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 identified 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].
Table 1 The sources and EPS yield of 22 suspecting LAB strains isolated from silage.
Source silage
|
Strains
|
Yield
(mg/L in MRS broth)
|
Napier grass
|
SPP-1
|
59.3 ± 2.9l
|
SPP-2
|
67.9 ± 4.9kl
|
SPP-3
|
62.4 ± 5.1l
|
SPP-5
|
87.5 ± 6.5jk
|
SPP-6
|
202.5 ± 17.9bc
|
SSP-9
|
172.6 ± 27.2def
|
Corn
|
SSC-1
|
51.0 ± 5.0l
|
SSC-3
|
200.5 ± 16.3bc
|
SSC-12
|
276.6 ± 12.1a
|
SSC-16
|
110.6 ± 6.3i
|
SSC-23
|
212.3 ± 10.5b
|
SSC-48
|
143.7 ± 16.7gh
|
Orchardgrass
|
SDJ-3
|
30.5 ± 5.2m
|
SDJ-6
|
188.5 ± 9.0cd
|
SDJ-8
|
203.7 ± 8.2bc
|
SDJ-16
|
131.1 ± 10.0h
|
Stylo
|
SS-9
|
90.5 ± 4.9j
|
SS-17
|
181.7 ± 10.2cde
|
Soybean
|
SGM-9
|
202.9 ± 11.5bc
|
SGM-16
|
164.8 ± 15.8ef
|
SGM-18
|
176.8 ± 19.9de
|
SGM-20
|
153.2 ± 7.3fg
|
Data with different letters within a column were significantly different at P < 0.05.
The EPS yield in the table was mean ± SD of three replicates.
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. SSC-12 exhibited fast growth during first 8 h of incubation. According to previous studies, L. reuteri SHA101 and L. vaginalis SHA110 reached maximum EPS production in 48 h [18] and L. plantarum WLPL04 in 24 h [1]. However, SSC-12 achieved maximum EPS production in 20 h, suggesting that SSC-12 would have a great application value.
Monosaccharide composition of EPS
High-performance anion exchange chromatography (HPAEC) determination showed that the EPS produced by SSC-12 (SSC-12 EPS) was a heteropolysaccharide. The total sugar content of SSC-12 EPS was 73.6 %, comprising glucose (42.6 %), mannose (28.9 %), galactose (16.2 %), arabinose (9.4 %), and rhamnose (2.9 %) (Table 2). According to the number of monosaccharide and the proportion of each monosaccharide, EPS produced by SSC-12 was different from that produced by P. pentosaceus as previously reported. For example, the EPS produced by P. pentosaceus M41 consisted of glucose (79.0 %), mannose (9.5 %), arabinose (6.2 %), and galactose (5.2 %) [19], and the EPS produced by P. pentosaceus DPS comprised glucose, mannose, and fructose in different ratios [20]. Therefore, SSC-12 produced a novel type of EPS.
Table 2
The monosaccharide composition of EPS produced by SSC-12.
Monosaccharide name
|
Molar ratio (%)
|
Glucose
|
42.7 ± 0.28
|
Mannose
Galactose
Arabinose
|
28.9 ± 0.39
|
16.3 ± 0.06
|
9.4 ± 0.36
|
Rhamnose
|
2.9 ± 0.02
|
The values are represented as mean ± SD (n=3). |
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 significantly 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 fighting 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. The antioxidant capacity of SSC-12 EPS was concentration-dependent and the gap between them at the same concentration became smaller and smaller. At 10 mg/mL, the DPPH scavenging ability (77.4 %), hydroxyl radical scavenging ability (97.5 %), superoxide radical scavenging ability (77.5 %), and reducing ability (1.3) of SSC-12 EPS reached maximum values. Rajoka et al [18] observed lower values than that of SSC-12 EPS (56.72 %) at a concentration of 4 mg/mL, with the superoxide radical scavenging rate of 40.5 % and 25.5 % for the EPS produced by L. reuteri SHA101 and L. vaginalis SHA110, respectively. Seo et al [26] also evaluated the antioxidant capacity of an EPS produced by L. plantarum YML009 and reported a lower DPPH radical scavenging (7.24 %) and reduction (0.15) activities at 10 mg/mL, compared with those of SSC-12 EPS.
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 significant (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 biofilm formation or destruction of membrane integrity and fluid 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.