121fc97dae90ee.png"},{"id":82611885,"identity":"01829f55-71f3-46c1-abe4-fc40f00deaf1","added_by":"auto","created_at":"2025-05-13 10:54:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":71880,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential trend (a), Particle size distribution (b), DSC (c) and TGA-DTG (d) of EPS-Z4.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/e52dccc6f84ed14324530ed5.png"},{"id":82611897,"identity":"96f8996f-6212-4c2c-bed0-1842cc4a3821","added_by":"auto","created_at":"2025-05-13 10:54:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":250127,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of EPS-Z4 at 1000 × (a) and 10000 × (b) magnification.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/7845f5823f9a771620bb37d6.png"},{"id":84152071,"identity":"e20fd1f2-0129-4f0c-a93f-2b180c7f64cd","added_by":"auto","created_at":"2025-06-08 08:54:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1874877,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6584835/v1/0356628a-e623-423b-9910-40821549cacb.pdf"}],"financialInterests":"","formattedTitle":"Production and partial characterization of exopolysaccharide from a newly isolated halophilic strain Halomonas sp. DT-Z4","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobial Exopolysaccharides (EPS) are high molecular weight polymers secreted by microorganisms, including bacteria and fungi. Owing to their distinctive rheological properties, biocompatibility, and functional versatility, EPS has demonstrated significant potential for applications in the food industry, biomedicine, and environmental remediation [1; 2; 3; 4]. In recent years, the exploration of microbial resources from extreme environments has advanced research on EPS derived from halophilic bacteria to a new level. These microorganisms produce polysaccharides that are rich in sulfate groups and uronic acids, resulting in EPS characterized by high thermal stability, salt tolerance, and exceptional emulsification capabilities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Such properties confer enhanced biological activity and functionality. For instance, their remarkable emulsifying characteristics, metal chelation ability, as well as antioxidant activities position them as ideal alternatives to traditional industrial polysaccharides such as xanthan gum.\u003c/p\u003e \u003cp\u003eAlthough studies have successfully isolated EPS-producing strains, such as \u003cem\u003eKocuria rosea\u003c/em\u003e ZJUQH [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and \u003cem\u003eHalorubrum\u003c/em\u003e sp. TBZ112 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], from extreme environments like saline lakes and saline-alkali soils, significant knowledge gaps remain in the understanding of EPS biosynthetic networks among strains derived from high-salt ecosystems. For instance, while \u003cem\u003eHalomonas sulphidaeis\u003c/em\u003e has been demonstrated to alleviate salt stress through EPS secretion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the structure-activity relationship between the proportion of fructose units in its linear branching structure and the emulsifying properties of \u003cem\u003eHalomonas sulphidaeis\u003c/em\u003e remains unclear. Furthermore, traditional lactic acid bacteria such as \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e BD170 can produce up to 32 g/L of EPS under low-salt conditions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; however, their adaptation to high-salt industrial fermentation environments is challenging due to their reliance on sucrose substrates and limited salt tolerance (1% NaCl). Therefore, developing new halophilic bacteria with high yield potential and exceptional environmental adaptability is crucial for overcoming the current technical bottleneck.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the microbiome of high-saline-alkali soil in Qinghai Province and successfully isolated a halophilic strain, designated DT-Z4, which exhibited significant EPS production. This strain was identified as \u003cem\u003eHalomonas\u003c/em\u003e sp. through 16S rDNA sequencing. Through single-factor optimization experiments, we achieved an EPS production level of 3.09 g/L under the following conditions: carbon source (sucrose at 9% w/v), nitrogen source (peptone at 12 g/L), pH 9.0, and a temperature of 25\u0026deg;C. The structural characterization of EPS-Z4 revealed that fructose constituted the main chain (97.6%), with trace amounts of glucose, uronic acid, and amino sugars contributing to its unique linear branching configuration. The surface morphology of EPS-Z4 was examined using scanning electron microscopy (SEM), revealing a dense layered network structure. Functional analyses demonstrated that this polysaccharide possesses excellent thermal stability (Td\u0026thinsp;=\u0026thinsp;275.9\u0026deg;C), high water solubility index (WSI\u0026thinsp;=\u0026thinsp;85.79%), and remarkable oil holding capacity (OHC\u0026thinsp;=\u0026thinsp;321%). These properties suggest its potential applications in high-temperature food processing and saline-alkali soil remediation. This study represents the first comprehensive analysis of the structure-function relationship for fructosyl EPS derived from Halomonas, providing theoretical support for targeted engineering and large-scale production efforts involving EPS from this genus while expanding our understanding of microbial resources in extreme environments.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eIsolation and screening of EPS-producing halophilic bacteria\u003c/h2\u003e\n \u003cp\u003eA gradient dilution coating method was employed to isolate strains from saline-alkali soil samples collected in Qinghai Province. A 1 g soil suspension was diluted in 9 mL of sterile saline containing 5% NaCl, followed by gradient dilutions ranging from 10⁻\u0026sup1; to 10⁻\u003csup\u003e8\u003c/sup\u003e. Subsequently, 100 \u0026micro;L of each diluted solution was evenly spread on solid medium composed of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (0.5 g/L), MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (0.15 g/L), peptone (1.5 g/L), yeast extract (2.5 g/L), sucrose (20 g/L), agar (10 g/L), NaCl (80 g/L) at a pH of 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2. The viscous and mucoid colonies, characterized by irregular edges and a wet metallic luster, were evaluated using a double-blind method.\u003c/p\u003e\n \u003cp\u003eThe target strains were inoculated into liquid fermentation medium and incubated at 37\u0026deg;C with shaking at 200 rpm for a duration of 48 hours, after which the content of EPS was quantitatively measured. The strains were preserved in a glycerol suspension at -80\u0026deg;C for future analysis. For EPS quantification, the absorbance value was determined at an optical density of 490 nm using the modified phenol-sulfuric acid method [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]; quantification was achieved through comparison with a glucose standard curve. Each experimental group included three biological replicates to ensure statistical validity.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMorphological and molecular characteristics\u003c/h3\u003e\n\u003cp\u003eSamples of strain DT-Z4 in logarithmic growth phase were washed with isotonic PBS buffer (pH 7.4), fixed in 2.5% (v/v) glutaraldehyde solution at 4 \u0026deg; C for 12 h, and then dehydrated in 30%, 50%, 70%, 80%, 90%, and 100% gradient ethanol (10 min per stage). After critical point drying, the cells were fixed on a copper mesh and coated with gold film. Imaging was performed using a field emission scanning electron microscope (Thermo Scientific Apreo 2C) equipped with a T2 secondary electron detector operating at 10 kV and representative images were captured at 20,000\u0026times; and 100,000\u0026times; magnification to resolve surface structure.\u003c/p\u003e\n\u003cp\u003eThe Coico staining method was employed for Gram stain identification, while the morphological characteristics of the cells were systematically characterized according to the Bergey\u0026apos;s Manual of Systemaic Bacteriology (9th edition, 2004). The DNA template of strain DT-Z4 was prepared utilizing the Chelex-100 boiling method [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Universal primers 27F (5 \u0026apos;-AGA GTT TGA TCM TGG CTC AG-3\u0026apos;) and 1492R (5 \u0026apos;-CTA CGG CTA CCT TGT TAC GA-3\u0026apos;) [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e] were used to amplify the 16S rDNA gene. The purified products were cloned into pUCm-T vector and subjected to Sanger bidirectional sequencing. The nearly complete sequences obtained were compared against multiple databases via BLAST on the EzBioCloud server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] to identify highly homologous sequences. A phylogenetic tree was constructed based on default parameters in MEGA version 7.0 software [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]; specifically, the Neighbor-Joining method was applied to calculate genetic distances, while bootstrap analysis was conducted to assess topology reliability.\u003c/p\u003e\n\u003ch3\u003eOptimization of the culture conditions for EPS production\u003c/h3\u003e\n\u003cp\u003eBased on the basic fermentation medium described above (pH 8.0, temperature 30\u0026deg;C, rotation speed 200 rpm, inoculum volume 5%), a single-factor optimization strategy was employed to systematically investigate the composition of the medium and fermentation parameters for EPS production by the strain. Following activation in LB medium for 24 hours, the strains were inoculated into the fermentation medium as seed solutions.\u003c/p\u003e\n\u003cp\u003eThe following variables were examined sequentially: first, carbon sources (sucrose, fructose, lactose, maltose, mannitol, sorbitol; at a concentration of 20 g/L) and nitrogen sources (yeast extract, peptone, tryptone, beef extract; at a concentration of 5 g/L). After identifying the optimal carbon and nitrogen sources, concentration gradients were established (carbon source: 1\u0026ndash;10 g/L; nitrogen source: 0\u0026ndash;20 g/L).\u003c/p\u003e\n\u003cp\u003eBuilding upon this optimized medium composition, we evaluated the effects of various fermentation parameters on EPS synthesis including fermentation time (ranging from 24 to 120 hours with intervals of 24 hours), inoculum size (from 1\u0026ndash;13%, with increments of 2%), agitation speed (50\u0026ndash;250 rpm with increments of 50 rpm), pH levels (from pH 5.0 to pH 10.0 with increments of pH 1.0), and temperature settings (ranging from 20\u0026deg;C to 40\u0026deg;C with increments of 5\u0026deg;C). Throughout each parameter test conducted during this study,the other variables were maintained at their baseline values.\u003c/p\u003e\n\u003ch3\u003eCrude extraction and purification of EPS\u003c/h3\u003e\n\u003cp\u003eBased on Kim\u0026apos;s methodological framework and the characteristics of halophilic bacteria, the fermentation broth of strain DT-Z4 was centrifuged at 7000 rpm for 10 minutes at 4\u0026deg;C to remove cellular debris. The supernatant was then mixed with absolute ethanol in a volume ratio of 1:3 (v/v) and thoroughly stirred before being allowed to stand at 4\u0026deg;C for 12 hours to facilitate polysaccharide precipitation [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. The resultant precipitate was collected via centrifugation at 7000 rpm for 20 minutes and subsequently redissolved in ultrapure water to yield a crude EPS solution.\u003c/p\u003e\n\u003cp\u003eDeproteinization was performed using a modified Sevage method: the crude extract was combined with chloroform-n-butanol (4:1, v/v) in a volume ratio of 2:1, vortexed for 20 minutes, and centrifuged at 7000 rpm for 15 minutes to separate denatured proteins from the aqueous phase into the organic phase. This procedure was repeated until no white flocculent material remained at the interface. The deproteinized solution was transferred into an dialysis bag with a molecular weight cut-off of 8\u0026ndash;14 kDa and dialyzed continuously against ultra-pure water for a duration of 72 hours at 4\u0026deg;C [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Finally, the product obtained after freeze-drying under vacuum conditions for 48 hours resulted in an off-white powder designated as EPS-Z4, which is intended for further characterization.\u003c/p\u003e\n\u003ch3\u003eUltraviolet\u0026ndash;visible and fourier transform infrared spectroscopy\u003c/h3\u003e\n\u003cp\u003eThe purity of EPS-Z4 was assessed using UV-visible spectrophotometry. Specifically, a 10 mg sample of the polysaccharide EPS-Z4 was dissolved in 10 mL of ultrapure water. The resulting aqueous solution was subjected to analysis with a UV-visible spectrophotometer (U-3900H, Hitachi, Japan). Scanning was conducted across wavelengths ranging from 200 to 700 nm at intervals of 0.5 nm. Peaks corresponding to protein and nucleic acid absorption were identified at wavelengths of 260 nm and 280 nm [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eTo evaluate the functional groups and glycosidic bonds present within EPS-Z4, the freeze-dried powder was mixed with potassium bromide and compressed into pellets using a hydraulic press. An FTIR spectrophotometer (Nicolet iS10, Thermo Scientific, USA) was utilized to record spectra over the range of 4000 to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, achieving a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Data acquisition and processing were performed using OMNIC spectral software.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eMonosaccharide composition analysis\u003c/h2\u003e\n \u003cp\u003eThe monosaccharide composition of EPS-Z4 was determined by high-performance anion-exchange chromatography (HPAEC) with some modifications as outlined by Gan et al [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Five mg of purified EPS-Z4 was hydrolyzed with 2 mL of 3 M trifluoroacetic acid (TFA) for 2 h at 121\u0026deg;C in a sealed glass ampoule. The acid hydrolysate was aspirated into a tube and dried under a stream of nitrogen (N2) atmosphere, and finally the residue was dissolved into ultrapure water. The released monomers were detected using an ion chromatography system (ICS5000, Thermo Fisher). Standard monosaccharides (rhamnose, arabinose, fucose, fructose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, mannuronic acid, gururonic acid, ribose, galactosamine hydrochloride, glucosamine hydrochloride and xylose) were used as references for the identification and quantification of the corresponding peaks.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eZeta potential, particle size, and scanning electron microscopy examination\u003c/h3\u003e\n\u003cp\u003eThe EPS-Z4 aqueous solution was filtered through a 0.22 \u0026micro;m membrane and subsequently dissolved in ultrapure water to achieve a final concentration of 1 mg/mL. The particle size distribution and zeta potential of EPS-Z4 were assessed using a Zetasizer Nano ZSP (Malvern, UK) at a temperature of 25\u0026deg;C [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe morphology of EPS-Z4 was examined using scanning electron microscopy (SEM; JSM-7500F, JEOL, Japan). Dried samples were affixed to copper stubs and coated with a conductive gold layer approximately 10 nm thick for SEM characterization. Observations were conducted at magnifications of 1000\u0026times; and 10000\u0026times; under an accelerating voltage of 7 kV [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetermination of thermal stability\u003c/h3\u003e\n\u003cp\u003eTo investigate the thermal properties of the obtained EPS-Z4, a 3 mg sample of EPS-Z4 was subjected to thermal stability testing in an Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e crucible. The experiments were conducted using a thermogravimetric analyzer (TG209F1, Netzsch, Germany) [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] and a differential scanning calorimeter (DSC214, Shanghai). The samples underwent TGA analysis with nitrogen as the carrier gas and were heated from 35\u0026deg;C to 800\u0026deg;C at a linear heating rate of 10\u0026deg;C/min. Concurrently, DSC measurements were performed within the temperature range of 20\u0026deg;C to 400\u0026deg;C to ensure consistency with other parameters according to DTG guidelines [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eWater solubility index (WSI)\u003c/h2\u003e\n \u003cp\u003eThe water solubility index (WSI) of the samples was determined using modified method of Yang. A total of 50 mg of EPS-Z4 was dissolved in 0.5 mL of distilled water. The solution was vigorously agitated at room temperature for 2 hours using a vortex mixer to achieve a uniform suspension. The precipitate was collected by centrifugation at 8000 rpm for 20 minutes. Subsequently, the supernatant was decanted, freeze-dried, and the weight of the tube was recorded [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. WSI was calculated according to Eq.\u0026nbsp;(1).\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"477\" height=\"35\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eWater holding capacity\u003c/h2\u003e\n \u003cp\u003eThe 50 mg sample was dissolved in water in 1 mL ultrapure solution and shaken in a vortex mixer for 1min. The solution was allowed to rest for 30 min, shaken every 10 min, centrifuged at 8000rpm for 20 min, the upper water was poured, weighed, and the holding power (WHC) of the polysaccharide was calculated using Eq.\u0026nbsp;(2) [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"463\" height=\"37\"\u003e\u003c/h2\u003e\n \u003ch2\u003eOil holding capacity\u003c/h2\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003cp\u003eOne mL of soybean oil was added to a centrifuge tube of known weight containing 50 mg of sample and dispersed by vortexing for minutes using a vortex mixer. The plates were allowed to stand for 30 min at room temperature, shaken every 10 min, and centrifuged at 8000 rpm for 20 min [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The supernatant was aspirated, weighed, and the oil holding power (OHC) of the polysaccharide was calculated using Eq.\u0026nbsp;(3).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAeEAAAAnCAYAAAA4jPATAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAAqJSURBVHhe7Z09bhRLFIXbbxkIIYRZAwECAgJgAQRARIQEMYKEkMSIGCQiIkzAAoCAACMC1gAIIcQ23uuv3Mdc31f9M2PPjAefTyqmu/673V2n6lZVs/FvS2OMMcaYpfNP92uMMcaYJWMRNsYYY1aERdgYY4xZERZhY4wxZkVYhI0xxpgVYRE2xhhjVoRF2BhjjFkRFmFjjDFmRViE15yzZ882379/b548edJsbGyU4wjhuEXy+vXrUja/hwH53Lt3rzv7P9euXSvlzQp5fvr0qTvbD/fvsOpvjDFT8Rez/iIQ2/fv3zdnzpzpfJoiyleuXGm+fv3a+SwGBO7SpUvNjRs3Op/FggjXHl3q8ezZs+7sD1Pqh7jfvn17addgjDEeCZu/hr6RrEa/Y+L69u3b5tGjR92ZMcYsHovwmsBIjtFfNPvKbwqKi9kVGCErP5mrFYewaPKVSVsmb+UByqOG8qC+pOcYQYymcx3rXPVSGTEcR56CfBWXfG/evNk8f/58Xxx49epVc+vWre7sTzo5XT9gNegTc2OMOWwswmuARnKYX799+1bEBrGqmV1rkAZTLOkfPnxY0iK4+OOH8HBOfpubmyUNo0KBiZu4sLOz03z48KEcI3ackwfilyEP8jt37lwxh3N84sSJ5vz58yUdkBfpt7e3m6dPnxZT+tbWVgkDwlVP0sd6ffz4sYS9ePGiuXDhQsnj7t27++IA10u5gnSq99WrV8v1idOnTzc/f/7szowxZrFYhNeAz58/F3EARArh+P37dzmfAuIlUyxp4d27d3tzx4wSEaohyOPBgwfd2S7kgfgB4lfjzp07zZs3b0pHArHnmOshHddAHoxG6VhEMRTqGOQRK0zthGRId/HixZInnYM4h26MMctksghnEx4uUgtXwy4zp1w2F9JA18JzvBqkVbyYT59JMTbkMe1R5tSpU2UEKxAOid8s8PeQ+CJsMvn++vWruXz5cjkWY6IM5ME9hJqAAqNeRrOUgdhrFA2MTukUMCLF1RaP8ewoPI9wp8L1xk5LtALkMn/8+FHu91FC75budSa/N5mxcGPMwaBNkeZkLYzk80LbEI3SNpQsQ+3Odtna2ip+29vbnc8ubcP8bzsq6s7+0DZ6Jf7Ozk7nswtxsz/H+FHuENSB8oTyUXrKjFBW9qP+xD3q6D7pGkF/F34VHu+H0N8Kp+vX30PpRcxHYTqOdSBP3WfF47cG/npOyCs+M7Fu5BHz5DiWiSO+ysp1U1r8I8pHxDKVVtTu3yrRteK4jkz0172JjIUbYw4GbQbvFuS2hvctvnM1HRxVHzWCWbxAjVkMo0KxEqJWuAQw+gn8hhrEHM55rAvHsbHnOJ5H8HfjdDTRwy3y+VR4JvX378szxjlK6NnO7wn1nlV0+943Y8zs0GbE9iTrJO1J1jFpoRg0R2OSZMFNW1B13uz69evllwU188B2kLaCVdMqfswh9sF+zsePH3dn47AYp2+Livz7TNhmNfD8YYaPph09c7PCPDDPACbdnCcmc0zzy9zn3DcVQn2mgmk/TyO0olvm3GEs3BgzP7zD6GNcK5N1kkWeWaeI0wp3MWHDoAhrHk2LgjKaW+ybDxyCBrbtEQwKbd/CGy6etLHBlJBTZ82dsSoXaOzGFvHQWL18+bI7209ssGtOc6vmcOH5ajuK+1ytMzgVngGek5wnfrxIyxJgoEw6oVqjwPvAs0R9psL8fm3+mnltGAs3xswPWx8ZoPahQV2tXaHjrx0lgyLMYpp5IPMsVIh1ZJbVvRl68jk/aIf+ZdUrjmMabARSH2CIC8TywiNuCo1WjdxoZ5dXDRszBYSYDizPI51RniVjzHrAu9s3QGXgx44PrG41i5cGjQwYB0X45MmT3dFs0DvIQsXI9bCgJ5+3qwA9DpXHsYSWC5bpgDBMAX0j8CzOh4XE3+54uKmoM3qY74cxZvHwzvbtpGAnB1rDYJHBnayzGQa6gyKsDxzEbSWRKSblPtQTWJToASNfjVLj6JlRL/WeWnatkY1uijlanQO74+GmwIvJfJHS8CzNAvO7+cMibLlS73ws3BizWGrbLjODIow5l1EtSl4TLD68APfv3y+/s8KIdKiXgIjWyqURGbs40o7NA9eozTnGxrXmbI42s8J8EQIc9z7zLCHEUzuHrGPIHWQ6l1q8NhZujJkfBnVTvq5HvPjFvkixNrcv/ihtJvuWVAPLsvHLWzqIy7LtTPvyl/h5ewRbKGr54J/9hLZs9EF4ThvTUHfqGcGPMo05aujZze8ORH/eu/zujYUbY+ZjyvuErtTiRD2aJMIg0Y0ugujlcIQXqET0z2JXy7vW4EQQ0ZpIk65PTGM5qpsYEn1jVkV+d/ILrc4trvbcj4UbY+YjCqnI7yuaUyOK82QRPmpwA/Jodl4OM69FQiPaV089EH1/9FVDvRctApSRO1fiIPdn6NlYh+fGGLMYENJ52hTaIrVVg3PCRxkWdjGnNutilgxzc2xpmjKBflC0ObsP5gKHFnkNzXFzP9qHoTs7evTtwT5M+Bv27SMeuj88A33rEni+hp4Nrqu2Ut8Y8/dDm8y6i7G2XdDG06a0g4K9tmptRRjYhtT2Jqr7sKZAw0sjutsxWSxTvsalhW5mufR1EHixeFmGUGfQH2wx5njC4koWC09p4zc3N4veaHdQoYyHzUKRKRQnkyy/8iM8zqlj4ohzC5o7wHxRM3/ip3iYRnCcqwz9ymwSj4XKx5/jWGccKJ8YV2UpP+qCU/3lT366diAMp2sTKpc8VI7Sxfi6ZoWpPMFxdOSr+iotKB2O8iLEiyhdjt/3dzHGmDEswkuCBlsCwnEUJxp0kEhEYgNfa+zJU2JAmNIjTjGvGE+/EdUtlw/kRT2BuhKXunBMfI4lhvJXGRzjF0WY9MqPOhMeIa3qE49VN36Vf64bcB7Tx3SqD+G1PES8JojnMX+hso0xZhbW2hy9rvAfCehLK5glWiGq7g3FxEnYEHw6Td/I5j/Qj/BREsH/5YvZFRO84kf4zwuYq4j/b7HmL/LnPDU3Td20RzrOm+Kv76W2wlV+I+Sr/1S/Fbfmy5cvXcgupNV3VSEeA19M45Nwqlv+Dwlk6iGcePE+tELcO288BGnYd0ue1J37ZYwxB8UivAKYP9D3rEUWBs1zI1JDsChIItb3ZTOQMPHR8ZoI6ZOflMdcKPMb+p5xTUingkjm8jhvR5Mlb5wEO0KZ1IHN7BzTIZGYcv/aUexe+vyxFDoaxFH4vvmXiVDH2LGg44D4K89YZ8LGOkvGGFOlbVDMEsCEye2WSZNfznGYOgHzKOeYOhXeNu57fvFYkBY/nNK0o9k9vwh1kAk2E+tDHNUFp3IVh99YF8XjONYHp/J0LtNwjJNNwUA8ygDy0LFQ+TjCY91yHXC6/7hYZ+LqXHUV+Me6KU1MC6TTdRljzCxs8E/boJhjAKPJRX9ik1EhI+hlbPnqgxE0JneNwPP5LGB+5hXRFiaNqnXO5+hWfb3GmPXF5uhjAOKLmCwDBKkdIU7eN7cImHNvR8rlmnGczyPAgABj8mfeWfPYOLYlIcgWYGPMQfBI2BhjjFkRHgkbY4wxK8IibIwxxqyEpvkPiEltMMCxLoYAAAAASUVORK5CYII=\" width=\"481\" height=\"39\"\u003e\u003c/h2\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eScreening and identification of EPS-producing strain\u003c/h2\u003e \u003cp\u003eStrain DT-Z4 was isolated from saline soil in Qinghai Province. The exopolysaccharide (EPS) production of DT-Z4 was measured at 1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 g/L under conditions of 48 hours fermentation, pH 8.0, and 8% NaCl, indicating that it is a typical moderate halophile. Scanning electron microscopy (SEM) observations revealed that DT-Z4 consists of Gram-negative short bacilli with a length ranging from 1.5 to 1.8 \u0026micro;m and a diameter between 0.4 and 0.7 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); the colonies appeared as thick milky white, round or oval shapes with slightly convex edges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic analysis based on the complete sequence of the 16S rDNA (1545 bp, GenBank accession number OR690787) demonstrated that DT-Z4 shares a remarkable sequence similarity of 99.58% with \u003cem\u003eHalomonas sulfidaeris\u003c/em\u003e ATCC BAA-803\u003csup\u003eT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), surpassing the bacterial species threshold of 98.7% [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The phylogenetic tree constructed using the neighbor-joining method (MEGA version 7.0, bootstrap 1000) indicated that DT-Z4 and \u003cem\u003eHalomonas sulfidaeris\u003c/em\u003e ATCC BAA-803\u003csup\u003eT\u003c/sup\u003e form an independent evolutionary branch with high confidence (99%), suggesting that DT-Z4 may represent a homologous species to \u003cem\u003eHalomonas sulfidaeris\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003eHalomonas sulphoides\u003c/em\u003e MV-19 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which was isolated from mud volcanoes, has also been shown to regulate soil osmotic potential through EPS secretion to mitigate salt stress effects on crops; this suggests potential applications for DT-Z4 in ecological restoration efforts aimed at saline soils. Although strain DT-Z4 exhibited lower EPS yield compared to the reported high EPS-producing strain \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e BD170 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] which produced up to 32 g/L EPS, its stable glucose production capability under extreme salinity conditions demonstrates greater environmental adaptability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOptimization analysis of fermentation conditions\u003c/h2\u003e \u003cp\u003eThrough systematic optimization of the type and concentration of carbon and nitrogen sources, as well as fermentation parameters, we identified the optimal culture conditions for EPS synthesis by the strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The carbon source screening experiment demonstrated that EPS production reached 1.83 g/L when sucrose was utilized as a substrate, significantly surpassing that observed with other carbon sources: fructose (0.40 g/L), lactose (0.51 g/L), mannitol (1.08 g/L), and sorbitol (0.73 g/L). This finding aligns with the metabolic characteristics of halophilic bacteria, which preferentially utilize disaccharide substrates [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Notably, EPS production was not detected in the experimental group using maltose as a carbon source (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This may be attributed to the strain's lack of α-1,4 glycosidase activity, rendering it incapable of effectively catabolizing maltose into usable monosaccharides. In addition, the sucrose gradient experiment showed that EPS production followed a bimodal curve with increasing concentration, reaching a peak of 2.91 g/L at 9% (w/v) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), and then decreased beyond the threshold (2.82 g/L at 10%), possibly related to the inhibition of sugar transporters activity by the hypertonic environment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn nitrogen source optimization, organic nitrogen sources significantly promoted EPS synthesis, which may be due to the important role of vitamins and cofactors present in organic nitrogen sources in the induction of growth and EPS production[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with the highest yield in the peptone group (2.29 g/L), which was higher than other nitrogen sources (yeast extract 1.70 g/L, tryptone 0.74 g/L, beef extract 0.63 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). When peptone concentration was increased to 12 g/L, EPS production reached 2.45 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), but further increasing concentrations (16\u0026ndash;20 g/L) resulted in a decrease in production, possibly related to the flow of carbon metabolism to TCA cycle rather than EPS synthesis caused by C/N imbalance.\u003c/p\u003e \u003cp\u003eThe dynamic study of fermentation parameters revealed that the synthesis of EPS exhibited typical growth-coupling characteristics. EPS production reached 2.76 g/L at a stirring speed of 200 rpm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which was significantly higher than the yield observed at lower speeds (50 rpm: 1.97 g/L). This finding indicates that moderate levels of dissolved oxygen can enhance glycosyltransferase activity. The yield peaked at 2.59 g/L after 96 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) but subsequently decreased to 2.53 g/L after 120 hours, likely due to substrate depletion or the accumulation of metabolic by-products, consistent with the typical fermentation kinetics associated with moderately halophilic bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe highest yield recorded was 3.09 g/L achieved with a 7% inoculum; however, an excessively high inoculum level (13%) led to increased competition for nutrients and resulted in a reduced yield of only 1.13 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Furthermore, temperature experiments indicated that an optimal condition was found at 25\u0026deg;C, yielding approximately 1.76 g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), aligning with the room temperature adaptability characteristic of most moderately halophilic bacteria. Additionally, it was observed that a neutral to weakly alkaline environment (pH range: 8.0\u0026ndash;9.0) favored EPS synthesis, achieving a maximum production rate of 1.99 g/L at pH 9.0; conversely, an acidic culture environment (pH5.0: 0.44 g/L) significantly inhibited bacterial metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eUV-Vis absorption spectroscopy and FTIR spectroscopy analysis\u003c/h2\u003e \u003cp\u003eThe purified EPS-Z4 was successfully obtained by ethanol gradient precipitation, Sevage deproteinization, 8\u0026ndash;14 kDa dialysis membrane purification and freeze-drying. Uv-vis spectrum analysis (200\u0026ndash;400 nm) showed that EPS-Z4 had no significant absorption peak at 260 nm (nucleic acid characteristic absorption) and 280 nm (protein characteristic absorption) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], indicating that its nucleic acid impurity content was negligible (A260/A280\u0026thinsp;\u0026lt;\u0026thinsp;0.5), which met the purity standard of food-grade microbial polysaccharides. This result is consistent with the evaluation system of the purity of biological macromolecules by the Lowry method [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFourier transform infrared spectroscopy (FTIR) revealed the molecular backbone characteristics of EPS-Z4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) : The broad absorption peak at 3370.9 cm⁻\u0026sup1; was attributed to the stretching vibration of O-H bond of polysaccharide [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while the strong peak at 1645.9 cm⁻\u0026sup1; might be due to the H-O-H bending vibration of bound water or the C\u0026thinsp;=\u0026thinsp;O stretching of amide I band [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], suggesting that EPS-Z4 may have trace amount of bound water or protein residue. The characteristic peak at 1080 cm⁻\u0026sup1; corresponded to the C-O-C stretching vibration of the galactopyranose ring [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], indicating that the monosaccharide unit might exist in the form of a pyranose ring. The absorption peaks at 850 cm⁻\u0026sup1; and 935.6 cm⁻\u0026sup1; locations indicated the C1-H out of plane bending vibration and C-O-C asymmetric stretching vibration of α-type pyarose ring, respectively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], suggesting that the glycosidic linkage of the main chain might be α-(1\u0026rarr;6). The weak peak (COO⁻ symmetric stretching vibration) at 1409.2 cm⁻\u0026sup1; [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] combined with the possible C\u0026thinsp;=\u0026thinsp;O signal at 1645.9 cm⁻\u0026sup1; [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] further supported that EPS-Z4 contains uronic acid components such as glucuronic acid, which is commonly found in microbial polysaccharides with immunomodulatory functions. The C-H stretching vibration at 2928.8 cm⁻\u0026sup1; and the α-terminal carbon vibration at 764 cm⁻\u0026sup1; [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] indicated that the molecule might have a complex branching structure.\u003c/p\u003e \u003cp\u003eCompared with \u003cem\u003eHalomonas\u003c/em\u003e EPS, EPS-Z4 showed significantly lower C-O-H bending vibration peak intensity at 1035.5 cm⁻\u0026sup1; compared with \u003cem\u003eHalomonas elongata\u003c/em\u003e EPS [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], suggesting differences in the distribution or connection mode of sugar ring substituents. A similar phenomenon has also been reported in \u003cem\u003eHalomonas sulfidaeris\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which may be related to the specificity of glycosyltransferases in the biosynthetic pathway. Nuclear magnetic resonance (NMR) is needed to further analyze the monosaccharide configuration and linkage order.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe monosaccharide composition of the EPS\u003c/h2\u003e \u003cp\u003eBy conducting a comparative analysis of HPLC retention times (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), the composition of EPS-Z4 was characterized. The primary components identified were fructose (97.60%) and glucose (1.40%). Additionally, trace amounts of glucuronic acid (0.50%), mannose (0.10%), xylose (0.10%), galactose (0.10%), arabinose (0.10%), glucosamine hydrochloride (0.06%), and galactosamine hydrochloride (0.04%) were also present in the sample. This compositional profile indicates that EPS-Z4 is classified as a heteropolysaccharide with fructose as its predominant component.\u003c/p\u003e \u003cp\u003eThe fructose-dominant composition pattern observed in EPS-Z4 markedly differs from that reported for exopolysaccharides derived from lactic acid bacteria in the existing literature; for instance, exopolysaccharides hydrolyzed by \u003cem\u003eLactobacillus casei\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] are primarily composed of glucose (89.4%) and mannose (10.6%). Furthermore, the EPS of most \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e isolates was dominated by glucose (58\u0026ndash;75%) and galactose (25\u0026ndash;40%) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is noteworthy that N-acetylaminocarbohydrate derivatives detected within EPS-Z4, totaling 0.1%, exhibit compositional characteristics similar to those described for exopolysaccharides produced by \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e DGCC 7785 and other strains documented in the literature [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The presence of such functional groups may influence both the physicochemical properties, such as charge distribution and biological activities, including immunomodulatory or antioxidant capacities of polysaccharides.\u003c/p\u003e \u003cp\u003eCompared to homopolysaccharides (HoPS), the intricate composition of EPS-Z4 aligns more closely with the classification of heteropolysaccharides (HePS). HePS are characterized by their inclusion of both neutral sugars, such as fructose and glucose, and acidic sugars like glucuronic acid within their structure [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This charge heterogeneity may influence the rheological properties and functional activities of EPS-Z4. Moreover, when compared to conventional heteropolysaccharides, the high fructose content (\u0026gt;\u0026thinsp;97%) observed in EPS-Z4 suggests that it may possess a unique glycosidic linkage or synthetic pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eParticle size and zeta potential examination\u003c/h2\u003e \u003cp\u003eThe charge characteristics of the solution system are a critical parameter for evaluating the stability of colloids, as they govern particle aggregation behavior through the dynamic balance between electric bilayer repulsive forces and van der Waals forces [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Systematic characterization of the colloidal properties of an aqueous solution of EPS-Z4 revealed that polysaccharide particles were uniformly distributed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), with a hydrodynamic diameter distribution ranging from 68.1 to 342 nm and a mean Z-average particle size of 172.9 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Notably, EPS-Z4 exhibited a zeta potential of \u0026minus;\u0026thinsp;21.4 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), indicating its negatively charged nature, which suggests that the surface of the polysaccharide is rich in ionizable groups (e.g., carboxylate or phosphate groups) and possesses electron-donor capacity consistent with the anionic properties typically associated with microbial exopolysaccharides [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the perspective of colloid stability, it is generally accepted that systems exhibit high stability when the absolute value of zeta potential exceeds 30 mV, while moderate or partial stability is observed when zeta potential falls within \u0026plusmn;\u0026thinsp;10\u0026ndash;30 mV range [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The measured value of -21.4 mV in this study indicates that the EPS-Z4 solution system falls into the medium stability category; its stability may be influenced by steric effects arising from physicochemical parameters such as pH and ionic strength, as well as by molecular weight distribution within the polysaccharide [46; 47]. Importantly, although EPS-Z4's absolute zeta potential did not reach levels indicative of high stability, its negative charge characteristics can still effectively inhibit rapid particle aggregation through electrostatic repulsion, thereby providing essential support for subsequent functional applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eThermal stability of EPS-Z4\u003c/h2\u003e \u003cp\u003eThe thermal stability of polysaccharides is a crucial factor influencing their practical applications. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, the thermal properties of EPS-Z4 were systematically evaluated using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The TGA curve indicates that EPS-Z4 underwent three distinct stages of thermal weight loss during linear heating from 35\u0026deg;C to 800\u0026deg;C. In the first stage (35 to 72.3\u0026deg;C), a weight loss of 6.2% was primarily attributed to the removal of adsorbed and bound water from the samples, which aligns with the dehydration behavior observed in glucan derived from \u003cem\u003epseudomesenteroides Leuconostoc\u003c/em\u003e YF32, as reported by Yang et al., who noted a similar weight loss of 6.4% [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. No significant weight loss was detected in the second stage (72.3 to 210.4\u0026deg;C), indicating that EPS-Z4 exhibits excellent structural stability within this temperature range; thus, it is advisable to maintain application temperatures below 210.4\u0026deg;C to prevent molecular chain degradation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In the third stage (210.4 to 461.2\u0026deg;C), a substantial weight loss rate of 56.3% was observed, likely resulting from depolymerization of the polysaccharide backbone and cleavage of glycosidic bonds. The subsequent weight loss recorded between 461.2\u0026deg;C and 800\u0026deg;C (11.6%) can be associated with oxidative decomposition processes affecting organic residues [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe DTG curve further indicated that the degradation temperature (Td) of EPS-Z4 was 275.9\u0026deg;C, which is significantly higher than the heat sterilization temperature commonly employed in food processing (typically\u0026thinsp;\u0026le;\u0026thinsp;121\u0026deg;C). This finding suggests its potential applicability as a stabilizer or thickener in hot-processed foods [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In contrast, Yang et al. reported that the Td of YF32 glucan reached as high as 307.62\u0026deg;C [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], while Guo et al. did not provide a clear report on the Td of Edwardsiella tarda exocellular polysaccharide ETW1 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, they noted that its antioxidant activity remained well-preserved at elevated temperatures. These results indicate that although EPS-Z4 exhibits lower thermal stability compared to other similar substances documented in the literature, it still fulfills industrial requirements effectively.\u003c/p\u003e \u003cp\u003eDSC analysis further revealed the phase transition properties of EPS-Z4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The Tg of EPS-Z4 was 72.2\u0026deg;C, indicating that EPS-Z4 had an amorphous structure in the solid state. The Tm was 213.4\u0026deg;C, which was consistent with the stability results of the second stage in TGA. Compared with commercial polysaccharides, the Tm of EPS-Z4 was significantly higher than that of xanthan gum (153.4\u0026deg;C) and locust gum (109.11\u0026deg;C), and slightly lower than that of guar gum (490.1\u0026deg;C) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], which highlights its potential as a heat resistant food additive.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eScanning electron microscopy\u003c/h2\u003e \u003cp\u003eThe microscopic morphology of EPS-Z4 was characterized using scanning electron microscopy (SEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which revealed a distinctive surface topology for this polysaccharide. At a magnification of 1000 \u0026times;, EPS-Z4 exhibited a complex network composed of densely arranged irregular lamellae and interwoven dendrites. This hierarchical porous structure bears resemblance to the \"multi-branched flat sheet structure\" observed in the polysaccharide mhEPS produced by the moderately halophilic \u003cem\u003eGracilibacillus\u003c/em\u003e sp. SCU50, as reported by Gan et al [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Upon further magnification at 10,000 \u0026times;, the surface of EPS-Z4 transitioned to a smooth and dense sheet morphology, whose specular reflection characteristics starkly contrast with the porous scintillation surface features of \u003cem\u003eLeuconostoc citreum\u003c/em\u003e NM105 exopolysaccharide studied by Yang et al [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These findings suggest that polysaccharides derived from different strains may exhibit morphological differentiation due to variations in molecular chain arrangement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ePartial functional properties of the freeze-dried EPS-Z4\u003c/h2\u003e \u003cp\u003eEPS-Z4 demonstrated remarkable water solubility, achieving a WSI of 85.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%. This high solubility may be attributed to the synergistic effect of its linear (1\u0026rarr;6)-α-D-glucan structure combined with short branched chains, which facilitate water permeation by reducing intermolecular hydrogen bond density. The WHC of EPS-Z4 was only 14.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.62%, which was significantly lower than that of Leuconostoc lactis KC117496 (117\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5%) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This relatively low WHC could be associated with its lower molecular weight and dense lamellar microstructure: smaller molecular sizes limit the network formation capability of molecular chains, while smooth surfaces decrease the adsorption sites for water molecules. This observation aligns with the proposition made by Gan et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], suggesting that low molecular weight and reduced porosity work synergistically to diminish WHC. Furthermore, EPS exhibiting low water holding capacity can enhance brittleness, minimize breakage, and improve the processability of extruded products.\u003c/p\u003e \u003cp\u003eThe OHC was 321.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.90%, which was three times higher than that of \u003cem\u003eBacillus licheniformis\u003c/em\u003e PASS26 (101.7%) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], but significantly lower than that of mhEPS (1023.34%) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, the OHC/WHC ratio of EPS-Z4 was 22.8, which was much higher than that of mhEPS (68.7). This unique property makes EPS-Z4 especially suitable for composite food systems where both water transport and oil oxidation need to be controlled, such as coating of fried food.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA novel exopolysaccharide (EPS-Z4) was isolated from \u003cem\u003eHalomonas\u003c/em\u003e sp. DT-Z4. Through the optimization of fermentation conditions, EPS production reached 3.09 g/L at 8% NaCl, pH 9.0, and a temperature of 25\u0026deg;C, demonstrating its potential for large-scale production in high-salinity environments. Structural characterization revealed that EPS-Z4 is a fructose-dominated heteropolysaccharide containing trace amounts of glucose, glucuronic acid, and amino sugars; these components collectively form a unique linear backbone with short branches. The main properties of EPS-Z4 include high thermal stability (Td\u0026thinsp;=\u0026thinsp;275.9\u0026deg;C), excellent water solubility (WSI\u0026thinsp;=\u0026thinsp;85.79%), significant oil-holding capacity (OHC\u0026thinsp;=\u0026thinsp;321%), and moderate colloidal stability (zeta potential = -21.4 mV). The distinctive lamellar microstructure observed via scanning electron microscopy (SEM), along with its fructose-rich composition, sets EPS-Z4 apart from conventional polysaccharides derived from lactobacillus species that are primarily composed of glucose or galactose. The thermoelasticity and solubility characteristics of EPS-Z4 render it suitable for use as a stabilizer or thickener in hot processed foods. Additionally, its salt tolerance presents promising applications in the ecological restoration of saline soils.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFengqian Yang:\u0026nbsp;\u003c/strong\u003eData curation, Writing-original draft. \u003cstrong\u003eFangyan Wang:\u0026nbsp;\u003c/strong\u003eConceptualization, Visualization. \u003cstrong\u003eLongzhan Gan:\u0026nbsp;\u003c/strong\u003eVisualization, Formal analysis, Conceptualization, Methodology, Supervision, Writing-review and editing. \u003cstrong\u003eChengyang Wang:\u003c/strong\u003e Data curation, Methodology, Formal analysis. \u003cstrong\u003eChunbo Dong:\u003c/strong\u003e Formal analysis, Supervision, Resources. \u003cstrong\u003eXiao Zou:\u003c/strong\u003eMethodology, Visualization, Writing - review and editing. All authors contributed to the article and approved the version as submitted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by National Natural Science Foundation of China (No. 32460033), Guizhou Provincial Basic Research Program (Natural Science) [No. QiankeheFoundation-ZK(2024) General 089], Guizhou Provincial Department of Education Youth Science and Technology Talent Development Project [No. Qianjiaoji (2024) 35] and Special Project of Natural Science Foundation of Guizhou University [No. Gui Da Te Gang He Zi(2023)16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data that support the fndings of this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has no ethical issue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBanerjee, A., Sarkar, S., Govil, T., Gonz\u0026aacute;lez-Faune, P., Cabrera-Barjas, G., Bandopadhyay, R., \u0026amp; Sani, R. 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Purification and structural-functional characterization of an exopolysaccharide from Bacillus licheniformis PASS26 with in-vitro antitumor and wound healing activities. \u003cem\u003eInternational Journal Of Biological Macromolecules\u003c/em\u003e, \u003cem\u003e120\u003c/em\u003e(Pt B), 1441\u0026ndash;1450.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Exopolysaccharides, Halophilic bacterium, Preliminary optimization, Structural characterization, Halomonas sp","lastPublishedDoi":"10.21203/rs.3.rs-6584835/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6584835/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eHalomonas\u003c/em\u003e sp. DT-Z4, a moderately halophilic strain isolated from saline and alkali soil in Qinghai Province, was used to characterize the structure and function of a novel extracellular polysaccharide (EPS-Z4). By optimizing the fermentation conditions (carbon source: sucrose 9% w/v; Nitrogen source: peptone 12 g/L; pH 9.0; 25\u0026deg;C), EPS production reached 3.09 g/L, indicating its adaptability to extreme salinity environment. Structural characterization showed that EPS-Z4 is a fructose-dominated heteropolysaccharide (97.6%) with small amounts of glucose (1.4%), glucuronic acid (0.5%) and amino sugar (0.1%). Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used to confirm its α-(1\u0026rarr;6) linkage and high thermal stability (Td\u0026thinsp;=\u0026thinsp;275.9\u0026deg;C). Scanning electron microscopy (SEM) showed that it had a dense lamellar network morphology and a zeta potential of -21.4 mV, indicating that it had moderate colloidal stability. Functional analysis showed that EPS-Z4 had excellent water solubility (WSI\u0026thinsp;=\u0026thinsp;85.79%) and oil retention (OHC\u0026thinsp;=\u0026thinsp;321%), which were better than most traditional microbial polysaccharides. EPS-Z4 could be used as a stabilizer for high-temperature food processing and as a biological agent for saline-alkali soil remediation.\u003c/p\u003e","manuscriptTitle":"Production and partial characterization of exopolysaccharide from a newly isolated halophilic strain Halomonas sp. 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