Characterization and Biological Activity of a Novel Exopolysaccharide Produced By Pediococcus Pentosaceus SSC-12 From Silage


 Background: The exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) are widely used in various fields because of their safety and various biological activities. In this study, we extracted and characterized the composition as well as antioxidant and antibacterial activities of EPS from Pediococcus pentosaceus SSC-12 isolated from the silage.Results: The LAB strain SSC-12 was screened and identified as Pediococcus pentosaceus, based upon 16S rDNA gene sequencing and Neighbor Joining (NJ) phylogenetic analysis. The analysis of the EPS production kinetics results of SSC-12 showed that the EPS production reached the maximum at 20 h of culture. High-performance anion exchange chromatography (HPAEC) analysis showed that the EPS produced by SSC-12 was a heteropolysaccharide comprising glucose (42.6 %), mannose (28.9 %), galactose (16.2 %), arabinose (9.4 %) and rhamnose (2.9 %). The EPS had good antioxidant activity, especially hydroxyl radical scavenging activity. When the concentration of the EPS produced by SSC-12 (SSC-12 EPS) was 10 mg/mL, its 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability, hydroxyl radical scavenging ability, superoxide scavenging ability, and reduction ability were 77.4 %, 97.5 %, 77.5 % and 1.3, respectively. At 10 mg/mL，SSC-12 EPS completely killed Staphylococcus aureus GDMCC 1.1220 and substantially inhibited the growth of Salmonella enterica subsp. enterica GDMCC 1.345; however, it had a weak inhibitory effect on Listeria monocytogenes GDMCC 1.347. Conclusions: Due to its strong antioxidant and antibacterial properties, EPS produced by LAB strain SSC-12 have potential application as a bioactive product in the feed, food, and pharmaceutical industries.


Background
Exopolysaccharide (EPS) is a carbohydrate secreted by microorganisms during growth and metabolism.
The EPS produced by different LAB have different bioactive functions. It has been identi ed that EPS produced by LAB has immunomodulatory properties, antioxidant activity, antibacterial activity, anti-tumor, anti-cancer, hypoglycemic and other biological properties [11]. EPS produced by LAB could enhance cellular defense mechanisms and prevent diseases by reducing reactive oxygen species and free radicals [12]. Therefore, it is a safe and harmless biologically active substance that has potential antioxidant property. Moreover, EPS produced by LAB could inhibit the growth of harmful microorganisms and even kill them, thereby preventing the occurrence of microbial infections [5]. Therefore, EPS can be used as an alternative to antibiotics to reduce drug resistance. With the increasing health hazards caused by food spoilage or bacterial infections, the search for EPS with antioxidant and antibacterial activities has attracted the attention of researchers.
Currently, the sources of EPS-producing LAB are relatively limited; hence, the identi cation of new sources and the determination of their functional activities have become the focus of several studies. Silage is a kind of roughage obtained through the fermentation of LAB under anaerobic conditions to inhibit the reproduction of various miscellaneous bacteria [13]. Silage is rich in a variety of LAB, and hence, it is potentially a good source of LAB. This study primarily aimed to screen out LAB with high EPS yield from silage, analyze the relationship between LAB growth and EPS production, and determine monosaccharide composition and biological activity of EPS produced. In this study, we not only identify a new source of EPS-producing LAB, but also provide theoretical support for EPS application in the feed, food, and pharmaceutical industries.

EPS-producing LAB and EPS yield
In this experiment, 22 EPS-producing LAB strains were screened out after the observation of colony morphology, Gram stain test, and EPS yield determination. As shown in Table 1, the EPS yield of 22 strains screened from silage ranged 30.5-276.6 mg/L. Of all the strains, 14 were considered as high-yield EPS strains (EPS yield > 100 mg/L), based on Smitinont [14]. SSC-12 exhibited the highest yield of crude EPS, producing 276.6 mg/L in De Man, Rugose, and Sharpe (MRS) broth; therefore, this strain was selected for further studies. The EPS yield of SSC-12 selected in this experiment was higher than that of Pediococcus pentosaceus F3 (99.53 mg/L) [15], but was similar to that produced by P. pentosaceus NR 042058.1 (263.6 mg/ L) [16]. The 16S rDNA results indicated that SSC-12 was closely related to P. pentosaceus (> 99 % identity); it clustered apart from other species of this genus, and thus, was identi ed as P. pentosaceus (Fig. 1). Pediococcus pentosaceus is a homofermentative LAB with physical characteristics and biological functions, which can be used in the production of fermented food [17].
Previously, it has been demonstrated that the EPS produced by P. pentosaceus had good antioxidant activity and could be used as food preservative and therapeutic agent [15].

Strain Growth and EPS Production
With increase in culture time, the number and EPS production of SSC-12 gradually increased and both reached the peak at 20 h of culture (Fig. 2). However, after 20 h, SSC-12 entered a decline stage, because its life activities slowed down, leading to a gradual decrease in its count and EPS production. SSC-12 had the strongest activity in the exponential phase because EPS was the secondary metabolite produced by bacterial activities, and EPS production was the highest at this time.

Antioxidant activity of EPS
Free radicals, such as reactive oxygen species, combine with biological macromolecules in the body to cause tissue damage and induce different diseases [21]. Certain EPSs can scavenge active oxygen free radicals in vivo and reduce the incidence of diseases. EPS from L. kimchi SR8 signi cantly improved the liver index, serum superoxide dismutase activity, and the survival rate of mice [22]. Moreover, some EPSs have antioxidant properties and can slow aging and deterioration by ghting off excess free radicals in vitro; for example, EPS produced by P. pentosaceus has good antioxidant activity and could prolong the shelf life of bananas [15]. The EPS produced by LAB is a natural and safe antioxidant, which could have a good application prospect in food preservation and health product industry.
DPPH radicals can accept free electrons into stable molecules, thus attacking cells and causing lesions [23]. Hydroxyl free radical is the most active free radical, which could cause oxidative damage to neighboring biological molecules and induce diseases [24]. Superoxide free radicals cause severe tissue damage by inducing lipid peroxidation and oxidative damage [25]. Antioxidants provide electrons to scavenge free radicals through their reducing action, and a high reducing power indicates a strong antioxidant power.
In this study, the antioxidant activity of SSC-12 EPS was assessed in terms of its ability to inhibit the formation of free radicals and its reducing ability. As shown in Fig. 3(c), the hydroxyl radical scavenging ability of SSC-12 EPS was higher than that of ascorbic acid, with 2 mg/ml of EPS (86.6 %) exhibiting roughly 1.4-times higher scavenging activity than ascorbic acid (63.7 %) at the same concentration. At low concentrations (1.0-4.0 mg/mL), the hydroxyl radical scavenging ability of SSC-12 EPS was substantially stronger than that of ascorbic acid. At 4 mg/mL, the hydroxyl radical scavenging ability of SSC-12 EPS (87.33 %) was also higher than the EPS produced by L. helveticus MB2-1 (56.30 %) [27]. However, the hydroxyl radical scavenging ability of SSC-12 EPS (95.30%) was similar to that of EPS produced by L. kimchi SR8 (96.58%) [22] at 8 mg/mL.
Our results indicated that EPS produced by SSC-12 had good antioxidant capacity and might serve as a good alternative to ascorbic acid. The degree of antioxidant capacity of EPS produced by different LAB differed, which may be attributed to the varying composition and structure, such as glycosidic linkages embodiment, functional group, and molecular weight [11]. For example, it has been reported that the EPS produced by L. delbrueckii ssp. bulgaricus SRFM-1 had more carboxyl functional groups than those produced by other LAB, which could provide an acidic environment to promote its hydrolysis and expose more hemiacetal hydroxyl groups for excellent antioxidant activity [28]. Moreover, low molecular weight of EPS indicates that few hemiacetal hydroxyl groups are exposed at the same mass concentration and consequently high antioxidant activity [11]. The strong antioxidant activity of SSC-12 EPS might be due to the presence of hydroxyl groups and other functional groups.

Antibacterial ability
Pathogens can cause food to decay in vitro, while pathogenic bacteria in the gastrointestinal tract can cause gastrointestinal infections in vivo [11]. Antibiotics are widely used for the control of bacterial infections. However, as the phenomenon of drug resistance becomes a growing concern, the search for safe and effective antibacterial drugs has also become a focus for researchers. It has been reported that some EPSs from LAB have good antibacterial activity. For example, the EPS of L. plantarum HM47 isolated from human breast milk had a strong inhibitory effect on pathogenic Escherichia coli and Salmonella typhimurium in vitro [29]. The EPS of L. fermentum S1 isolated from traditional fermented Fuyuan pickle had good antibacterial activity against E. coli and Staphylococcus aureus, with the highest inhibition rates of 32 % and 43 %, respectively [30].
The absorbance of a bacterial liquid indicates its turbidity degree. The higher the absorbance, the more the number of bacteria, and the determination of absorbance facilitated the qualitative analysis of the antibacterial property of SSC-12 EPS in this study. The inhibition rate of SSC-12 EPS was quantitatively analyzed by measuring the number of harmful bacteria cultivated in the culture medium after adding SSC-12 EPS. The inhibitory effect on Staphylococcus aureus and Salmonella enterica subsp. enterica increased with increase in SSC-12 EPS concentration, but the inhibitory effect on Listeria monocytogenes was not signi cant (Fig. 4). Although the inhibitory effect of SSC-12 EPS on the three harmful bacteria was concentration-dependent, SSC-12 EPS had a considerable inhibitory effect on S. aureus, Salmonella enterica subsp. Enterica, and Listeria monocytogenes at 2, 6, and 8 mg/mL respectively. At 10 mg/mL, the inhibitory effect of SSC-12 EPS on Staphylococcus aureus, Salmonella enterica subsp. Enterica, and Listeria monocytogenes reached the maximum, which were 100 %, 71.9 % and 14.9 %, respectively. These results suggest that the SSC-12 EPS has the strongest inhibitory effect on Staphylococcus aureus, followed by Salmonella enterica subsp. enterica, and the worst inhibitory effect on Listeria monocytogenes. Liu et al [1] observed that the EPS produced by L. plantarum WLPL04 had good inhibitory effect on Staphylococcus aureus and Listeria monocytogenes and its inhibitory ability increased gradually with increasing concentrations of EPS. At 2 mg/ml, the inhibitory rate of EPS produced by L. fermentum S1 [30] against Staphylococcus aureus did not exceed 12 %, while SSC-12 EPS reached 17.8 % at the same concentration. When the EPS produced by P. pentosaceus M4 [19] was 5 mg/ml, its inhibitory effect on Staphylococcus aureus reached 56.5 %, which was similar to that of SSC-12 EPS.
Different types of EPS have varying inhibitory effects on harmful microorganisms. According to previous studies, the antibacterial mechanism of EPS might be attributable to the prevention of bio lm formation or destruction of membrane integrity and uid soluble protein, which are mediated by signal molecules or sugar receptors [31,32]. SSC-12 EPS had good antibacterial activity (Fig. 4), providing a theoretical basis for its application in feed production and clinical treatment. However, the associated mechanism is unclear and needs further exploration.

Conclusions
In this study, a novel SSC-12 EPS isolated from silage was identi ed as P. pentosaceus. SSC-12 had strong vitality and fast EPS production rate. EPS produced by P. pentosaceus SSC-12 was a heteropolysaccharide and consisted of glucose (42.6 %), mannose (28.9 %), galactose (16.2 %), arabinose (9.4 %), and rhamnose (2.9 %). The EPS had strong antioxidant ability and antibacterial ability. The present study identi ed a new strain of EPS-producing LAB, and the EPS showed good biological activity, which can potentially be applied in the feed, food, and pharmaceutical industries as well as in the development of new natural antibiotic substitutes.

Materials And Methods
Screening and identi cation of EPS-producing LAB LAB was isolated from silage and puri ed by culturing on MRS agar medium under anaerobic condition for 24-48 h at 37 ℃ [1]. After morphological observation, a single colony, having milky white color, sticky surface, surrounding diffusion phenomenon, protruding round shape, and obvious viscosity when picked by inoculation ring [11], was suspected to be the EPS-producing LAB.
The above strains were inoculated into 50 ml MRS liquid medium and statically cultured at 37 ℃ for 24 h. Next, the broth was collected, centrifuged at 8000 r/min for 10 min, and the supernatant was collected, followed by the addition of 95 % ethanol (3×volume) and incubation at 4 ℃ overnight [1]. The mixture was centrifuged at 8000 r/min for 10 min, and the precipitate was collected and dissolved in distilled water, followed by dialysis for 3 days to obtain crude EPS solution [33]. The EPS production was measured by phenol sulfuric acid method, and the strains with the highest EPS production were identi ed.
The screened strains were puri ed and cultured for two generations to obtain bacterial suspension, and total DNA was extracted from cell precipitate using TIANamp Bacteria DNA Kit. The 16S rDNA was ampli ed with primer pair 27F / 1492R using polymerized chain reaction (PCR) procedure [34]. Puri ed PCR products puri ed from each strain were sequenced by ABI3730-XL DNA Analyzer, and we used the Blast (http://www.ncbi.nlm.nih.gov.blast) to compare the spliced sequence le with the data in the NCBI 16S database [5]. The MEGA-X software was used to construct phylogenetic evolutionary tree, and the species with more than 99% similarity were identi ed. The identi ed LAB was stored in MRS liquid medium with 20% glycerol at -80 ℃ [35].

Analysis of strain growth and EPS production
The identi ed strains were inoculated into MRS liquid medium and cultured for two generations. After the LAB was cultured for different periods, the number of LAB was counted by the plate counting method and EPS production was determined by using the phenol sulfuric acid method. The peak time for EPS production was determined, and the extraction and puri cation of EPS was carried out. The puri cation steps of EPS were repeated to obtain EPS aqueous solution. The EPS solution was freeze-dried (Thermo Modulyo Freeze Dryer, Thermo Fisher Scienti c, USA) at low temperature, and the ocs were stored at ambient temperature in a sealed state [35].

Determination of EPS monosaccharide composition
After the EPS samples were acid hydrolyzed, their monosaccharide composition was analyzed by high performance anion exchange chromatography (HPAEC) (DIONEX ICS-6000, USA) equipped with Dionex™ CarbopacTM PA-20 anion exchange chromatography column (3 × 150nm) and electrochemical detector. NaOH (1) where "A 11 " is the absorbance of the sample, "A 01 " is the absorbance of the control group, and "A 10 " is the absorbance of the blank. Ethanol (0.4 mL) and sample solutions of different dilutions (1.0 mL) were used as the control, and DPPH radical-ethanol (0.4 mL) and water (1.0 mL) were used as the blank.

Hydroxyl radical scavenging ability
The hydroxyl radical scavenging ability of EPS was measured according to the method described by Xu et al [34]  (2) where "A sample " was the absorbance when the sample contains different concentrations of EPS, "A black " was the absorbance of the sample when EPS and H 2 O 2 were replaced by water, and "A" was the absorbance of the sample when EPS and H 2 O 2 were replaced by water.
Superoxide radical scavenging activity The superoxide radical scavenging activity of EPS was measured according to the method described by Xu et al [34]. The superoxide radical was generated in 50 µL of Tris-HCl buffer (pH 8.0, 150 mM) containing 25 µL of pyrogallol (1.50 mM, dissolved in 10 mM HCl) and 100 µL of EPS samples (1.0-10.0 mg/mL). Next, the mixture was incubated at 25 ℃ for 30 min and the absorbance was measured at 325 nm. Different concentrations of ascorbic acid in equal volumes (1.0-10.0 mg/mL) were used as a positive control. Each experiment was carried out in triplicate. The superoxide radical scavenging activity of EPS was calculated as follows (3): (3) where "A s " is the absorbance of samples containing EPS and pyrogallic acid, "A c " is the absorbance of samples containing EPS but were used 10 mM HCl was used instead of pyrogallic acid, "A b " is the absorbance of deionized water instead of EPS but containing pyrogallic acid, "A 0 " is the absorbance of EPS sample and pyrogallic acid sample replaced with deionized water and 10 mM HCl, respectively.

Reducing ability
The reduction ability of EPS was measured according to the method of Rajoka et al [18]. A solution containing 1.5 mL sodium phosphate buffer (0.2 M, pH 7.3), 1.5 mL K 3 Fe(CN) 6 (1 %, w/v), then 1.5 mL EPS samples (1.0-10.0 mg/mL) was incubated at 50°C for 25 min. After cooling, 1.5 ml trichloroacetic acid (12 %, w/v) was added, and the mixture was centrifuged at 6000 × g/min for 10 min. Next, 0.5 ml FeCl 3 (0.2 %, w/v) was added to the supernatant, and the absorbance was measured at 700 nm. Different concentrations of ascorbic acid in equal volume (1.0-10.0 mg/mL) were used as a positive control. The experiment was carried out in replicates. The reducing ability of EPS was calculated as follows (4): Reducing ability = A 1 -A 0 (4) where "A 1 " is the absorbance of EPS sample, "A 0 " is the absorbance of FeCl 3 replaced by deionized water.

Measurements of EPS antibacterial ability
In this experiment, Staphylococcus aureus GDMCC 1.1220, S. enteritidis subsp. enteritidis GDMCC 1.345, Listeria monocytogenes GDMCC 1.347 were selected as indicator bacteria. These bacteria were separately inoculated into LB broth medium and cultured at 37°C for 24 h with shaking, which was repeated twice to obtain the second-generation strain [1]. Next, the bacterial suspension was adjusted to an estimated concentration of 10 5 -10 6 CFU/mL based on the absorbance at 600 nm. The puri ed EPS was diluted with deionized water into EPS solutions of different concentrations (1.0-10.0 mg/mL), followed by ltration and sterilization with a 0.45-µM microporous lter. Next, 1 mL of the EPS solutions of different concentrations and 10 µL of indicator bacteria solution were added to 1 mL of fresh LB broth medium and cultured for 24 h at 37 ℃ with shaking [5], with equal amount of sterile water used as a blank instead of EPS. The inhibition rate and bacterial count indicated the antibacterial activity of EPS. The experiment was carried out in triplicate. The formula for the inhibition rate is as follows (5): (5) where "A EPS " is the number of colonies of EPS sample, "A Black " is the number of colonies of positive control.

Statistical analysis
All statistical analyses were conducted using IBM SPSS Statistics 22 software for Windows (IBM Corp, New York, USA), and means were compared for signi cance by Duncan's multiple range method.

Authors' contributions
Authors' contributions were introduced in order of name. FY was responsible for experimental design, experiment execution, data analysis, and writing (manuscript). XL preformed the experiment and collected data. RT contributed to experiment execution and data analysis. RT was participated in the experiment operation. JZ contributed to experimental design and writing (review and editing).

Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 31971764).

Availability of data and materials
All data generated or analysed during this study are included in this article.  Figure 1 Neighbor-joining phylogeny of SSC-12 with closely related Lactobacillus strains according to 16S rRNA gene sequence.

Figure 2
The dynamic relationship between the SSC-12 counts and its EPS yield. The values are expressed as mean ± SD of three independent analyses.

Figure 3
Antioxidant ability of SSC-12 EPS and standard antioxidant reagents ascorbic acid. SSC-12 EPS, EPS produced by SSC-12. Antioxidant ability includes DPPH radical scavenging activity (a), Hydroxyl radical scavenging activity (b), Superoxide radical scavenging activity (c) and Reducing power (d). The values are expressed as mean ± SD of three independent analyses. Different lowercase letters indicate signi cant differences among SSC-12 EPS concentrations (P < 0.05). Different capital letters indicate signi cant differences among ascorbic acid concentrations (P < 0.05). The differences between SSC-12 EPS and ascorbic acid at the same concentration was represented by "** P < 0.01, * P <0.05, NS P >0.05".