In Vitro Assessment of Probiotic Properties for Lactic Acid Bacteria Isolated from Korean Traditional Fermented Food, Kimchi

Homemade kimchi was collected and serial dilutions were made in saline. The saline mixtures were spread on de Man, Rogosa and Sharpe (MRS) agar followed by overnight incubation at 30°C. To obtain a pure colony, a single representative colony was transferred to a new MRS agar plate at least three times. The pure isolates were preserved at –80°C in 25% (w/v) glycerol suspension for long-term storage.

To determine the tolerance level of isolated LAB in low pH conditions and bile salt conditions we used the method described by Pieniz et al. (2004) with slight modifications. An overnight culture of potential probiotics was centrifuged at 12,000 ×g for 5 min at 4°C. The cell pellet washed with 0.85% NaCl (w/v) solution and suspended in 0.85% NaCl (w/v) solution adjusted to pH 2.0, prior to being incubated at 37°C for 2 h. After incubation viable cell count was determined on MRS agar plate. For bile salt tolerance, a similar protocol was followed. Cell pellet was dissolved in 0.85% NaCl (w/v) solution supplemented with 1.5% and 2.0% bile salt (BD) and further incubated at 37°C for 4 h. After incubation the cell viable count was determined on MRS agar plate. The strains with acid and bile salt tolerance were primarily selected and the following experiments were conducted on the selected strains.

BATH test was performed according to an established protocol with simple modifications (Collado et al., 2007). Bacterial cells were washed with 0.85% NaCl (w/v) solution and re-suspended in the same solution. Initial OD₆₀₀ adjusted to 0.2 to 0.3. One milliliter of toluene was added to bacterial cells and vortexed well for about 5 min, prior to being incubated for 1 h at room temperature. When two distinct phases were observed, the aqueous phase was removed and the reading was noted. BATH% was calculated as follows:

where A and A₀ represent absorbance readings after and before mixing with toluene.

The antibiotic susceptibilities of strains isolated from kimchi were tested using paper disc diffusion methodology (Bauer et al., 1996). Overnight culture of isolates was spread onto the MRS agar plate and discs containing specific antibiotic concentrations were placed onto them. The plates were incubated overnight at 37°C for 24 h and the inhibition zones were identified. Antibiotic concentrations used in this experiment were as follows: Ampicillin (100 μg/mL), ciprofloxacin (10 μg/mL), erythromycin (25 μg/ mL), gentamicin (30 μg/mL), lincomycin (15 μg/mL), novobiocin (10 μg/mL), streptomycin (50 μg/mL) and tetracyclin (30 μg/mL).

The isolates were inoculated into 20 mL of MRS broth (n=3) and incubated overnight at 37°C. After centrifugation (15,000 ×g for 10 min at 4°C), the scavenging activity of the isolates was determined using the supernatant. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was added to the supernatant and the antioxidant activity was quantified using a colorimetric microplate assay described by Kim et al. (2019). Prepared DPPH solution (0.2 mM in ethanol; Sigma Aldrich, USA) was mixed with bacterial supernatant. The reaction mixture was incubated in the dark at room temperature for 30 min. The DPPH radical scavenging activity was then detected by measuring the absorbance at 517 nm using a microplate reader. The scavenging ability was calculated as follows:

Identification of isolates was performed at the 16S rRNA gene sequencing facility of Macrogen Co., Ltd. (Seoul, Korea). The obtained DNA sequence was analyzed by using BLAST program provided by GenBank (https://www.ncbi.nlm.nih.gov/) and EzBioCloud server (http://www.ezbiocloud.net/) (Yoon et al., 2017) for phylogenetic analysis. Phylogenetic analysis was conducted by MEGA 7 (Kumar et al., 2016) after a gap deletion and multiple alignments of data via the CLUSTAL W program (Thompson et al., 1994). The evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1980), and neighbor-joining (NJ) algorithm was used to construct the phylogenetic trees.

Thirty-eight isolates of LAB isolated from kimchi were tested for their abilities to resist high acidity at the low pH of 2.0 and respective bile salt concentrations of 1% and 2% (w/v). All isolates were observed to have survival rates above 75% with tolerance to 1% and 2% bile salt (w/v), and of the isolates that were screened for tolerance to pH 2.0, twenty-two had an adequate survival rate of between 20 and 60% (Table 1). The bile and pH tolerance of microbiota within the gastric juice are considered among the main factors affecting the survival of probiotic bacteria upon passage through the stomach to the intestine. Interestingly, all of the strains showed high survival rate in bile salts at 1% and 2%, particularly the strain KCCP 11349, which also presented adequate viability in pH 2 and was comparable to the reference strain. Collectively, twenty-two strains of LAB recorded probiotic potential and were selected for further study.

The selected strains on the basis of their survival under in vitro GIT conditions, were evaluated for their hydrophobicity toward toluene that may reflect the colonization potential of the organism in the intestinal lumen. The degree of hydrophobicity was high for KCCP 11356 with the highest hydrophobicity value of 98.8% (Table 2). This value was significantly higher than Lactobacillus rhamnosus GG (82.8%). The hydrophobicity of the outer surface of microorganisms has been implicated in the attachment of bacteria to host tissue. This property may provide an important competitive advantage in maintaining bacteria in the human gastrointestinal tract (Singh et al., 2012). Several tested strains exhibited weak hydrophobicity values for toluene, while hydrophobicity values of KCCP 11349 and KCCP 11356 presented strong hydrophobicity (63.9± 0.6 and 98.8±0.3%, respectively). The variation in hydrophobicity has been reported in other probiotic strains and has been elucidated by the fact that attachment depends upon the origin of the strains as well as their surface properties (Abushelaibi et al., 2017).

The selected strains were resistant to erythromycin but only four strains were resistant to novobiocin (Table 3).

While the resistances to ampicillin and gentamicin was similar to that of the LAB isolates reported previously (Argyri et al., 2013), the other antibiotics showed different patterns for each strain.

The DPPH-scavenging activity of the strains is shown in Table 4. With the exception of JK226, the potential probiotic strains had a higher antioxidant potential when compared to the reference strain (L. rhamnosus GG). The maximum DPPH radical inhibition activity was observed the strain KCCP 11303 (86.2±1.4). From the above results we could conclude that of the antioxidant potential of probiotic strains depends on the type of strain. These results imply that the metabolic products of LAB might be contributing to the higher antioxidant potential.

Twenty-two potential probiotics were identified by 16S rRNA gene sequence. The isolates underwent molecular phylogeny analysis and phylogenic tree construction to identify the LAB to a species level based on the 16S rDNA sequences from evolutionary distances by neighbor-joining method. The phylogenetic tree of the 22 isolates with reference strain is shown in Fig. 1. Almost all the isolates belonged to the genera Pediococcus and Lactobacillus, while some belonged to Leuconostoc and Weissella (Fig. 1).

Fig. 1. Neighbor-joining phylogenetic tree, based on the 16S rRNA gene sequences, showing the position and relationship of strains isolated from kimchi with probiotic potential. Numbers at nodes indicate bootstrap values (>70%) calculated based on the NJ algorithms for the branch point based on 1000 replications. Bar, 0.01 substitutions per nucleotide position.

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