Functional and genomic evaluation of novel exopolysaccharide produced by marine Pediococcus pentosaceus E3 with antidiabetic, anticancer, and anti-inflammatory potentials.

Introduction
Exopolysaccharides (EPS) are high molecular-weight extracellular carbohydrate biopolymers produced by microorganisms, including lactic acid bacteria (LAB) [28]. EPS is produced as a slimy layer that can be discharged into the surrounding environment or adhered to the cell's surface to form a capsule [48]. The production of EPS significantly influences the physicochemical properties of the cell surface, thereby conferring enhanced resilience against dehydration, detrimental environmental factors, phagocytic engulfment, and phage infection [28, 32]. In addition, EPS polymers play a significant role in biofilm formation, facilitating cell adhesion and influencing strain-specific host interactions [7, 24]. The diversity of the EPS biopolymer is due to the variations in the sugar building blocks, anomeric configuration, glycosidic linkage, and molecular weight [50]. Otherwise, the unique physicochemical properties of the EPS contribute to a spectrum of potential applications [23]. For instance, EPS produced by LAB is associated with numerous functional foods, therapeutic agents, and health benefits [23]. Thus, the potential EPS production by a safe probiotic strain is necessary to avoid the pathogenicity of the producing strains during large-scale processes [51]. Moreover, the underexplored marine environment provides a viable setting for novel LAB strains that can produce novel EPS with distinctive structures and varied biological activities, as they thrive in a harsh and challenging habitat subjected to extreme conditions [49].
The diversity of glycosyltransferases found in the gene clusters responsible for exopolysaccharide (EPS) biosynthesis is indicative of the various structures of EPS. Additionally, bacterial genomes may contain multiple gene clusters that encode for polysaccharide biosynthesis [50]. These genes play essential roles in regulation, chain-length determination, repeat-unit assembly, polymerization, and the export of EPS [3]. The primary mechanisms for bacterial polysaccharide production include Wzx/Wzy-dependent pathways, ATP-binding cassette transporter-dependent pathways, synthase-dependent pathways, and extracellular synthesis pathways [40].
Inflammation is an innate defensive reaction to illness, disorder, injury, and stress that negatively impacts the host’s immune system. Cytokine storm is a potentially fatal condition characterized by the uncontrolled and increased release of cytokines that promote inflammation, such as interleukin IL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), etc. It is associated with various diseases that might lead to death, including chronic hepatitis, rheumatoid arthritis, colon cancer, atherosclerosis, multiple sclerosis, and inflammatory bowel disease (IBD) [52]. IBD, such as ulcerative colitis or Crohn's disease, are long-lasting inflammatory disorders that affect the gastrointestinal system. IBD is believed to result from multiple factors, such as an unhealthy diet and immunological reactions. IBD is characterized by symptoms such as abdominal pain, weight loss, and bloody stool, as well as inflammatory responses in the intestinal mucosa, which involve an influx of neutrophils and macrophages that release cytokines [17, 26].
Prior studies have indicated a strong association between the development of IBD and cytokines that promote inflammation, including IL-1β, IL-6, and TNF-α. Thus, the use of antibodies that counteract pro-inflammatory cytokines has been explored as a potential treatment for IBD. Multiple studies have proposed different biological variables (such as anti-TNF-α, anti-integrins, or anti-ILs) and small molecules (such as tofacitinib, a non-selective Janus kinase (JAK) inhibitor) as potential candidates for treating IBD. Nevertheless, because of the safety concerns associated with these products, such as headaches, dizziness, dermatologic effects, and acute allergy, additional research is necessary to explore the creation of medications and nutritious functional foods for IBD [26].
Recently, the rising significance of inflammation in the pathogenesis of both type 1 and type 2 diabetes, along with related metabolic illnesses, has sparked growing interest in targeting inflammation to enhance disease prevention and management. Following a comprehensive examination of the processes influencing the metabolic patterns in Type 1 and Type 2 Diabetes, as well as the associated inflammatory pathways, it is more evident that future research should prioritize a model of concurrent suppression for several inflammatory response pathways [37, 43].
Pediococcus pentosaceus E3 is a safe, fully sequenced marine lactic acid bacterium (LAB) with promising probiotic potential. It can produce metabolites, including lactic acid, EPS, and bacteriocin, that confer utility in preserving food and pharmaceutical potentials [47]. Therefore, this study focuses on investigating the genome of the marine P. pentosaceus E3 to identify gene clusters associated with EPS biosynthesis. Additionally, the production and characterization of its novel EPS and the evaluation of the cytotoxicity, antidiabetic, anti-colon cancer, and anti-inflammatory potentials of E3-EPS. Moreover, it determines the relation between the EPS biosynthetic genotype and phenotype.

Materials and methods

Bacterial strain and culture conditions
The marine probiotic isolate identified as Pediococcus pentosaceus E3 (NCBI Accession no. JAVLVE000000000) was isolated from the gut of marine shrimp samples collected from the Mediterranean Sea. P. pentosaceus E3 whole genome sequence was analyzed [47]. It was activated at 37 °C for 24 h in De Man Rogosa and Sharpe broth medium (MRS; Merck, Germany).

E3-EPS biosynthesis gene cluster identification
The EPS biosynthesis gene cluster in the marine strain P. pentosaceus E3 was identified using the egg-NOG Mapper tool (Version 5.0) (http://eggnog-mapper.embl.de). Protein sequence homologies were analyzed using the protein Basic Local Alignment Search Tool (BLASTp). The Nucleotide sugar biosynthesis pathway was predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.kegg.jp).

Exopolysaccharide (EPS) production
For EPS production, the marine isolate P. pentosaceus E3 was cultured in 1 L MRS broth containing 1.0% sucrose for 48 h at 37 °C [12]. After incubation, the bacterial cells were separated by centrifugation at 4000 rpm for 20 min at 4z °C. The supernatant was treated with 10% (w/v) trichloroacetic acid (TCA) for 30 min for protein degradation. After that, it was centrifuged at 3500 rpm for 20 min at 4 °C to remove proteins. Three times the volume of ice-cold absolute ethanol was added to the protein-free supernatant and left overnight at 4 °C for EPS precipitation. Following centrifugation, the pellets were collected, dissolved in deionized water, and then subjected to dialysis (10–12 kDa, Sigma-Aldrich, USA) for 48 h at 4 °C, and the water was changed twice [48, 49].

Physicochemical characterization

Total carbohydrate content
The total carbohydrate content of E3-EPS was determined following dialysis using the phenol sulfuric acid assay [11]. Briefly, 0.5 mL of phenol (2.5% w/v) was added to 0.5 mL of E3-EPS (5 mg/mL), followed by the addition of 2.5 mL of concentrated sulfuric acid. The reaction mixture was incubated at room temperature for 30 min; after that, the absorbance was measured at 490 nm using a spectrophotometer (Jenway 6305, UK). A standard plot was generated using glucose concentrations (10–100 µg/mL).

Fourier Transform Infrared Spectroscopy (FTIR)
The dried E3-EPS was investigated with an FTIR spectrophotometer (Bruker Tensor 27, Germany) to identify the main functional groups. The E3-EPS powder (10 mg) was mixed with potassium bromide (KBr) to form a pellet, and then it was loaded onto the single-crystal germanium of the FTIR spectrometer. The FTIR spectra were obtained within the frequency range of 4000—400 cm−1 with a resolution of 4.0 cm−1 and 64 scans [20].

Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX)
E3-EPS surface morphology and EDX elemental analysis were examined with a scanning electron microscope spectrometer (SEM; JSM-IT 200, JEOL, Japan). Before imaging, the dried E3-EPS biopolymer (10 mg) was coated with a layer of gold (15 Å) for 2 min at an accelerating voltage of 20.0 kV using physical vapor deposition, then it was subjected to SEM visualization at 2000x, 5000x, and 10,000 × magnifications. The elemental composition analysis of E3-EPS was subsequently conducted, using a scanning electron microscope-energy dispersive X-ray (SEM–EDX) spectrometer. The emitted X-rays were used to determine the atomic composition and weight of the detected elements [46].

High-Performance Liquid Chromatography (HPLC)
The monosaccharide composition of the E3-EPS was determined after hydrolysis through HPLC analysis. A 10 mg of E3-EPS was treated with 2 mL of 2 M trifluoroacetic acid (TFA) in a sealed glass tube and heated at 100 °C for 6 h. The hydrolysate was evaporated, dissolved in deionized water, and filtered through a 0.22 μm filter. Chromatographic analysis was performed using high-performance liquid chromatography (HPLC; Shimadzu 1100, Singapore). The EPS hydrolysate was injected into a reversed-phase C18 column (50 mm × 3 mm, 2.7 μm, Agilent, Poroshell 120EC). The UV detection wavelength was 355 nm. The mobile phase, composed of solvent A, contained phosphoric acid (0.5%), 1-butylamine (0.2%), and tetrahydrofuran (1.0%) mixed in water, while solvent B contained acetonitrile (50%) and solvent A (50%) with a 1 mL/min flow rate and the temperature was maintained at 25 °C. The elution program consisted of a 5% B isocratic phase for 25 min, followed by a linear increase to 15% B at 50 min. The column was washed for 15 min with 100% B and equilibrated for 15 min at the initial conditions to guarantee reproducibility across runs [2].

Structural Studies Using Nuclear Magnetic Resonance Spectroscopy (NMR)
The E3-EPS sample (20 mg) was dissolved in 1 mL of DMSO-d₆ and analyzed using a Bruker Avance III 400 MHz NMR spectrometer (Germany) at 25 °C. 1D 1H and 13C NMR spectra were recorded to identify the proton and carbon environments. All spectra were processed using TopSpin (Version 3.6), and peak assignments were made based on chemical shift data and literature comparison for common saccharides.

Thermogravimetric (TGA) and Differential Scanning Calorimetric (DSC) analyses
Thermogravimetric and differential colorimetric analyses were performed using the TGA and DSC (V20.9; SDT Q600, USA). To determine weight loss, 14 mg of dried E3-EPS was subjected to a platinum crucible at temperatures ranging from 50 to 500 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. The relation between temperature and weight loss was recorded to investigate the response of the E3-EPS biopolymer to heating.

Biological activities of E3-EPS

Cytotoxic activity
The cytotoxic impact of E3-EPS was evaluated using a 3-(4,5-dimethylthiazole-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay against the Caco-2 colon cancer cell line (ATCC HTB-37) responsible for colorectal adenocarcinoma. A 96-well tissue culture plate was inoculated with 1 X 105 cells/mL (100 μL/well) and incubated at 37 °C for 24 h. The formed cell monolayer was washed twice with the wash media, and 0.1 mL of two-fold dilutions of E3-EPS, prepared in RPMI medium with 2.0% serum (maintenance medium), was added to each well and then incubated at 37 °C. A 20 μL of MTT solution (Bio Basic, Canada) was added to each well, shaken at 150 rpm for 5 min, and incubated at 37 °C for 4 h in the presence of 5% CO2. The plate was dried to remove media, and the MTT metabolic product was resuspended in 200 μL DMSO and shaken at 150 rpm for 5 min. Finally, the optical density was recorded at a wavelength of 560 nm.

Antidiabetic activity
The α-amylase inhibitory activity of different concentrations of the E3-EPS was performed using the α-amylase Inhibitor Screening Kit (BioVision, USA) according to the manufacturer’s protocol, and acarbose was used as a reference. The absorbance was measured at 405 nm, and the inhibition percentage was calculated according to the following equation:where A is the absorbance of the enzyme control, and B is the absorbance of the sample.

Anti-inflammatory activity
The anti-inflammatory properties of E3-EPS were in vitro assessed against lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells, which stimulate the release of inflammatory cytokines, including TNF-α and IL-6. The total RNA was extracted from the cells treated with E3-EPS using an RNA extraction kit (Qiagen, USA) and reverse-transcribed to generate cDNA. The expression of cytokine genes was determined using the Script One-Step RT-PCR Kit (Bio-Rad, USA) according to the manufacturer’s protocol using a real-time PCR cycler (Rotor-Gene, India). The primer sequences used are listed in Table 1. The gene expression analysis data were quantified as fold change and compared to a calibrator (control) after normalization using the housekeeping gene, β-actin. The quantity of target genes was determined using the comparative threshold cycle (Ct) approach.

Statistical analysis
The trials were performed three times, and the results were reported as the mean ± standard deviation (SD). The data was processed utilizing Microsoft Excel 2010, and statistical significance was determined by one-way variance analysis (ANOVA). The observed differences were statistically significant at a significance level of p < 0.05.

Results and discussion

Prediction of EPS Biosynthesis-Related Genes Based on Genome Analysis
Limited research has been undertaken on the EPS of probiotic bacteria; nevertheless, new findings indicate that they are significant substances due to their health-promoting properties and economic utility across several food and pharmaceutical sectors [9]. P. pentosaceus E3 is derived from a marine habitat and has been recognized for its probiotic properties. The whole genome sequence examination of the marine-safe probiotic P. pentosaceus E3 revealed the presence of chromosomally located eps gene clusters encoding the Wzx/Wzy-dependent biosynthetic pathway. This pathway allows the production of heteropolysaccharides containing two or more different monosaccharide units [50]. Previously, a study reported the occurrence of eps gene clusters in P. pentosaceus LP28 and P. pentosaceus LL-07 [27, 44]. The eps gene clusters in P. pentosaceus E3 contain 13 genes organized into four parts, including the transcriptional regulatory region of EPS expression (epsA), the chain length determination region (epsBCD), genes encoding glycosyl transferase (GTF) that catalyze the biosynthesis of repeated units and polymerization of the EPS (epsEFGHIJK), and gene encoding export/flippase that occur within the EPS operon and is employed in the transportation of the EPS outside the cell membrane (wzx) (Table 2, Fig. 1). A previous study reported the presence of the conserved epsBCD gene cluster in P. pentosaceus SL4 [10], similar to that in Lactococcus lactis B40. Moreover, the CpsD/CapB family tyrosine-protein kinase has a significant regulatory role and acts as a domain protein involved in EPS biosynthesis [16]. Additionally, GTFs catalyze the transfer of activated monosaccharides to carbohydrates during the biosynthesis of EPS [5]. Moreover, the Wzy protein catalyzes polymerization in the periplasmic region [3, 40]. Once synthesized, the EPS is transported across the cell membrane and secreted into the extracellular environment by the action of the Wzx protein (flippase) [29].

Biosynthesis of Nucleotide Sugars of P. pentosaceus E3
In this study, a total of 16 key enzymes involved in the nucleotide sugar biosynthesis pathway were predicted according to the KEGG metabolic pathways (Table 3). LAB consumes glucose as substrate, and glucokinase (glk) catalyzes the phosphorylation of the substrate to glucose-6-phosphate, which serves as a central metabolite in the glycolysis pathway [30]. The phosphorylated sugars are converted into nucleotide sugars, which are important precursors for exopolysaccharide synthesis [54]. For instance, the phosphoglucomutase enzyme, encoded by the pgm gene, functions in converting glucose-6-phosphate into glucose-1-phosphate in the glycolysis pathway [15]. Glucose-1-phosphate uridylyltransferase (galU) catalyzes the formation of UDP-glucose (an essential precursor for EPS synthesis) from glucose-1-phosphate. Moreover, UDP-glucose 4-epimerase (galE) and galactokinase (galK) catalyze the reversible conversion between UDP-glucose and UDP-galactose [54].
Glucose-6-phosphate isomerase (pgi) catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate [22, 25]. Furthermore, UDP-N-acetylglucosamine synthesis is catalyzed by three enzymes, namely, Glutamine-fructose-6-phosphate transaminase (GlmS), Phosphoglucosamine mutase (GlmM), and Glucosamine-1-phosphate N-acetyltransferase (GlmU) [36]. Furthermore, mannose-6-phosphate isomerase is a critical enzyme that is involved in the reversible conversion of mannose-6-phosphate and fructose-6-phosphate through the production of mannose-containing polysaccharides [42]. Thus, E3-EPS is composed mainly of repeating units of glucose, galactose, and mannose. This is in accord with Ayyash et al. [4], who indicated that the EPS-M41 produced by P. pentosaceus M41 is composed mainly of glucose, galactose, mannose, and arabinose.

Characterization of E3-EPS
Unlike Lactiplantibacillus plantarum, only a few studies have been conducted on the characterization of EPS produced by P. pentosaceus. To the best of our knowledge, this is the first study to produce EPS from marine P. pentosaceus and evaluate its biological activities. P. pentosaceus E3 successfully produced 400 mg/L of EPS upon being grown for 48 h at 37 °C in MRS broth medium supplemented with 1.0% sucrose. The E3-EPS yield obtained in this study is much higher than the EPS yield obtained by the isolate P. pentosaceus SSC–12, which produced 276.6 mg/L [13]. The marine environment provides a viable setting for novel LAB strains as they thrive in a harsh and challenging habitat subjected to extreme conditions such as high salinity, fluctuating temperature, and lack of nutrients. In response, the marine LAB tend to produce structurally unique EPS adapted to function under such harsh conditions [49]. These polymers aid in cell defense, biofilm formation, and integrity preservation in challenging environments [38]. Since EPS synthesis usually increases in response to nutritional or physical stress, the marine P. pentosaceus E3 is a promising source for novel EPS with potentially improved stability and bioactivity.

Total carbohydrate contents
The phenol–sulfuric acid technique was employed to quantify the total carbohydrate content in the produced EPS. The carbohydrate percentage was determined by UV–VIS measurement, which revealed a significant carbohydrate content of approximately 93.2% using glucose as the reference standard. The high carbohydrate contents indicate the purity of E3-EPS. The carbohydrate content acquired in this investigation significantly exceeds that reported by Fan et al. [13], where the total sugar content of the isolate P. pentosaceus SSC–12 was determined as 73.6%.

FTIR spectral analysis of the E3-EPS
The specific peaks in the FTIR spectrum were used to identify the functional groups present in E3-EPS (Fig. 2). The broad peak at 3371 cm−1 corresponds to the presence of -OH stretching vibrations [21]. The presence of a peak at around 2932 cm−1 revealed the presence of C-H stretching vibrations characteristic of polysaccharides [4]. The absorption at 1686 cm−1 corresponds to the carbonyl group (C = O) stretching vibrations. In addition, the peak at 1420 cm−1 is attributed to the bending vibrations of C-H bonds. The 1233.51 cm−1 indicates that E3-EPS contains a pyranose ring [4]. Moreover, the absorption peak at 1056 cm−1 corresponds to the C–O–C stretch. Finally, the peak at 672–812 cm−1 is associated with the stretching vibrations of α- and β-glycosidic linkages between sugar moieties. The IR spectrum's signature in this study aligns with prior reports regarding exopolysaccharides [46, 48].

Morphological studies by SEM and elemental analysis using EDX spectroscopy
The E3-EPS underwent structural characterization using SEM and EDX. The SEM micrographs of the E3-EPS biopolymer produced by P. pentosaceus E3 revealed an irregular porous morphology and an amorphous texture (Fig. 3). EDX was employed to assess the elements present in terms of weight percentage. The qualitative elemental analysis by EDX revealed the predominance of oxygen and carbon with mass ratios of 55.17 ± 0.41 and 43.05 ± 0.19 (w/w%), respectively, reflecting the high carbohydrate contents of the EPS (Fig. 4). The mass ratio of phosphorus was 1.45 ± 0.05 (w/w%), while nitrogen was not determined in the sample, indicating the purity of the EPS. Moreover, the analysis demonstrated traces of other elements, including sodium, magnesium, calcium, and potassium. These elements might be involved in how monosaccharide hydroxyl and carboxyl groups interact with one another [33].

Determination of monosaccharide composition by HPLC
HPLC analysis revealed that E3-EPS was a heteropolysaccharide that contained four monosaccharides, including galactose, glucose, mannose, and fucose, in the mass percentages 27.17, 39.92, 12.10, and 9.15%, respectively. Monosaccharide peaks estimated by the HPLC method were observed in the chromatogram (Fig. 5). The results suggest that glucose and galactose were the predominant monosaccharides in the E3-EPS sample. Interestingly, fucose was detected in E3-EPS biopolymer, a rare sugar that may provide EPS with extra biological properties such as anti-inflammatory, anticancer, antioxidant, and wound healing activity [35]. Unlike the obtained results, the monosaccharide composition of P. pentosaceus M41 was determined by Ayyash et al. [4] as arabinose, mannose, glucose, and galactose [4]. A further study reported that EPS produced by P. pentosaceus LP28 acidic hydrolysate contained galactose, glucose, glucosamine, and mannose [44]. Moreover, P. pentosaceus SSC–12 EPS contained glucose (42.6%), mannose (28.9%), galactose (16.2%), arabinose (9.4%), and rhamnose (2.9%) [13]. The differences in monosaccharide composition and ratio within the same species are due to various factors, including culture medium and conditions [21]. Consequently, the E3-EPS produced by the marine P. pentosaceus E3 is a unique EPS with unique bioactivities.

1H and 13C NMR spectroscopy analyses
The structural characterization of E3-EPS using 1H NMR spectroscopy revealed signals in the range of δ 3.0–4.0 ppm, characteristic of the protons of hexose and pentose sugar units (Fig. 6a). A significant signal was observed at δ 4.860 ppm corresponding to the anomeric proton, characteristic of α-linked hexoses, and indicating the presence of glycosidic linkages of sugar units. An additional signal at δ 3.1 and δ 3.9 ppm is assigned to ring protons of hexopyranose residues. The 13C NMR spectrum (Fig. 6b) supported this with ring carbon signals in the δ 39–49 ppm range. The lack of well-resolved signals above 100 ppm could be attributed to overlapping or line-broadening effects common in polysaccharide analysis. These patterns are consistent with spectra reported for α-glucans in EPS-E8 [21] and EPS-4412 [8]. These observed chemical shift patterns are characteristic of glycosidic linkages and further confirm that the polymer is a heteropolysaccharide with an α-configuration [1].
The EPS produced by P. pentosaceus E3 is a branched heteropolysaccharide, which is mainly made up of glucose residues linked by α-(1 → 6) and side chains connected by α-(1 → 3) that contain galactose, mannose, and fucose. Integrated FTIR, HPLC, 1H and 13C NMR spectroscopy, and comparative genomic analysis strongly support the E3-EPS structure. Similar sugar profiles have been reported previously for EPS from P. pentosaceus E8 (glucose, mannose, galactose) [21] and M41 (glucose, mannose, galactose, arabinose) [4], showing a branched heteropolysaccharide composed primarily of α-(1 → 6)-linked glucose units with galactose, mannose as branching residues [4, 21].
The comprehensive analysis of the P. pentosaceus E3 genome revealed the presence of sixteen key genes associated with the nucleotide sugar biosynthesis pathway, which strongly supports the produced E3-EPS chemical characterization. The detected genes glk, pgm, and galU are responsible for converting glucose to UDP-glucose, the primary precursor for the glucan backbone formation [15, 30]. The genes galE and galK enable the reversible interconversion between UDP-glucose and UDP-galactose, which permits galactose to be incorporated into the EPS side chains [54]. Likewise, manA facilitates mannose-derived branching by allowing the routing of fructose-6-phosphate to mannose-6-phosphate [42]. The EPS chemical structure revealed from NMR and HPLC confirmed the dominant presence of glucose, along with galactose, mannose, and trace fucose. This composition is entirely in line with the anticipated enzymatic capabilities. Thus, the combination of genomic and chemical characterization data provides a consistent and coherent model for a branched heteropolysaccharide structure, confirming that P. pentosaceus E3 has functional EPS production machinery.

Thermostability of E3-EPS
The evaluation of thermal properties plays a significant role in understanding the physicochemical characteristics of EPS. As illustrated in Fig. 7, the E3-EPS biopolymer degrades in a stepwise manner. The initial step revealed that the weight loss at temperatures between 75.37 and 267.26 °C was about 9.15% and 29.62%, respectively. The initial loss is owing to the loss of surface-bond water molecules and the fast decomposition of the EPS [21, 45]. In the second step, weight loss was about 21.57% at 485.15 °C. This was caused by high temperature, which led to the breakage of carbon chains in the ring unit and hydrogen bonds in the EPS [21]. The remaining weight of the E3-EPS at 500 °C was about 38.65% (5.45 mg) of the original weight. The complexity of the E3-EPS molecular structure and its capacity to withstand higher degradation temperatures indicated the thermostability of the EPS produced by P. pentosaceus E3 and supported its use in pharmaceutical industries.

Biological activities
The functions of the EPS are significantly influenced by its molecular weight (Mw). High Mw, for example, increases viscosity, while low Mw is useful as a bioactive molecule. P. pentosaceus EPS is characterized by moderate Mw, making it suitable for diverse pharmaceutical and industrial applications [4]. Therefore, the current study investigated different biological activities of E3-EPS.

Assessment of anticancer activity of E3-EPS
The MTT assay was used to assess the anticancer efficacy of E3-EPS against colon cancer cell lines in vitro. The E3-EPS revealed a cytotoxic effect on the investigated cell lines, with the value of IC50 77.05 ± 0.24 µg/mL (Figs. 8 and 9). The toxicity was approximately evaluated as 87.9% at a 125 µg/mL concentration of E3-EPS. This is in accordance with the results obtained by Ayyash et al. [4], who reported inhibition percentages of 77.89 ± 1.66 and 87.37 ± 2.44 against colon cancer cell lines at concentrations of 5 and 10 mg/mL of P. pentosaceus EPS-M41, respectively. Moreover, the anticancer efficacy of dextran derived from P. pentosaceus CRAG3 on colon cancer (HT29) cells has been shown. EPS (500 μg/mL) inhibited 65.76% of HT29 cells [41]. The precise anticancer mechanism(s) of EPS remain unclear. Multiple factors may account for E3-EPS anticancer effects, including the induction of apoptotic processes in cancer cells and the competition with growth promoters (e.g., tumor necrosis factor) for cellular receptors [4]. L-fucose is unusual and has been shown to possess anticancer, antioxidant, and immunomodulatory properties [6]. The prohibitive expense of chemically synthesizing fucose-containing polysaccharides, which hampers large-scale commercial manufacturing, has led to a growing interest in naturally occurring fucose-rich EPS. An elevation in fucose content in EPS corresponded with an enhancement in the antioxidant activity of EPS. The anticancer mechanism of fucose-rich LAB-EPS resembles that of other LAB-EPS, namely, facilitating tumour cell apoptosis and cell cycle arrest. Research indicated that fucose-enriched EPS exhibited anti-tumor bioactivity in human leukemia K562 cells via influencing mitochondria-mediated pathways. Fucose-rich EPS may decrease cytokine release by activating TLR4 and Dectin-1 receptors and create morphological alterations in dendritic cells while enhancing the expression of surface characteristic markers in murine dendritic cells [53].

Influence of E3-EPS on α-amylase inhibitory activity
Inhibition of α-amylase activity is related to reducing hyperglycemia in diabetic patients. The IC50 of E3-EPS was evaluated as 3.85 ± 0.14 µg/mL, and the inhibition rate was 58.3% at a concentration of 10 µg/mL. The inhibitory effect was increased to 82.8% by raising the concentration of E3-EPS to 100 µg/mL. Similar results were obtained by Ayyash et al. [4], who reported that the EPS produced by P. pentosaceus M41 at a 100 μg/mL concentration showed 86.8% and 90.8% inhibition of α-amylase and α-glucosidase activities, respectively. Moreover, Sasikumar et al. [39] showed 10% and 67% inhibition of α-amylase and α-glucosidase activities at concentrations of 100 μg/mL and 300 μg/mL of EPS generated by L. plantarum BR2, respectively. The inhibition of α-amylase and α-glucosidase activity is a significant indirect strategy for diabetes management, as it diminishes sugar absorption from carbohydrate hydrolysis by these enzymes. Ayyash et al. [4] proposed that the mechanism of action of the EPS may be correlated to the EPS blocking both enzymes’ active sites. Moreover, Robyt [34] revealed that the glycosidic bonds may limit the activity of α-amylase and α-glucosidase enzymes.

Anti-inflammatory activity
Pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and anti-inflammatory cytokines (IL-4, IL-10, IL-11, and IL-13) exemplify cytokines that modulate inflammatory responses through the activation or suppression of immune cells. Pro-inflammatory cytokines are particularly prevalent in immune cells, such as macrophages, monocytes, T cells, and B cells, and are crucial in the development of inflammatory disorders. In cases of inflammation, injured tissues exhibit upregulation of pro-inflammatory proteins and cytokines, with their expression levels correlating to the extent of the injury [26]. The expression levels of TNF-α and IL-6 were determined in the LPS-induced RAW264.7 macrophage cells, which stimulate the release of inflammatory cytokines. The mRNA expression levels of cytokines TNF-α and IL-6 were decreased after treatment with E3-EPS (concentration 20 µg/mL), compared to the LPS-induced level (Fig. 10). These findings indicate that E3-EPS exerts anti-inflammatory effects by decreasing pro-inflammatory mediators.
The LAB EPSs are potent bioactive agents capable of modulating the immune system. The β-D-glucose and mannose present in the EPS exhibit a triple-helix shape, resulting in higher stiffness. This distinctive shape enables interaction with immune cell receptors innovatively, demonstrating its capacity to diminish inflammation. The anti-inflammatory efficacy is considerably affected by parameters including types of sugars, molecular weight, types of glycosidic linkages, and their positions. Nonetheless, the precise mechanism by which the EPS mediates its anti-inflammatory effects remains incompletely elucidated [18].
Similar results were obtained by Lee et al. [26], who documented the anti-inflammatory properties of an EPS fraction derived from P. pentosaceus KFT18 in mice. These EPS successfully decreased the elevated expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1) in the colonic tissue of mice with induced colitis. Moreover, Zhou et al. [55] declared the mechanism and structural function relation of the polysaccharides of Dendrobium officinale, which consist of mannose, glucose, galactose, and rhamnose, to reduce CRP production and downregulate blood levels of IL-6 and TNF-α by blocking the activation and proliferation of CD6 + T cells. Huang et al. [19] indicated that the EPS-LP2, which has a high concentration of glucose from L. plantarum DMDL 9010, suppresses the overactive immunological response via the MAPK/NF-κB pathways.

Conclusion
The marine probiotic strain P. pentosaceus E3 genome studies revealed a gene cluster responsible for EPS production of 13 genes organized into four regions, including the regulatory region of EPS expression, chain length determination, EPS repeat units’ biosynthesis, and polymerization and transportation of the EPS. A total of sixteen key enzymes implicated in the nucleotide sugar biosynthesis pathway were identified through KEGG metabolic pathway analysis. E3, cultured in MRS broth with 1.0% sucrose, yielded 400 mg/L of EPS. The purified E3-EPS was found to be a heteropolysaccharide characterized by a substantial carbohydrate content (~ 93.2%), composed of five prevalent monosaccharides: galactose, glucose, mannose, and fucose linked by α-glycosidic bonds and able to withstand high temperatures. Moreover, E3-EPS showed promising bioactivities during testing against colon cancer cell lines, with an IC50 value of 77.05 ± 0.24 µg/mL and inhibited α-amylase activity by 58.3% and 82.8% at 10 and 100 µg/mL concentrations, respectively. E3-EPS also reduces the expression of inflammatory cytokines (TNF-α and IL-6). The findings of this study indicate that P. pentosaceus E3 isolated from the underexplored marine environment is a promising, safe source of a unique bioactive EPS for pharmaceutical purposes.