Isolation of a Potential Probiotic Levilactobacillus brevis and Evaluation of Its Exopolysaccharide for Antioxidant and α-Glucosidase Inhibitory Activities

The probiotic properties of ten lactic acid bacteria and antioxidant and α-glucosidase inhibitory activities of the exopolysaccharide (EPS) of the selected strain were investigated in this study. Levilactobacillus brevis L010 was one of the most active strains across all the in vitro tests. The cell-free supernatant (50 g/l) of L. brevis L010 showed high levels of both α-glucosidase inhibitory activity (98.73 ± 1.32%) and 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (32.29 ± 3.86%). The EPS isolated from cell-free supernatant of L. brevis L010 showed 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activity (80.27 ± 2.51%) at 80 g/l, DPPH radical-scavenging activity (38.19 ± 9.61%) at 40 g/l, and ferric reducing antioxidant power (17.35 ± 0.20 mg/l) at 80 g/l. Further, EPS exhibited inhibitory activities against α-glucosidase at different substrate concentrations. Kinetic analysis suggests that the mode of inhibition was competitive, with a kinetic constant of Km = 2.87 ± 0.88 mM and Vmax = 0.39 ± 0.06 μmole/min. It was concluded that the EPS might be one of the plausible candidates for possible antioxidant and α-glucosidase activities of the L. brevis L010 strain.

Lee and Kim [8] reported that Leuconostoc mesenteroides MKSR possesses α-glucosidase inhibitory activity and cholesterol-lowering effects.Treatment of Latilactobacillus sakei OK67 ameliorated high-fat diet-induced blood glucose intolerance [9].In addition, Levilactobacillus brevis KU15006 exhibited antimicrobial activity against foodborne pathogens and anti-diabetic properties [10].For a potential probiotic to be able to exert its beneficial effects, it must be able to survive in the gastrointestinal environment.Therefore, the essential characteristics for a LAB to be functional as a probiotic include the following: (i) tolerance to low pH and bile salt stress conditions, (ii) ability to adhesion to intestinal surfaces, and (iii) inhibitory activity against pathogenic bacteria [11].Lacticaseibacillus rhamnosus GG (LGG) is a classic example of a probiotic strain with all three characteristics: acid and bile tolerance and excellent adhesion to human tissue.LGG has been used as the control in many studies with which other Lactobacillus spp.were compared [12][13][14].
Chen et al. [14] examined antioxidant and α-glucosidase inhibitory activities to identify potential anti-diabetic The probiotic properties of ten lactic acid bacteria and antioxidant and α-glucosidase inhibitory activities of the exopolysaccharide (EPS) of the selected strain were investigated in this study.Keywords: Levilactobacillus brevis, probiotic, exopolysaccharide, antioxidant activity, α-glucosidase inhibitory activity and probiotic LAB.L. rhamnosus Z7 showed significantly high antioxidant and α-glucosidase activities.A recent study has suggested that exopolysaccharides (EPS) might result in inhibitory activity [19].

Levilactobacillus brevis
Ten lactic acid bacteria were screened for their probiotic properties in this study.The antioxidant and αglucosidase inhibitory activities of the EPS of selected strains were investigated.

LAB Strains
Ten LAB strains isolated from Korean fermented foods [20] were used in this study and are shown in Table 1.
Preparation of Cell Lysates and Cell-Free Supernatant LAB strains were incubated in MRS broth (MB Cell, Korea) at 30°C for 48 h and centrifuged at 10,000 ×g for 1 min.A cell-free supernatant was obtained by filtering the supernatant using a membrane (0.22 μm, Sartorius, Germany).Cells were washed three times with phosphate-buffered saline (PBS, pH 7.4; Welgene, Korea), disrupted using an ultrasonic homogenizer (Sonics, USA), and centrifuged at 10,000 ×g for 1 min to obtain clear cell lysates.

α-Glucosidase Inhibition Assay
Inhibition of α-glucosidase was determined following a previously described method [14] with minor modifications.The cell-free supernatant or cell lysates (50 μl) were mixed with 50 μl of α-glucosidase (1.0 U/ml, Sigma-Aldrich, USA).After incubation at 37°C for 5 min, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside (p-NPG; Sigma-Aldrich) was added.The reaction proceeded at 37°C for 30 min and was terminated by adding 100 μl of 0.1 M sodium carbonate.Inhibitory activity was determined by measuring the absorbance at 405 nm to determine the amount of p-nitrophenol released from p-NPG [14].

DPPH Radical-Scavenging Activity Assay
The cell lysates or cell-free supernatant (20 μl) were mixed with 180 μl of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical solution (0.4 M) and incubated in the dark at 25°C for 30 min.After centrifugation at 10,000 ×g for 1 min, the absorbance was measured at 517 nm.The DPPH free radical-scavenging activity was determined as previously described [21].

Adhesion to Caco-2 Cells
Caco-2 cells (Korea Cell Line Bank, Korea) were cultured according to a previously described method [22]; LGG (KCTC5033; Korean Collection for Type Cultures, Korea) was used as a positive control.The Caco-2 cells were inoculated at a cell density of 1.0 × 10 5 cells/ml in a six-well plate and maintained for two weeks.The LAB were inoculated into Caco-2 cell-containing medium at a concentration of 1.0 × 10 8 CFU/ml following incubation at 37°C for 2 h.After removing unbound LAB by washing three times with PBS, adhered LAB was released by treating with Triton X-100 (0.05%, Sigma-Aldrich).The mixture of Caco-2 cells and LAB was plated on MRS agar using the pour plate method [23] and incubated at 30°C for 2 days.The adhesion rate was estimated using a previously reported equation [12].

Tolerance to Artificial Gastric Juice and Bile Juice
The tolerance of the strains to artificial gastric juice and bile juice was determined according to a previously described method [24] with minor modifications.LAB growing exponentially in MRS were harvested when the absorbance at 600 nm (OD 600 ) reached 1.0, and 50 μl of culture was inoculated into a sterile 96-well plate.Subsequently, 150 μl of artificial gastric juice (pH 2.5, adjusted using pepsin) or bile juice (1% ox gall in MRS) was added.After incubation at 30°C for 2 h, cell growth was determined by measuring the OD 600 .

Antimicrobial Activity Assay
The disk diffusion method [25] was used to determine antimicrobial activity against foodborne pathogens (Bacillus cereus KCTC1012, Escherichia coli O157: H7 KCCM40406, and Staphylococcus aureus KCTC1916).Pathogens growing exponentially (OD 600 = 0.8-1.0) in nutrient broth (MB cell) were spread onto nutrient agar.A paper disk was placed on the surface of the agar, and 10 μl of LAB showing exponential growth in MRS was loaded on each paper disk.The plates were incubated at 37°C for 24 h.Ampicillin (50 mg/l, Sigma-Aldrich) and LGG were positive controls.

Identification of LAB
Genomic DNA was extracted as previously described [26].The 16S rRNA gene sequences were compared using BLAST (National Center for Biotechnology Information, USA).A phylogenetic tree of the retrieved sequences was constructed using the neighbor-joining method in MEGA 11.0 software (version 11.0.8,USA) [27].

Determination of Specific Growth Rate of L. brevis L010
The specific growth rate of L. brevis L010 was determined under various culture temperatures (25, 30, 37, 40, and 45°C) and medium acidity (pH 4, 5, 6, 7, and 8).The culture medium acidity was adjusted using a buffer (pH 4 and 5: citrate buffer; pH 6 and 7: potassium phosphate buffer; pH 8: Tris-HCl buffer).L. brevis L010 pre-cultured in MRS at 30°C for 12 h was inoculated into 200 ml MRS broth at an initial OD 600 of 0.1 and then incubated in shake flasks (200 rpm).The specific growth rate (h -1 ) was determined during the exponential growth phase [28].

EPS Extraction
EPS was extracted using a previously described method [29].L. brevis L010 cultured at 30°C for 48 h was boiled for 10 min to inactivate enzymes following trichloroacetic acid addition at a final concentration of 10%.The mixture was incubated at 4°C for 4 h, and the precipitate was removed via centrifugation (9,000 ×g) for 10 min.Two volumes of ice-cold ethanol were added to the supernatant, and the mixture was stored at 4°C for 12 h.EPS was precipitated via centrifugation at 9,000 ×g for 20 min, dissolved in sterile water, dialyzed against water using a dialysis tube (molecular weight cut-off of 3,500 Da; Thermo Fisher Scientific., USA), and then lyophilized.

Antioxidant Activity of EPS
Antioxidant activity of the EPS was determined based on DPPH radical-scavenging, 2,2-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS)-scavenging, and ferric reducing antioxidant power (FRAP) activities.The EPS (500 μl) was mixed with DPPH radical solution (500 μl) and incubated in the dark at 25°C for 30 min.After centrifugation at 10,000 ×g for 1 min, absorbance was measured at 517 nm.The DPPH free radicalscavenging activity of the EPS was determined as previously described [21].
The ABTS solution was mixed with the same volume of EPS.After incubation at 37°C for 30 min, the absorbance at 734 nm was measured.The ABTS radical-scavenging activity was calculated as previously described [30].
The FRAP activity of EPS was determined as described previously [30].After treating the EPS (100 μl) with FRAP reagent (900 μl) at 37°C for 30 min, the absorbance was measured at 593 nm.Ascorbic acid was the positive control.

Statistical Analysis
Using Duncan's multiple range test, statistical analysis based on at least three independent experiments was performed [33].

DPPH Radical-Scavenging Activity
The DPPH radical-scavenging activity was determined to evaluate the antioxidant activity of the LAB (Fig. 2).All LAB strains exhibited DPPH radical-scavenging activity ranging from 27.14-35.35%for the cell-free supernatant and 3.18-11.05%for the cell lysates.DPPH radical-scavenging activity of the cell-free supernatant was higher than those of the cell lysates in all tested strains.The L003 strain showed the highest DPPH radicalscavenging activities of 11.05 ± 0.89% and 35.35 ± 3.84% for cell lysates and cell-free supernatant, respectively, followed by the cell-free supernatants of L001 and L010, both of which showed approximately 32% DPPH radicalscavenging activity.L. plantarum B2 and L. brevis D7 have been reported to show radical-scavenging activities of 30.3 and 44.9%, respectively [36].The authors suggested that the DPPH radical-scavenging activity is associated with EPS in the cell-free supernatant.

Tolerance to Artificial Gastric Juice and Bile Juice Activity of LAB
The effects of artificial gastric juice and bile juice on LAB cell viability are shown in Fig. 3.The viability of the L010 strain was the highest after exposure to artificial gastric juice, followed by that of L004.However, the L005, L006, L008, and L009 strains did not tolerate exposure to artificial gastric juice.Hassanzadzar et al. [24] reported that incubation at pH 2 and 3 decreases LAB viability.Among the ten LAB exposed to bile juice, five strains (L004, L005, L006, L007, and L010) showed tolerance, whereas the other five (L001, L002, L003, L008, and L009) did not.The tolerance of the L010 strain was considerably higher than that of the other strains.

Antimicrobial Activity of LAB
The antimicrobial activity against foodborne pathogens was tested for the LAB strains (Fig. 4).LAB inhibited  the growth of all pathogens with inhibition zones ranging from 7.09-18.60mm in diameter (Table 1).Among the ten LAB, seven strains showed antimicrobial activity against B. cereus with inhibition zones ranging from 7.09-8.54mm in diameter.Nine strains inhibited the growth of E. coli with inhibition zones ranging from 9.10-12.15mm in diameter.S. aureus was inhibited by nine LAB strains with inhibition zones ranging from 12.04-18.60mm in diameter.The L001, 002, 003, 005, 007, and 010 strains showed antimicrobial activity against all three pathogens.The LGG strain, a positive control, also showed inhibitory activity against B. cereus, E. coli, and S. aureus.A recent study [37] reported that L. plantarum isolated from Ethiopian fermented food exhibits antimicrobial activity against S. aureus and E. coli.In agreement with the findings of this study, L. brevis KU15153, isolated from kimchi, shows distinct antimicrobial activity against E. coli and S. aureus [38].

Identification of LAB
The L010 strain demonstrated antimicrobial activity and acid tolerance and was identified as L. brevis.To determine the molecular differences between the L010 strain and other strains, we constructed a 16S rRNA gene phylogenetic tree (Fig. 5).The L010 strain showed the highest similarity with an L. brevis (NR116238) strain at 100%, and it showed 99% similarity with Lactiplantibacillus mudanjiangensis (NR125561).

Inhibition of α-Glucosidase Activity by EPS
The α-glucosidase activity was inhibited when 4 mM substrate was added; L010-derived EPS exhibited 7.05 and 23.30% inhibitory activity at 10 and 20 g/l, respectively (Table 2).To define the mode of inhibition, kinetics analysis was performed using optimal concentrations (Fig. 8, Table 3).The results indicate that EPS competitively inhibited α-glucosidase, and the K m values were 1.10 ± 0.03 and 2.87 ± 0.88 at 10 and 20 g/l, respectively (Table 3).Acarbose showed a mixed-type inhibition of αglucosidase, and its K m and V max values increased as the compound concentration increased.Few recent studies similarly reported acarbose as a mixed-type inhibitor [44,45].Bajpai et al. [46] reported that crude EPS purified from Probio 65 shows inhibition rates of 7.05% at 10 g/l and 18.83% at 25 g/l in the presence of 5 mM substrate.The EPS was used in its crude form in the present study, and it is necessary to purify EPS via further purification and characterization.
In this study, L. brevis L010 isolated from jangajji showed a greater adhesion to Caco-2 cells than LGG.In addition, the strain displayed antimicrobial activity against foodborne pathogens, including B. cereus, E. coli, and S. aureus.It also showed greater tolerance to artificial gastric and bile juice than LGG.The EPS isolated from   L. brevis L010 showed concentration-dependent antioxidant activity, and it competitively inhibited α-glucosidase.These results suggest that L. brevis L010 represents a probiotic candidate, and its EPS is attributed to the probiotic properties observed.The L010 strain was deposited in KCTC with Accession NO.KCTC14151BP.

Fig. 1 .
Fig. 1. α-Glucosidase inhibitory activity of cell-free supernatant ( ■ ) and cell lysates ( □ ) of LAB.All values are averages and standard errors determined from three independent experiments.Different letters indicate a significant difference between averages (p < 0.05).Acarbose was used as a control.ND: not detected.

Fig. 2 .
Fig. 2. DPPH radical-scavenging activity of cell-free supernatant ( ■ ) and cell lysates ( □ ) of LAB.All values are averages and standard errors determined from three independent experiments.Different letters on error bars indicate a significant difference between averages (p < 0.05).Ascorbic acid was used as a control.

Fig. 3 .
Fig. 3. Tolerance of LAB in (A) artificial gastric and (B) bile juices.Relative tolerances were determined by comparing the OD 600 of the strains with that of the L010 strain.All values are averages and standard errors determined from three independent experiments.Different letters on error bars indicate a significant difference between averages (p < 0.05).ND, not detected.

Fig. 5 .
Fig. 5. Phylogenetic relationships of L. brevis L010 with other strains.The relationship was constructed using the 16S rRNA gene sequence and the neighbor-joining method [47].The GenBank accession numbers are shown in parentheses.

Fig. 6 .
Fig. 6.Antioxidant activity of EPS of L. brevis L010.(A) ABTS radical-scavenging activity, (B) DPPH radicalscavenging activity, and (C) FRAP activity.All values are averages and standard errors determined from at least three independent experiments.Different letters on error bars indicate a significant difference between averages (p < 0.05).Ascorbic acid was used as a control.ND, not detected.

Fig. 7 .
Fig. 7. Effects of (A) temperature (at pH 6) and (B) medium acidity (at 30°C) on the specific growth rates of L. brevis L010.All values are averages and standard errors determined from three independent experiments.Different letters on error bars indicate a significant difference between averages (p < 0.05).ND: not detected.

Table 3 . Kinetic constants of α-glucosidase reaction inhibited by EPS from L. brevis L010*.
Averages and standard errors determined from three independent experiments are shown.Different letters indicate a significant difference between averages (p < 0.05). *