Probiotic properties of novel probiotic Levilactobacillus brevis KU15147 isolated from radish kimchi and its antioxidant and immune-enhancing activities

Abstract

This study was conducted to evaluate the probiotic properties and antioxidant activities of lactic acid bacteria strains including Levilactobacillus brevis KU15147 isolated from kimchi to determine their potential as a probiotic. The tolerance of all strains to gastric acid and bile salts was more than 90%. The strains did not produce a β-glucuronidase and survived following treatment with gentamicin, kanamycin, streptomycin, and ciprofloxacin. L. brevis KU15147 showed greater adhesion activity to HT-29 cells (6.38%) and its antioxidant activities were higher than those of other tested strains, showing values of 38.56%, 22%, and 23.82% in DPPH, ABTS, and β-carotene bleaching assays, respectively. Additionally, the relative expression intensities of induced nitric oxide synthase and tumor necrosis factor-α of L. brevis KU15147 were greater than those of other strains, suggesting that this strain can be applied in the health food or pharmaceutical industry as a novel probiotic strain.

Introduction

Consumers are becoming increasingly interested in functional foods that contain natural ingredients for their health benefits and disease prevention effects (Choi et al., 2018; Shori, 2016). Probiotics are commonly defined as living microorganisms that have various health benefits through symbiotic associations with the intestinal microflora (Silva et al., 2017). Lactic acid bacteria (LAB) are considered as representative probiotics and have gained attention for preventing or treating various human diseases or disorders. Accordingly, many studies have reported that LAB-mediated therapy was helpful for diseases such as foodborne illness-related diarrhea, irritable bowel syndrome, and immune malfunction (Feleszko et al., 2007; Kim et al., 2020).

However, to be applied as probiotics, microbes should be non-virulent and non-pathogenic to human (Lee et al., 2012; 2019). They should also survive under the digestive conditions of the intestinal tract in the presence of low pH and high concentrations of bile salts, as well as adhere to intestinal epithelial cells (Lee et al., 2015). Probiotics strains have various biofunctional activities such as antioxidant, anti-inflammatory, immune-enhancing, anticancer, and hypolipodemic effects (Hills et al., 2014) as well as antimicrobial activities (Li et al., 2012). Moreover, probiotics have been reported to produce short-chain fatty acids, which may modulate the intestinal mobility and have beneficial effects on colon inflammation and disorders of the neuronal system (Unger et al., 2016). Therefore, the characteristics and biofunctional activities of probiotics are very important for their practical application as functional food materials (Yu et al., 2019). However, the probiotic characteristics and activities differ by strain.

Kimchi is a representative traditional Korean food manufactured by naturally fermenting vegetables using various natural microorganisms including LAB strains. Major LAB strains isolated from kimchi include Lactobacillus spp., Leuconostoc spp., Lactococcus spp., and Pediococcus spp. (Son et al., 2017).

The aims of this study were to screen several LAB from kimchi, to evaluate the probiotic characteristics of isolated LAB strains, and to investigate the antioxidant activities and immune-enhancing activity of these strains as a functional food material in vitro. Our results may facilitate the development of new fermented cereal beverages in the food industry.

Materials and methods

Isolation and identification of LAB strains

The strains used in this study were screened and isolated from home-made radish kimchi. After diluting the liquid part of kimchi sample, 1 mL was serially diluted and spread onto de Man, Rogosa, and Sharpe (MRS; BD Biosciences, Franklin Lakes, NJ, USA) agar and incubated at 37 °C for 24 h. An isolated colony was inoculated into MRS broth at 37 °C for 24 h. Four potential probiotic LAB strains were selected and coded as KU15147, KU15148, KU15154, and KU15156. These strains were identified by 16S rRNA sequencing performed by Bionics, Inc. (Seoul, Korea). The sequencing results were analyzed using the GenBank database with the Basic Local Alignment Search Tool website (http://blast.ncbi.nlm.nih.gov). The KU15147, KU15148, and KU15154 strains were identified as Levilactobacillus brevis and the KU15156 strain was identified as Lactobacillus plantarum. Lactobacillus rhamnosus GG (LGG, KCTC 12202BP) from the Korean Collection for Type Cultures (KCTC; Daejeon, Korea) was used as a reference strain to evaluate antioxidant and immune-enhancing activities.

Resistance to artificial gastric juice and bile salts

The resistance of the isolated strains to artificial gastric juice and bile salts was evaluated as described by Guo et al. (2012) with some modifications. The resistance to artificial gastric juice was determined by incubation of the strains in 50 mM sodium carbonate buffer (pH 2.5) containing 0.3% (w/v) pepsin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 3 h. Resistance to bile salts was assessed after incubation in MRS broth containing 0.3% (w/v) Oxgall (BD Biosciences) at 37 °C for 1 day. The survival rate (%) of the strains treated with artificial gastric juice or bile salts was calculated by counting the surviving cells on the MRS plates compared to the initial cell numbers before incubation as follows:

$$\text{Survival rate}\left(\%\right)=\frac{{\text{Log N}}_1}{{\text{Log N}}_2}\times 100$$

where N₁ and N₀ are the total counts of viable cells after treatment with artificial gastric juice or bile salts and viable cells before treatment, respectively.

Adhesion activity to HT-29 cells

HT-29 cells (KCLB 30038, human colon adenocarcinoma cell) were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) to assess the adhesion activity of the four strains as described by Yang et al. (2019). HT-29 cells (2 × 10⁵ cells/mL) were incubated in a 24-well plate at 37 °C for 1 day. The intact cells of each strain at 1 × 10⁷ CFU/mL were mixed with HT-29 cells. After incubation at 37 °C for 2 h, the cell culture plate was washed three times with phosphate-buffered saline (Gibco, Grand Island, NY, USA) to removed non-adherent bacterial cells. All adherent cells were harvested using 1% Triton X-100 (Sigma-Aldrich). After dilution with 0.1% peptone solution, the cells were cultured on MRS agar at 37 °C for 24 h. Adhesion activity was defined as follows:

$$\text{Adhesion activity}\left(\%\right)=\frac{V_1}{V_0}\times 100$$

where V₀ and V₁ are numbers of initial cells and viral adherent cells to HT-29 cells, respectively.

Enzyme production

The API ZYM^® kit (BioMérieux, Marcy-l’Étoile, France) was used to assess the types of enzymes produced by the strains. Each strain was suspended in phosphate-buffered saline at a concentration of 10⁶ CFU/mL. The cell samples were added to each cupule and incubated at 37 °C for 4 h and added with one drop of each ZYM test reagent (A and B). The mixtures were inoculated more according to the manufacturer’s instructions. The value of nM means an approximate amount of each produced enzymes and the level of enzyme activity was evaluated by scoring the samples as 0–5 based on the degree of color changes according to company’s manual.

Antibiotic susceptibilities

The susceptibility of the strains to various antibiotics was determined by using the disk diffusion method (Yang et al., 2019). Firstly, 100 μL of 7-h cultured broth of each strain was spread onto MRS agar. Paper disks soaked in different antibiotics solutions were then placed on the MRS agar and incubated at 37 °C for 24 h. The diameter (mm) of the clear zone of each antibiotic disc was measured and compared with standard values established by the Clinical and Laboratory Standards Institute. The eight antibiotics and their concentrations were as follow: ampicillin (0.2 g/L), chloramphenicol (0.6 g/L), ciprofloxacin (0.1 g/L), doxycycline (0.6 g/L), gentamicin (0.2 g/L), kanamycin (0.6 g/L), streptomycin (0.2 g/L), and tetracycline (0.6 g/L).

Antioxidant activities

2,2,-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

DPPH radical scavenging activity was performed as described by Das and Goyal (2015 ) with some modifications. Firstly, 2 mL of 0.4 mM DPPH in methanol was added to 2 mL of cultured broth samples. The negative control was distilled water and the positive control was ascorbic acid (1.0 mg/mL). The mixtures were reacted at 37 °C for 30 min in the dark. DPPH radical scavenging activities were determined by measuring the absorbance of the reactant of sample and control at 517 nm and using the following equation:

$$\text{DPPH radical scavenging activity}\left(\%\right)=\left(1-\frac{{\text{A}}_{\text{sample}}}{{\text{A}}_{\text{control}}}\right)\times 100\%$$

where A_sample and A_control are the absorbance values of the sample and control, respectively.

2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging activity

ABTS radical scavenging activity was evaluated by a modified method reported by Verón et al. (2017). First, 14 mM ABTS and 5 mM potassium persulfate were dissolved in 0.1 M potassium phosphate buffer (pH 7.4) and diluted until the absorbance at 734 nm was 0.7 ± 0.02. In next, 150 μL of sample or control (distilled water) was mixed with the same amount of ABTS solution and incubated at 37 °C for 10 min. Ascorbic acid (1.0 mg/mL) was used as a positive control. ABTS radical scavenging activity was calculated by measuring the absorbances at 734 nm and using the following equation:

$$\text{ABTS radical scavenging activity}\left(\%\right)=\left(1-\frac{{\text{A}}_{\text{sample}}}{{\text{A}}_{\text{control}}}\right)\times 100\%$$

where A_sample and A_control are the absorbance values of the sample and control, respectively.

β-Carotene bleaching assay

The β-carotene bleaching assay was performed by using a modified method of Kassim et al. (2019). Firstly, 132 μL of linoleic acid, 6 mg of β-carotene, and 600 μL of Tween 80 in 20 mL of chloroform were mixed and used as β-carotene solution. Chloroform in the solution was evaporated with a rotary vacuum evaporator. The solution was mixed with distilled water until that the absorbance of the solution at 470 nm was 1.20. A 200-μL sample was dissolved in distilled water, mixed with 4 mL of β-carotene solution, and reacted in a water bath at 50 °C for 2 h. Ascorbic acid (1.0 mg/mL) was used as a positive control. The absorbance of the samples was determined at 470 nm after 0- and 2 h-reactions and the inhibitory activities on β-carotene and linoleic acid oxidation were calculated as follows:

$$\text{Inhibition on}\beta -\text{carotene and linoleic acid oxidation}\left(\%\right)=\frac{{\text{A}}_{\mathrm{sample},2\text{h}}-{\text{A}}_{\mathrm{control},2\text{h}}}{{\text{A}}_{\mathrm{control},0\text{h}}-{\text{A}}_{\mathrm{control},2\text{h}}}\times 100\%$$

where A_{sample, 2 h} and A_{control, 2 h} are the absorbance values of the sample and control after the 2 h-reaction, respectively. A_{control, 0 h} is the absorbance of distilled water at the initial reaction time.

Immune-enhancing effect

Nitric oxide (NO) production on RAW 264.7 cells

RAW 264.7, a murine macrophage cell line, was obtained from the Korean Cell Line Bank (code no.: KCLB 40071, Seoul, Korea) and cultured in Dulbecco’s Modified Eagle Medium (Hyclone, Logan, UT, USA) containing fetal bovine serum (10%, v/v) and streptomycin/penicillin (1%, v/v) (Hyclone). The amount of nitric oxide in the cell culture medium was determined as described by Lee et al. (2015). RAW 264.7 cells (2 × 10⁵ cells/well) were plated into a 96-well plate and mixed with 10 ng/mL of lipopolysaccharide (LPS) and the sample strains (1 × 10⁵ CFU/mL) and incubated for 24 h. Next, 100 μL of the cell culture medium was mixed with the same amount of Griess reagent. Nitric oxide (NO) production by the samples was determined by measuring the absorbance of the mixtures at 540 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) and sodium nitrite (NaNO₂) used as a standard.

Immune-enhancing activity

The immune-enhancing activity of the strains was evaluated by using a modified method reported by Chang et al. (2015). RAW 264.7 cells (1 × 10⁶ cells/mL) were cultured in a 6-well plate for 24 h and then the sample strains (1 × 10⁵ CFU/mL) with or without LPS (10 ng/mL) were added and incubated for 1 day. Total mRNA of RAW 264.7 cells was separated with an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The expression levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α were determined by using SYBR Green PCR Master mix reagent with semi-quantitative real-time PCR (PikoReal 96, Pierce, Rockford, IL, USA). The following primers were used:

iNOS sense: 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3‘

iNOS antisense: 5′-GGCTGTCAGAGTCTCGTGGCTTTGG-3′

TNF-α sense: 5′-GCAGAAGAGGCACTCCCCCA-3′

TNF-α antisense: 5′-GATCCATGCCGTTGGCCAGG-3′

Semi-quantitative real-time PCR was performed as follows: polymerase activation at 95 °C for 2 min, denaturation 95 °C for 5 s, and annealing/extension at 60 °C for 15 s. Denaturation and annealing/extension were performed for 40 cycles The ΔΔCq method was used to analyze the PCR results, and the reaction specificity was determine by analyzing the melting curve.

Statistical analysis

All experiments were performed in triplicate, and significant differences among samples were determined by one-way analysis of variance. Duncan’s multiple range tests were performed using SPSS software (version 24; SPSS, Inc., Chicago, IL, USA).

Results and discussion

Tolerance to artificial gastric juice and bile salts

In general, food substances remain in the stomach for approximately 3 h and are digested under the strong acidic conditions of pH 1.0–4.5 (Guo et al., 2009). The human intestinal tract is a weakly basic environment in which the concentration of bile salts is 0.3% (w/v) (Jeon et al., 2016). Under these conditions, probiotics should survive as much as possible when the living cells pass from mouth to colon. As shown in Table 1, three probiotic strains (L. brevis KU15147, KU15148, and KU15154) showed survival rates of over 99% in artificial gastric juice, with no significant differences among these strains (p > 0.05), but the survival rate of L. plantarum KU15156 was slightly decreased.

Table 1.

Artificial gastric juice and bile salt tolerance (%) and adhesion ability (%) of the strains

Strains	Survival rate (%)		Adhesion ability (%)
	Gastric acid tolerance (0.3% pepsin, pH 2.5)	Bile salt tolerance (0.3% oxgall)
L. brevis KU15147	98.94 ± 1.04a1)	105.99 ± 0.87a	6.38 ± 0.19a
L. brevis KU15148	100.34 ± 3.22a	103.78 ± 2.34a	1.21 ± 0.52d
L. brevis KU15154	100.54 ± 3.02a	104.05 ± 0.22a	4.12 ± 0.51b
L. plantarum KU15156	97.23 ± 1.07b	74.63 ± 0.37b	1.79 ± 0.35d
L. rhamnosus GG	97.88 ± 2.04b	100.23 ± 1.87a	2.71 ± 0.25c

^1)a–cDifferent superscript letters in the same column indicate significant differences in each characteristic (p < 0.05). All values are the mean ± standard deviation of triplicate experiments

Following exposure to bile salts, the survival rates of L. brevis KU15147, KU15148, and KU15154 in 0.3% oxgall were over 100%, suggesting that these strains are not affected by bile salts in the intestine. However, L. plantarum KU15156 survived as much as approximately 75% showing that this strain was affected more by bile salts than any other strains tested in this study. These results demonstrate that the growth of all strains tested except for KU15156 was not significantly affected at the concentration of bile salts found in the intestine. Vidhyasagar and Jeevaratnam (2013) found that the survival rate of Pediococcus pentosaceus VJ13 decreased by as much as approximately 50% under human gastric juice conditions (pH 2 for 4 h) and Osmanagaoglu et al. (2010) reported that the number of P. pentosaceus OZF cells was reduced from 8.7 to 7.2 log CFU/mL when this strain was treated with 0.3% oxgall. Some researchers also reported that the cell count of Lactobacillus reuteri L4/1 in artificial gastric juice (pH 2.5) containing 0.3% pepsin and in bile salts (0.3% oxgall, pH 7) was reduced by as much as 84% and 90%, respectively (Bujnakova et al., 2014). Based on these results, the survivals of four strains tested in this study (L. brevis KU15157, KU15158, KU15154, and L. plantarum KU15156) may be affected in the human gastrointestinal condition.

Ability to adhere to HT-29 cells

To exert various bio-functional activities in the intestine, the adhesion to intestinal epithelial cells is an important prerequisite for probiotics (Vidhyasagar and Jeevaratnam, 2013). As shown in Table 1, the adhesion abilities of the four strains to HT-29 cells varied (1.21–6.38%) by strain. L. brevis KU15148 showed a higher adhesion rate of 6.38% than any of the other strains tested in this study, whereas L. brevis KU15154 showed the lowest adhesion ability. Some researchers found that adherence ability of Lactobacillus strains to the gastrointestinal tract was generally as much as 2–10% (Jeon et al., 2016). Lee et al. (2016) reported that the adhesion ability of L. plantarum C182 to HT-29 cells was as high as 1.2% and Monteagudo-Mera et al. (2012) also showed that L. lactis ATCC 11454, L. paracasei 34, L. paracasei ATCC 27092, and L. casei ATCC 393 had adherence rates of 2-4% to Caco-2 cells.

Many researchers have shown that important factors affecting the adhesion ability of microorganisms to epithelium cells in the gastrointestinal tract include the hydrophobicity and auto-aggregation ability of microbial cells, as adhesion between epithelium cells and the bacterial cell surface occurs via electrostatic interactions (Bengoa et al., 2018; Han et al., 2017; Hernández-Alcántara et al., 2018). Specifically, they showed that auto-aggregation is related to the electron charge and some compounds such as proteins (SlpA) and exopolysaccharides on the probiotic cell surface. Some researchers reported that many Lactobacillus spp. have high adhesion ability relatively comparing to other probiotics. For instance, L. brevis R4 and Lactobacillus acidophilus AD1 strongly adhere to the Caco-2 cell surface (Han et al., 2017). The results of measuring the adhesion ability of L. brevis KU15154 to HT-29 cells indicate that this strain can colonize the human gastrointestinal tract.

Enzyme production by LAB strains

To estimate the probiotic properties of the LAB (Bujnakova et al., 2014), metabolite enzyme production was examined and were showed in Table 2.

Table 2.

Analysis of enzyme production by the strains with the API ZYM kit

Enzymes	Enzyme activities1)
	KU151472)	KU15148	KU15154	KU15156
Control	0	0	0	0
Alkaline phosphatase	0	0	0	0
Esterase	1	1	1	0
Esterase lipase	1	1	0	0
Lipase	0	0	0	0
Leucine arylamidase	3	3	3	2
Valine arylamidase	2	2	2	2
Cystine arylamidase	1	1	0	0
Trypsin	0	0	0	0
α-Chymotrypsin	0	1	0	0
Acid phosphatase	1	1	1	0
Naphthol-AS-BI-phosphohydrolase	1	1	1	1
α-Galactosidase	1	1	1	0
β-Galactosidase	4	4	2	2
β-Glucuronidase	0	0	0	0
α-Glucosidase	2	2	1	0
β-Glucosidase	4	4	3	1
N-Acetyl-β-glucosaminidase	0	0	0	2
α-Mannosidase	0	0	0	0
α-Fucosidase	0	0	0	0

¹⁾0, 0 nM; 1, 5 nM; 2, 10 nM; 3, 20 nM; 4, 30 nM; 5, ≥ 40 nM

²⁾KU15147, KU15148, and KU15154 are Levilactobacillus brevis strains and KU15156 is Lactobacillus plantarum strain

According to the results in Table 2, the various enzymes produced showed different productivities according to the strain. All strains produced leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, and β-glucosidase. Particularly, β-galactosidase and β-glucosidase levels were higher than those of the other enzymes in four strains. β-Galactosidase hydrolyzes lactose into glucose and galactose, which is helpful for relieving lactose intolerance (Bujnakova et al., 2014). α-Glucosidase also is a major enzyme that hydrolyzes disaccharides to produce glucose (Son et al., 2017). β-Glucosidase is used mainly in the bioconversion of bioactive glycoside compounds to improve biofunctional activities or increase the absorptivity in the human body (Lee et al., 2016). Specifically, all strains tested in this study may be safe because they lack β-glucuronidase. Generally, microorganisms that produce glucuronidase show limited practical applications because toxic compounds such as carcinogens can bind to glucuronic acid in the liver during detoxification through phase II. Therefore, β-glucuronidase is detrimental to health because toxin complexes (glucuronides) may be dissociated to form glucuronic acid and toxins that are recycled in the body (Ljungh and Wadstrom, 2006).

Susceptibility to antibiotics

As shown in Table 3, all strains were resistant to ciprofloxacin, gentamicin, kanamycin, and streptomycin but showed similar sensitivities to ampicillin, tetracycline, and doxycycline. In particular, L. brevis KU15156 showed slight resistance to tetracycline.

Table 3.

Antibiotic susceptibility of LAB strains

Antibiotics	Strains
	KU151471)	KU15148	KU15154	KU15156
Ampicillin	S2)	S	S	S
Gentamicin	S	S	S	S
Kanamycin	R	R	R	R
Streptomycin	R	R	R	R
Tetracycline	S	S	S	I
Ciprofloxacin	R	R	R	R
Chloramphenicol	S	S	S	S
Doxycycline	S	S	S	S

¹⁾KU15147, KU15148, and KU15154 are Levilactobacillus brevis strains and KU15156 is Lactobacillus plantarum strain

²⁾S, susceptible (> 5 mm); I, intermediate (0–5 mm); R, resistant (not detected)

The susceptibility of LAB strains to antibiotics has been evaluated as a probiotic property, and resistance to antibiotics differs by both strain and even subspecies. Vidhyasagar and Jeevaratnam (2013) reported that P. pentosaceus VJ13 was resistant to ampicillin, ciprofloxacin, gentamicin, and streptomycin, and Osmanagaoglu et al. (2010) showed that P. pentosaceus OZF is resistant to kanamycin. Wang et al. (2018) showed that ten L. brevis strains were resistant to ciprofloxacin, chloramphenicol gentamicin, kanamycin, and streptomycin. The resistance of probiotics to antibiotics is beneficial for antibiotics-remedied patient.

Antioxidant activities

To evaluate antioxidant activities, L. brevis KU15147 and KU15154 were selected as probiotics, as both L. brevis KU15148 and L. plantarum KU15156 showed lower adhesion abilities than these strains. In this study, three methods were used to assess the antioxidant effects of the strains, with the commercial strain L. rhamnosus GG used as a reference strain (Table 1).

The results of the DPPH assay showed that the DPPH radical scavenging activities of L. brevis KU15147 and KU15154 were 38.56 ± 2.03% and 35.19 ± 0.30%, which were significantly higher (p < 0.05) that of L. rhamnosus GG (33.12 ± 2.03%). However, in the ABTS assay, L. brevis KU15147 (22.30 ± 2.22%) exhibited a slightly higher antioxidant effect than two other strains (L. brevis KU15154; 20.57 ± 2.06%, L. rhamnosus; 20.15 ± 1.06%); however, this difference was not significant (p < 0.05) (Table 4).

Table 4.

Antioxidant activities of L. brevis KU15147, L. brevis KU15154, and L. rhamnosus GG

Antioxidant assays	Strains			Ascorbic acid
	L. brevis KU15147	L. brevis KU15154	L. rhamnosus GG
DPPH radical scavenging activity (%)	38.56 ± 2.03a	35.19 ± 0.30b	33.12 ± 2.03c	31.12 ± 1.03c
ABTS radical scavenging activity (%)	22.30 ± 2.22a	20.57 ± 2.06a	21.15 ± 1.06a	20.15 ± 2.06a
β-Carotene bleaching inhibitory activity (%)	23.82 ± 2.91a	21.42 ± 0.47a	19.85 ± 2.06ab	17.85 ± 1.06b

^{2) a–c}Different superscript letters in the same raw indicate significant differences in each characteristic (p < 0.05). All values are shown as the mean ± standard deviation of triplicate experiments

The β-carotene bleaching assay results showed that the inhibition rates of L. brevis KU15147 and L. brevis KU15154 were 23.82 ± 2.91% and 21.42 ± 0.47%, respectively, whereas L. rhamnosus GG (19.85 ± 2.06%) showed lower inhibitory activity. Therefore, L. brevis KU15147 was superior in antioxidant activity to any other two strains tested in this study. In general, many researchers have measured free radical scavenging activity, metal-chelating activity, lipid anti-peroxidation, and antioxidation-related enzyme activities, such as that of superoxide dismutase, to assess the antioxidative effects of probiotics (Han et al., 2017; Tang et al., 2017). These studies showed that the antioxidative compounds of LAB are antioxidative enzymes, such as superoxide dismutase; coenzymes, such as NADH and NADPH; metal ions, such as Mn²⁺, bioactive compounds, such as glutathione; and exopolysaccharides. Particularly, Li et al. (2012) reported that the free radical scavenging ability of probiotic cells is related to components on the cell surface, e.g. lipoteichoic acid, proteins, and exopolysaccharides. Das and Goyal (2015) reported the high DPPH radical scavenging activity of L. plantarum DM5, L. plantarum B-4496, and L. acidophilus B-4495. Han et al. (2017) also showed that the ABTS radical scavenging activity of intact cells of P. pentosaceus R1 and L. brevis R4 was greater than that of the cell-free extract and supernatant. Tang et al. (2017) also showed that intact cells of L. plantarum MA2 strongly inhibited lipid peroxidation. Based on the results of the DPPH assay and β-carotene bleaching assays, L. brevis KU15154 had higher antioxidant ability than the other two strains.

Immuno-enhancing activities

To evaluate the immune-enhancing activity of two strains (L. brevis KU15147 and KU15154) in this study, NO production was determined in RAW 264.7 cells (Fig. 1A). RAW 264.7 cells stimulated with LPS (10 ng/mL) were used as a positive control. All treated strains produced more NO compared to cells not treated with LPS(–).LPS (+) cells and L. brevis KU15147 cells showed higher NO concentrations of 42.18 and 16.38 μM, respectively, compared to cells cultured with L. brevis KU15154 (6.23 μM) and L. rhamnosus GG (7.81 μM). NO generated by macrophage RAW 264.7 cells, iNOS and TNF-α expression was determined (Fig. 1B and 1C). As shown in Fig. 1B, iNOS and NO production by the strains showed similar patterns.

Fig. 1.

Immuno-enhancing effect of L. brevis KU15147, KU15154, and L. rhamnosus GG on RAW 264.7 cells. (A) Nitric oxide(NO) production, (B) relative intensity of iNOS expression, and (C) relative intensity of TNF-α. LPS (–), not treated with lipopolysaccharide; LPS(+), treated with 10 ng/mL lipopolysaccharide. Different letters on each bar indicate significant differences among samples (p < 0.001)

Cytokines such as interleukin (IL)-1β, IL-6, and TNF-α are small proteins (m.w. approximately 25 kD) produced in response to external stimuli. They activate other cells immunologically by binding to specific receptors on the surfaces of other cell surfaces (Parham, 2015). As shown in Fig. 1, these results indicate that the strains exert immune-enhancing effects in the stimulation of RAW 264.7 cells and all samples showed higher cytokine production of iNOS and TNF-α than control cells (not stimulated with LPS). In addition, the relative expression intensities of iNOS and TNF-α of L. brevis KU15147 were greater than those of L. brevis KU15154 and L. rhamnosus GG.

Some researchers showed that NO production and cytokine induction are dependent the concentration and time following treatment with lipoteichoic acid (LTA), and LTAs isolated from LAB induce TNF-α production (Jeong et al., 2015). Therefore, the immune-enhancing activity of these strains may occur through an interaction of LTA on RAW 264.7 cells.

In conclusion, L. brevis KU15147 was isolated from kimchi and showed high resistance to gastric acid and bile salts, as well as the ability to adhere to HT-29 cells, demonstrating that this strain can be used as a probiotic strain. Additionally, L. brevis KU15147 is safe because it does not produce harmful enzymes that negatively affect health and cause antibiotic resistance. The results of three in vitro antioxidant assays showed that L. brevis KU15147 had greater antioxidant activity (radical scavenging activity and inhibitory effect on lipid peroxidation) than the other strains examined in this study. Additionally, L. brevis KU15147 enforces immune activity on macrophages by increasing NO production and the mRNA expression of iNOS and cytokines.

This strain was deposited in Korean Culture Center of Microorganisms (Seoul, Korea) as Lactobacillus brevis KCCM12434P and approved by Ministry of Food and Drug Safety (Korea) as a food gradient (https://www.foodsafetykorea.go.kr/portal/safefoodlife/foodMeterial/foodMeterialDB.do). In our future studies, we will evaluate the biofunctional activity of this strain in clinical tests in animals. Based on our results, L. brevis KU15147 may be used as a novel probiotic in the food or pharmaceutical industry to promote human health.

Compliance with ethical standard

Conflict of interest

The authors declared that they have no conflict of interest.