Currently, fermented non-dairy sources are preferred to derive novel EPS from LAB which improves the texture, rheology, and viscosity of functional foods and provides extraordinary health-promoting benefits such as anti-inflammatory, anticancer, antioxidant, antibacterial, and enhanced intestinal colonization[36]. Our previous communicated work reported the isolation of Ped. pentosaceus 4412 from fermented Manilkara zapota juice with excellent probiotic properties. Further, the physicochemical characterization of purified EPS 4412 revealed the presence of β-D-glucose and α-D-mannose connected by with α-(1 → 6) and with α-(1 → 3) glycosidic linkages. The prominent features of EPS produced by LAB are enhanced texture, good mouth-feel, and stability which results in the development of novel food products in recent years. Besides, EPS plays a cardinal role in elevating nutritional value, and functional and sensory properties of food products during fermentation[37, 38]. In the present work, the SEM, AFM, XRD, and TGA of EPS 4412 provide detailed information on its microstructure, physical properties, and thermal stability. To authenticate the health-promoting attributes of EPS 4412, anti-biofilm, anti-oxidant, and in vivo anti-inflammatory activity in LPS-induced C57BL/6 mice were demonstrated.
The SEM is the most widely employed technique to understand three-dimensional and physical properties that correspond to the technological and functional roles of EPS[11]. According to SEM, the smooth, fibrous, spherical, and porous irregular of EPS 4412 can be involved in food industries to enhance the texture, thickening, stabilizing, gelling, and emulsifying purposes[16]. Therefore, all this information confirms that the microstructure of EPS 4412 could play a significant role in ameliorating the physicochemical properties of food products. EPS produced by Ped. acidilactici MT41-11 has shown identical characteristics such as smooth, porous, and flaky structures[39]. Further, EPS produced by Ped. pentosaceus E8 has disclosed analogous details like smooth, spherical, and flaky appearance [11]. AFM has been extensively used to observe the supramolecular structure and microscopic surface morphology of polysaccharides. Besides, AFM is an advantageous technique to learn about the conformation of individual molecules and the molecular structure of EPS[40]. EPS 4412 was observed to have large amounts of spherical clusters with complex structures. These results are analogous to the earlier reports on EPS structure from Ped. pentosaceus E8[11]. Also, the α-glucan produced by Lact. reuteri E81 showed similar features which are comparable with the EPS 4412 [25]. The observations of SEM and AFM correspond with each other.
XRD is a most significant analytical technique and is used for qualitative and semi-quantitative evaluation of amorphous and crystalline components[40]. The partial crystalline nature of EPS obtained from B. tequilensis FR9 was lower (15.6%) than EPS 4412[14]. Further, the semi-crystalline behavior of EPS purified from Ped. pentosaceus E8 and Lact. kefiri MSR101 are closely related to EPS 4412[40]. This kind of semi-crystalline structure is responsible for viscosity, solubility, swelling, and flexibility in aqueous solutions. Overall, these EPSs could be exploited as edible films and food coatings in manufacturing food products[16]. TGA of the EPS was implemented dynamically to understand the relationship between weight loss and temperature. The thermostability and degradation behavior of EPS 4412 may be probably due to its molecular structure and monosaccharide composition which majorly relies on growth medium, environmental factors, and species- and strain-dependent factors. The degradation temperature of EPS from Ped. pentosaceus E8 was 257°C which is slightly higher than EPS 4412[11]. However, the melting point of EPS 4412 is higher than that of EPS extracted from Ped. pentosaceus M41 (158.82°C)[10] and Ped. pentosaceus DPS (232°C)[41]. Therefore, this EPS 4412 would play an inevitable role in thermally processed foods[42].
There was increasing scientific evidence that various EPS extracted from LAB could reduce or inhibit microbial biofilms and control bacterial biofilm-associated infections[43]. These EPSs hinder early adhesion and auto-aggregation of the pathogenic bacteria by governing cell surface modification or decreasing cell-to-cell surface interaction. There are a few studies on the antibiofilm activity of EPS from LAB but no reports yet on EPS from Pediococcus pentosaceus. The anti-biofilm activity of purified EPS 4412 was recorded against four enteric pathogens. The earlier findings on EPS produced by Ped. acidilactici MT41-11 exhibited good antibiofilm activity against Salm. enterica subsp. enterica (55.18%) and Staph. aureus (54.22%) at 2 mg/mL[39]. The EPS obtained from Weissella confusa MD1 showed excellent antibiofilm activity against various pathogens like Staph. aureus (74 ± 2.31%), L. monocytogenes (65.73 ± 3.30%), Salm. enterica (57.67 ± 2.70%), and Salm. typhi (56.81 ± 2.37%) at a concentration of 1.25 mg/mL[16]. EPS from Streptococcus phocae PI80 inhibited L. monocytogenes, Salm. typhi, Pseudomonas aeruginosa, B. cereus, and Staph. aureus biofilm formation at optimum EPS (1 mg/mL) in a dose-dependent manner[44]. Based on the above results, it is believed that EPS produced by Ped. pentosaceus 4412 inhibits biofilm formation in pathogenic bacteria[39]. Most of the LAB-EPS have manifested antioxidant potential to prevent oxidative stress provoked by free radicals or ROS. Several in vitro assays performed have reported that heteropolysaccharides have more radical scavenging power than homopolysaccharides[43]. Among the various antioxidant activity assays, DPPH radical scavenging and reducing power assays are considered to be highly sensitive in detecting ROS. The antioxidant activity is strongly associated with molecular mass and EPS with low molecular mass possesses high scavenging potential[39]. The principal role of antioxidants is to scavenge free radicals which is predominantly demonstrated using DPPH radical. The functional group and monosaccharides of EPS are responsible for the scavenging action due to the presence of the hydroxyl group which donates hydrogen or electrons during interaction with free radicals [8]. At 10 mg/mL concentration, EPS produced by Ped. pentosaceus E8 from cereal vinegar samples was observed to have a lower DPPH scavenging ability at 50.62 ± 0.5% than the current results[11]. On the other hand, EPS retrieved from Ped. pentosaceus M41 of marine origin was found to exert higher DPPH radical scavenging activity at 76.5%[10]. Therefore, it is confirmed that low molecular mass EPS produced by Ped. pentosaceus 4412 could exhibit good radical scavenging potential equivalent to other LAB EPSs[39].
The reducing ability of metal ions is regarded as one of the predominant features of antioxidants known as reducing agents. These reducers have the potential to reduce ferric ions (Fe3+/ferricyanide complex) into the ferrous form which enables the conversion of free radicals to stable products. In the reducing power assay, the yellow color of the test solution changes to various shades of green and blue, depending on the reducing power of each compound. The Fe2+ concentration can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Commonly, polysaccharides donate electrons owing to the existence of reducing sugars comprised of free aldehyde or ketone groups to the reactant[8]. The reducing power of EPS was higher when compared to the EPS of Lact. reuteri SHA101 and Lact. vaginalis SHA110 measured as 1.03 ± 0.04 and 0.8 ± 0.06[17]. The presence of reducing sugars like glucose, mannose, and their functional groups has exhibited reducing activity through electron donation[8]. The antioxidant activity of the EPS could be enhanced by the sulfated modifications[45].
DEX is a robust anti-inflammatory and immunosuppressive drug that participates in the inhibition of pro-inflammatory factors. Regardless of its outstanding benefits, it is accountable for side effects like cardiovascular diseases, hypertension, hormonal imbalance, diabetes, etc.[46]. EPS belonging to LAB can prevent immunodeficiency and inflammatory diseases by regulating cytokine production[47]. According to the literature, C57BL/6 mice were chosen as an inflammatory model to evaluate the anti-inflammatory potential, reduction of oxidative stress, and organ damage by histopathology. In our previous communication, sufficient explanation had been provided on the structure-anti-inflammatory activity relationship. The thymus and spleen are major immune organs and their mass indices signify the degrees of inflammation[48]. The body weight and immune organ indices (thymus and spleen) were determined by EPS 4412 and LPS. The EPS treated groups gained more weight whereas LPS treated groups were noticed to have reduced weights. Similar results were observed in EPS from Ped. pentosaceus KFT8 that increased thymus and spleen indices in cyclophosphamide-induced immunosuppressed mice[49]. The IL-6 and TNF-α production was considerably elevated in the LPS group compared to the control group which designates that LPS has activated the in vivo inflammatory response through the cytokine secretion. Here, IL-10 is a potent anti-inflammatory mediator[50]. LPS recognizes toll-like receptor 4 (TLR-4) to activate systemic inflammatory response involving nuclear factor kappa B (NF-κB) signaling pathways. Upon LPS interaction with TLR-4, IκBα attached to NF-κB gets degraded via phosphorylation which leads to the migration of NF-κB into the nucleus and eventually initiates transcription of inflammation-related factors[5, 19]. The inhibition of pro-inflammatory cytokines production by the suppression of NF-κB activation is the major mechanism of polysaccharides proven by many researchers so far[50]. The proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 play a remarkable role in the inflammatory process [5]. Specifically, the chief contributors to the interplay of the cytokine storm are TNF-α and IL-6 [3]. TNF-α is one of the pre-eminent regulators of the inflammatory response, and its measure corresponds to the degree of infection and inflammatory response in serum and tissues[51]. In addition, the treatment of inflammation by anti-inflammatory cytokine IL-10, a potent immune regulator that controls inflammatory responses by suppressing the production of pro-inflammatory cytokines transcriptionally regulated by NF-κB [5, 50]. It is indispensable to maintain the balance between pro-inflammatory and anti-inflammatory cytokines to prevent sustained inflammatory response which leads to tissue damage [52].
From the literature, it is confirmed that IL-10 cytokines are produced by M2 macrophages or T lymphocytes whereas IL-6 and TNF-α cytokines are secreted by M1 macrophages[1]. It has been reported that EPS from B. subtilis, a probiotic bacterium protects from intestinal inflammation by stimulating the development of M2 macrophages and binding peritoneal macrophages exclusively[53]. These findings implied that the EPS can suppress inflammatory response by the induction of macrophage proliferation followed by the production of cytokines. Likewise, EPS fractions from Ped. pentosaceus KFT18 were reported to possess an immunostimulatory effect with the enhancement of nitric oxide (NO), and cytokine production via NF-κB activation in IFN-γ-primed RAW 264.7 macrophages[49]. This EPS was also investigated in a dextran sulfate sodium-induced colitis animal model which reduced the expression of inflammatory responses such as iNOS, COX-2, and other pro-inflammatory cytokines (TNF-α, IL-6, and IL-1) by suppressing the STAT-1/NF-κB pathway[12].
The overproduction of proinflammatory cytokines accelerates the accumulation of reactive oxygen species (ROS) often results in the damage of DNA, lipids, and proteins, mitochondrial dysfunction, and apoptosis, which can lead to age-related diseases such as chronic inflammatory diseases and cancer[34]. This oxidative stress created by ROS could be efficiently prevented by antioxidative defense enzymes such as GSH-Px and CAT under normal physiological conditions[54]. The MDA content can reflect the degree of lipid peroxidation and cell damage by oxidative stress[48]. Most of the LAB-EPSs manifest antioxidant potential to prevent oxidative stress provoked by free radicals or ROS[43]. Based on the current findings, it is obvious that EPS 4412 has reduced the MDA contents and increased the GSH-Px and CAT activities compared with LPS treated group. The high concentration of low molecular mass EPS 4412 has played an effective role in reducing oxidative stress compared to lower concentration EPS + LPS and DEX + LPS. Consequently, it leads to the suppression of ROS-induced inflammation by eliminating free oxygen radicals and enhancing antioxidative enzymes. Correspondingly, a high dose of EPS (50 mg/kg per day) from Lact. plantarum YW11 effectively relieved the oxidative stress in aging mice, with increased serum levels of GSH-Px, SOD, CAT, and T-AOC, but decreased MDA levels [55]. Our results are in agreement with EPS from B. subtilis xztubd1 which had decreased the H2O2 and MDA contents, increased SOD and GSH-Px activities, and reduced expression of IL-4 and IL-5 preventing ROS-induced inflammation in the lungs of asthmatic mice [53].
The overexpression of the pro-inflammatory cytokines induces oxidative damage which is responsible for chronic inflammation and aggravates tissue injury[18]. The LPS-treated groups were observed to possess edema and mononuclear cell infiltration. Besides, the EPS treatment has amended the damages caused by LPS displaying mild inflammatory cell infiltration depending on their concentration. Therefore, oral administration of EPS 4412 with a high dose reduced the severity of inflammation, inflammatory cell infiltration to the colonic mucosa, and the degree and extent of epithelial damage which demonstrates satisfactory intestinal anti-inflammatory activity[32]. The earlier findings denote that the oral administration of EPS extracted from Ped. pentosaceus KFT-8 protects against DSS-induced colonic damage by restoring villus loss, disruption of crypts, and edema of the muscle layer[12]. The low molecular weight β-D-glucan from Ganoderma lucidum attenuated the secretion of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and decreased the chronic intestinal inflammation in DSS-induced colitis mice[33]. The higher production of TNF-α increased the extravasation of leucocytes from sinusoids into the liver parenchyma, which exacerbates the hepatic injury in LPS alone, and low-dose EPS + LPS when compared to other groups[31]. Moreover, the reduced tissue injury in high-dose EPS + LPS was due to the production of IL-10. EPS isolated from Pleurotus geesteranus showed potential hepatoprotective effects against alcohol-induced liver injury specifically by enhancing the antioxidative status and escalating anti-inflammatory effects[34]. When compared to the intestine and liver, the ultrastructure of the spleen has been preserved because the low-dose, high-dose EPS + LPS, and LPS + DEX groups have exhibited mild disfigurement than LPS-treated groups. The histopathological analysis indicated polysaccharides produced from Helvella leucopus had a protective function against cyclophosphamide-induced immunosuppression[6]. These results disclosed that the pretreatment of EPS 4412 has remediated the damages incorporated by the LPS administration except for control and DEX-treated groups.
Overall, the intestine, liver, and spleen tissues of the control and EPS-only group displayed normal with no destruction and inflammatory cell infiltration. The pathological impact on both high and low-dose EPS-treated groups was found to be low with mild tissue damage. However, low-dose EPS + LPS were investigated with higher tissue injury compared to high EPS-treated groups which indicates the increase in concentration has a stronger protective effect. The EPS from B. amyloliquefaciens Amy-1 had reduced the expression of proinflammatory cytokines, phagocytic activity, and oxidative stress in LPS-stimulated THP-1 cells. Further, the anti-inflammatory activity of EPSs in mice model has been confirmed by the inhibition of the NF-κB pathway, activation of the mitogen-activated protein kinase-p38, and prohibition of the extracellular signal-regulated kinase 1/2, but did not affect the c-Jun-N-terminal kinase 2[28]. Therefore, our results satisfy the fact that low molecular mass EPS 4412 can regulate innate and adaptive immunity, including effector T-cells, lymphocytes, natural killer cells, and macrophages[35]. From the histopathological investigation, it is apparent that the EPS from Ped. pentosaceus 4412 functions as a shield on the intestine, liver, and spleen tissues. The results of cytokine production, biochemical analysis, and histopathological observations were inter-related with each other.