Antioxidant Activity and Probiotic Properties of Lactic Acid Bacteria

: Oxidative stress, which can cause imbalance in the body by damaging cells and tissues, arises from the immoderate production of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Therefore, external supplements having antioxidant activity are required for reducing oxidative stress. In our study, we investigated DPPH and ABTS radical scavenging ability, and the inhibition effect on the nitric oxide (NO) production of 15 food-derived bacterial strains in LPS-activated RAW264.7 cells. Among these LAB strains, eight strains with an excellent inhibition effect on NO production were selected through comparisons within the same genera. Moreover, the selected strains, including Leuconostoc mesenteroides MG860, Leu. citreum MG210, Pediococcus acidilactici MG5001, P. pentosaceus MG5078, Weissella cibaria MG5090, Levilactobacillus brevis MG5306, Latilactobacillus curvatus MG5020, and Latilactobacillus sakei MG5048 diminished the inducible nitric oxide synthase ( iNOS )/cyclooxygenase-2 ( COX-2 ) expression. In addition, the stability and adhesion ability of the eight LAB strains in the gastrointestinal tract were determined. In conclusion, the selected strains have potential as new probiotics with antioxidant effects.


Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are usually generated during normal metabolic processes and are unavoidable products for aerobic organisms [1]. Appropriate ROS/RNS levels play a critical role in immunoreaction against invading microbes and regulating intercellular communication [2]. However, the excessive production of ROS/RNS causes oxidative stress, which leads to protein oxidation, lipid peroxidation, and DNA damage [3,4].
Probiotics are living microorganisms-mainly lactic acid bacteria (LAB) such as Lactobacillus, Bifidobacterium, Streptococcus, and Enterococcus-and they produce beneficial effects on the host when ingested at an appropriate level [12,13]. LAB have been known to improve intestinal transit, balance intestinal flora, and maintain the acid-base equilibrium in the colon, resulting in immune system regulation and the lowering of the serum cholesterol level [14]. Research on LAB is being actively conducted due to increasing interest in their antioxidant ability. For example, bacteria belonging to genera Bifidobacterium and Lactobacillus, and Enterococcus durans LAB 18 have been shown to decrease the levels of 2,2diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals [15][16][17][18]. In a previous study, heat-killed L. salivarius MG242, L. plantarum MG989, B. bifidum MG731 and B. lactis MG741 were confirmed to reduce oxidative stress by inhibiting NO production through a decrease in iNOS/COX-2 gene expression in LPS-activated RAW264.7 [19]. Although functional activities of various LAB strains have been reported, studies on some genera are insufficient.
Therefore, we evaluated DPPH and ABTS radical scavenging activity and NO inhibition of LAB to identify various LAB strains with antioxidant ability. Moreover, we investigated the gene expression of iNOS/COX-2, which affects NO production, in LPSactivated RAW264.7 treated with LAB. In addition, to assess the practical application of LAB as probiotics, the survival rate and adhesion ability of LAB in the gastrointestinal tract were investigated.

DPPH and ABTS Radical Scavenging Activity
The effect of bacterial strains on DPPH and ABTS radical scavenging activity was performed with slight modifications to the method of Kim et al. and Lee et al. [20,21]. Samples for antioxidant evaluation were live cells and were prepared as follows. After culturing the strains in MRS medium at 37 • C for 18 h, it was rinsed twice with phosphatebuffered saline (PBS). After centrifugation at 4000 rpm for 5 min, the pellet was resuspended in PBS to OD 600 1.0.
In the DPPH method, 0.2 mM DPPH (Sigma-Aldrich, St. Louis, MO, USA), and sample were mixed at a ratio of 1:2 (v/v) in the amber tube and reacted for 30 min at RT. In the ABTS method, first, ABTS + solution was prepared by mixing 7 mM ABTS (Sigma-Aldrich) and 2.45 mM potassium persulfate (Daejung Chemical and Metals, Siheung, Korea) in 1:1 (v/v) ratio. The ABTS + solution was adjusted by adding distilled water, so that the absorbance at 734 nm became 0.7 ± 0.01. Next, 300 µL of the sample and 600 µL of ABTS + solution were mixed in the amber tube and reacted for 30 min at RT. Absorbance was measured at 517 nm for DPPH method and at 734 nm for ABTS method. PBS and L-ascorbic acid were used as the control and positive control groups, respectively. The radical scavenging activity was computed using the following equation: Radical scavenging activity (%) = (1 − A sample /A CTL ) × 100 (1) where A sample = absorbance of sample group; A CTL = absorbance of control group.

NO Production and Cell Viability
NO production was analyzed by some modification to the method of Kang et al. [22]. RAW264.7 were seeded in 96-well plate at 2 × 10 6 cells/mL and incubated for 24 h. The seeded cells were treated with FBS-free DMEM diluted with 10% CFS and 500 ng/mL of E. coli LPS (Sigma-Aldrich) and further incubated for 24 h. After transferring the cell culture supernatant (100 µL) to a new plate, the equal amount of fresh Griess solution was added and reacted at RT for 10 min. Absorbance was then determined at 550 nm using a microplate spectrophotometer (Biotek, Winooski, VT, USA). NO was quantified using a standard curve of NaNO 2 (Sigma-Aldrich). Cytotoxicity was measured by the MTT (Sigma-Aldrich) method [23].

Simulated Gastrointestinal Tract Stability
LAB survival in the stomach and intestine tenue is crucial to reach the colon and to be used as probiotics. Therefore, in this study, viability of LAB was evaluated under conditions similar to those of the intestinal environment [24]. The LAB strain was cultured for 18 h, rinsed twice with PBS (pH 7.4), and resuspended at OD 600 of 1.0. The suspension solution of 1 mL was collected, mixed with 9 mL of PBS (pH 2.5) containing 0.3% pepsin (simulated gastric fluid, SGF; Sigma-Aldrich), and incubated at 37 • C for 2 h, with stirring at 200 rpm. Then, 10 mL of PBS adjusted to pH 7.4, containing 1% pancreatin and 1% bile salt (stimulated intestinal fluid, SIF; Sigma-Aldrich) was added to the pellet obtained by centrifugation at 4000 rpm for 5 min and incubated at 37 • C for 2.5 h, with stirring at 200 rpm. The viable cell count was analyzed by the plate count method using MRS agar, and the survival rate was represented as the percentage of viable cells after 4.5 h compared to the initial number.

Adhesion to HT-29 Cells
The adhesion ability of the selected LAB strain was assessed in HT-29 cells (human colorectal adenocarcinoma cell line), as described previously [25]. HT-29 were seeded in a 12-well plate at 1 × 10 6 cells/mL and incubated in 5% CO 2 at 37 • C for 24 h. The strain was cultured in MRS medium at 37 • C for 24 h. Then, LAB strain was resuspended at an OD 600 of 1.0 in FBS and P/S-free DMEM, and inoculated into cells. After 2 h, the cells were rinsed with PBS (pH 7.4) and then scrapped. The number of LAB attached to the cells was counted by the plate count method using MRS agar and expressed as log CFU/mL.

Statistical Analysis
All experimentations were measured in triplicate and represented as the mean ± standard deviation (SD). For statistical analysis, one-way ANOVA was carried out using the SPSS statistical software (version 25; IBM Inc., Armonk, NY, USA), and the Tukey's multiple comparison test was used to settle the significance of differences (p < 0.05) among treatments.

Antioxidant Activity of the LAB Strains
DPPH and ABTS radical scavenging activity were analyzed to estimate antioxidant activity of 15 food-derived bacterial strains, and the results are shown in Table 1. DPPH radical scavenging activity of LAB was in the range 2.55-6.88%, and ABTS radical scavenging activity was 19.69 to 86.26%.

LAB Strains Suppressed NO Production in LPS-Activated RAW264.7 Cells
The suppression of NO production was investigated in LPS-activated RAW264.7 treated with LAB CFS. LPS and MRS as a vehicle treatment increased NO production to 62.50 and 52.20 µM, respectively, but treatment with all bacterial strains significantly reduced it by 19.38-38.74 µM (inhibition rate, 38.01-69.00%) (Figure 1a). In particular, the NO inhibitory effect of L. sakei MG5048 (69.00 ± 0.02%) was the highest in RAW264.7 compared with the LPS-treated group. No cytotoxicity (cell viability > 90%) was observed upon LAB CFS treatment (Figure 1b). multiple comparison test was used to settle the significance of differences (p < 0.05) among treatments.

Antioxidant Activity of the LAB Strains
DPPH and ABTS radical scavenging activity were analyzed to estimate antioxidant activity of 15 food-derived bacterial strains, and the results are shown in Table 1. DPPH radical scavenging activity of LAB was in the range 2.55-6.88%, and ABTS radical scavenging activity was 19.69 to 86.26%.

LAB Strains Suppressed NO Production in LPS-Activated RAW264.7 Cells
The suppression of NO production was investigated in LPS-activated RAW 264.7 treated with LAB CFS. LPS and MRS as a vehicle treatment increased NO production to 62.50 and 52.20 μM, respectively, but treatment with all bacterial strains significantly reduced it by 19.38-38.74 μM (inhibition rate, 38.01-69.00%) (Figure 1a). In particular, the NO inhibitory effect of L. sakei MG5048 (69.00 ± 0.02%) was the highest in RAW264.7 compared with the LPS-treated group. No cytotoxicity (cell viability > 90%) was observed upon LAB CFS treatment (Figure 1b).  Based on the results of antioxidant activity and NO inhibition, Leu. mesenteroides MG860, Leu. citreum MG210, P. acidilactici MG5001, P. pentosaceus MG5078, W. cibaria MG5090, L. brevis MG5306, L. curvatus MG5020, and L. sakei MG5048 were selected, and the gene expression of iNOS/COX-2 was confirmed using these strains.

LAB Strains Reduced iNOS/COX-2 Gene Expression in LPS-Activated RAW264.7 Cells
The effect of the selected LAB strains on the gene expression of iNOS/COX-2 was assessed using qRT-PCR. Although the iNOS/COX-2 expression was significantly increased in LPS-activated RAW264.7, LAB treatment markedly suppressed gene expression. iNOS expression level was most significantly decreased by L. sakei MG5048 to 0.23fold; other strains also showed inhibitory effects ranging from 0.32-0.50-fold (Figure 2a). COX-2 expression level was the lowest after treatment with P. pentosaceus MG5078 and L. sakei MG5048, 0.13-fold and 0.12-fold, respectively; other strains showed COX-2 expression level from 0.41-0.70-fold (Figure 2b).

Viability of LAB Strains in Simulated Gastrointestinal Fluid
In this study, the viability of the strains was determined by culturing the selected strains in SGF and SIF for a certain period of time ( Table 2). The results confirmed that the viability of the selected strains ranged from 38.02-87.17%. Among them, P. acidilactici MG5001 (82.42%) and L. brevis MG5306 (87.17%) showed higher survival rates than those of other strains.

Viability of LAB Strains in Simulated Gastrointestinal Fluid
In this study, the viability of the strains was determined by culturing the selected strains in SGF and SIF for a certain period of time ( Table 2). The results confirmed that the viability of the selected strains ranged from 38.02-87.17%. Among them, P. acidilactici MG5001 (82.42%) and L. brevis MG5306 (87.17%) showed higher survival rates than those of other strains.

Adhesion of the LAB Strains on HT-29 Cells
The intestinal cell adhesion ability of the LAB strains was investigated using HT-29 cells (Table 3). The results revealed that the adhesion ability of the selected strains ranged from 63.02-84.91%. Among them, P. pentosaceus MG5078 (84.91%) and L. brevis MG5306 (82.35%) showed higher adhesion rates than those of other strains.

Discussion
Oxidative stress results from a disequilibrium between oxidant and antioxidant actions. Humans have an antioxidant defense system in the body, and when the accumulated ROS exceeds its intrinsic antioxidant capacity, excessive oxidation causes damage to cells or tissue. Thus, external antioxidants are required to decrease oxidative stress by improving individual antioxidant ability. Various antioxidant assays, involving stable non-biological radicals, have been conducted to determine the free-radical scavenging activity of probiotics [26]. LAB are safe bacteria with the ability to produce several compounds, such as organic acid and bacteriocin, and have the advantage of having few side effects [27]. When LAB are attached to the intestinal lumen, LAB and metabolites of LAB increase to remove ROS, thereby maintaining the intestinal oxidation-reduction balance. In addition, it is known that LAB constitute a strong natural xenogeneic antioxidant in consideration of nutritive value, safety, and functions of probiotic [28]. Therefore, it was attempted to confirm the antioxidant activity using various LAB.
Recently, much attention has been paid to CFS containing biologically active metabolites secreted by living bacteria [29]. Moreover, unlike CFS, which contain only extracellular metabolites, probiotics may be more effective in improving health through a broader range of mechanisms [30]. CFS of probiotics is used as an in vitro model for the purpose of evaluating functional effects [31]. In our study, the antioxidant activity of CFS from 15 food-derived bacterial strains was measured through the DPPH and ABTS radical scavenging method ( Table 1). All LAB has DPPH and ABTS radical scavenging activity. Among them, MG5020 is the highest radical scavenging activity. The protective ability of probiotics against oxidative stress has been elucidated by ROS scavenging, chelation of metal ion, and the reduction of ascorbate autoxidation [32]. The antioxidant components in LAB are bacterial exopolysaccharides, bioactive peptides, antioxidant enzymes, and manganese ions. Moreover, the gut microflora can produce bioactive dietary antioxidants through bioconversion processes using various enzymatic reactions [33].
NO is produced by various immune cells via the gene expression of iNOS, and these cells are stimulated by substrates of the microbe and various cytokines. The major functions of NO are antimicrobial, antitumor, tissue-damaging, anti-inflammatory, and immunosuppressive effects [34]. However, NO, which plays a physiologically important role, is a kind of reactive radical, and when excessively generated reacts with an anion radical to form peroxynitrite, a more powerful oxidizing agent, causing an inflammatory reaction [5]. Therefore, reducing NO production is one of the effective treatment strategies. In our study, 15 food-derived bacterial strains showed no cytotoxicity and inhibited NO production in RAW264.7 cells (Figure 1). Based on these results, MG860, MG210, MG5001, MG5078, MG5090, MG5346, MG5020 and MG5048 were selected for evaluating iNOS/COX-2 gene expression. All LAB reduced iNOS/COX-2 gene expression: among them, MG5048 showed the highest inhibitory activity of iNOS/COX-2 ( Figure 2). iNOS/COX-2 is expressed in LPS-activated macrophages and is known to be a factor related to NO production [10]. In addition, Choi et al. and Wang et al. reported that LAB treatment reduced NO production by decreasing the iNOS/COX-2 expression in LPS-activated cells [35,36]. Lactobacillus rhamnosus GG (LGG) was used as positive control primarily in probiotics study. Yang et al. reported that NO inhibition rate of LGG was 37% [29]. Our LAB strains showed higher efficacy compared with LGG with a NO inhibition rate of 38 to 69%. Therefore, the selected LAB can exhibit antioxidant potential by inhibiting NO production, by reducing the expression of iNOS/COX-2 in LPS-activated RAW264.7.
Probiotic bacteria are exposed to an acidic environment of the stomach (pH 1.5-3.5) and bile salt (pH 5.0-6.0), and should be able to survive under these conditions [37]. The results confirmed that the viability of the selected strains ranged from 38.02-87.17% (Table 2). Due to the difference in the pH of the gastric and intestines and the detergent action of bile salts, bacterial viability is limited. Bacterial strains belonging to the genera Bifidobacterium and Lactobacillus present in the intestine possess the bile salt hydrolase that converts primary bile acids into relatively less active secondary bile salts [38]. Therefore, the selected LAB showed the survival rate of 40% in the resistance of bile salt (Table 2). Overall, the strains MG5306 and MG5001 showed the highest survival rate in the gastrointestinal tract.
The adhesion ability of LAB is a strain-specific property, which indicates its probiotic potential. The health benefits of probiotics are associated with the composition of gut microbiota and their adhesion to the epithelial and mucosal surfaces. Therefore, the intestinal adhesion ability of probiotics is an important parameter that can reveal their functional property [39]. The adhesion process associates complex interactions between the host cell surfaces and the bacterial cell membrane. It has been suggested that bacteria can improve colonization in intestinal epithelial cells through the surface layer binding of proteins [40]. Generally, the Lactobacillus genus is known to have adhesion ability in the range of 2 to 10% [41]. In this study, the selected bacterial strains showed more than 60% adhesion ability; particularly, MG5078 and MG5306 strains showed excellent adhesion ability of 84.91% and 82.35%, respectively (Table 3). Therefore, these strains are expected to have excellent survival and adhesion in the gastrointestinal tract, suggesting that they will be useful for improving the intestinal environment; however, further research on the safety of these strains is needed before being used as probiotics.

Conclusions
Our results suggested that LAB treatment relieved oxidative stress by scavenging DPPH and ABTS radicals. In addition, LAB treatment suppressed NO production and reduced the levels of iNOS and COX-2 in LPS-activated RAW264.7 cells. Thus, LAB, including Leu. mesenteroides MG860, Leu. citreum MG210, P. acidilactici MG5001, P. pentosaceus MG5078, W. cibaria MG5090, L. brevis MG5306, L. curvatus MG5020, and L. sakei MG5048, have a strong potential for being used as probiotics and antioxidant functional foods.