제품화된 18종 동결건조 프로바이오틱스의 동정을 위한 RAPD-PCR 분석법 평가

The bacterial strains used in this study are listed in Table 1. The bacterial cells were incubated in de Man, Rogosa, and Sharp (MRS) broth (BD Difco, Franklin Lakes, NJ, USA) at 37°C aerobically in a static incubator. Bacillus coagulans IDCC 1201 and Lactococcus lactis IDCC 2301 were incubated in a shaking incubator at 200 rpm and 45°C and 37°C, respectively.

Genomic DNA from cellular and lyophilized form of probiotics was isolated using the Wizard^TM genomic DNA purification kit (Promega, Madison, USA). Briefly, lyophilized probiotics were washed twice with ddH2O to remove excipients prior to DNA isolation. PCR was performed with the universal primers: 5'-ACGCAGGCA-3' (Sesena et al., 2004); 5'-ACGAGGCAC-3' (Tilsala-Timisjarvi and Alatossava, 1998); and 5'-ACGCGCCCT-3' (Johansson et al., 1995). Randomly amplified PCR products were analyzed and visualized using a GelDoc imaging system (Bio-Rad, Hercules, CA). Finally, the products were quantified using Image Lab software (Bio-Rad, version 6.1) for further analysis.

Prior to identification using the RAPD-PCR analysis, identification of eighteen probiotic strains was verified by 16S rRNA sequence analysis (data not shown). Next, three universal primers were evaluated and the most suitable primer was selected. Unique and distinctive band patterns of the eighteen species were clearly observed based on band intensities and gene size (Fig. 1). However, when performing PCR using P1 primer, faint bands were observed for Enterococcus faecium IDCC 2102, Streptococcus thermophilus IDCC 2201, L. lactis IDCC 2301, Bifidobacterium longum IDCC 4101, B. breve IDCC 4401, and Lactobacillus fermentum IDCC 3901 (Fig. 1A). When performing the analysis using P2 primer, it was difficult to distinguish L. salivarius IDCC 3551 from B. bifidum IDCC 4201 due to the absence of a major band. Finally, P3 primer was selected as the suitable primer as it showed significant differences between the eighteen probiotic strains tested in the present study (Fig. 1C). These results are consistent with previous reports which showed differences or similarities among interspecies through RAPD-PCR analysis (Jarocki et al., 2020; Sharma et al., 2020).

Fig. 1. Evaluation of three universal primers for RAPD-PCR analysis. A, P1 (5'-ACGCAGGCA-3'); B, P2 (5'-ACGAGGCAC-3'); C, P3 (5'-ACGCGCCCT-3') primers. The figure is the representative from three independent experiments.

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Based on the RAPD-PCR patterns using primer P3, a blind test for the rapid identification of lyophilized eighteen probiotics was performed. The RAPD-PCR profiles of the lyophilized form of probiotics were consistent with those of the cellular form (Fig. 1C and 2). For example, the two bands (∼2.5 kb and ∼0.3 kb) in lane 3 of sample No. 3 were identified as E. faecium IDCC 2102. One major band (∼1.8 kb) and three minor bands (∼4.0 kb, ∼1.2 kb, and ∼0.8 kb) in lane 12 were identified as L. lactis IDCC 2301. Two major bands (∼3.0 kb and ∼2.7 kb) and one minor band (∼5.0 kb) in lane 16 were identified as L. helveticus IDCC 3801. Finally, the correlation between cellular and lyophilized forms of probiotics was determined bassed on the intensities of the major bands observed only for a specific strain (Fig. 1C and 2) using Image Lab software (Bio-Rad, version 6.1). The coefficient of determination (R²) was 0.91, indicating the RAPD-PCR method used in this study was reliable for the identification of the eighteen lyophilized probiotic strains commercially available (Fig. 3).

Fig. 2. Blind test for identification of eighteen lyophilized probiotics commercially available using RAPD-PCR analysis. The figure is the representative from three independent experiments.

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Fig. 3. orrelation of RAPD-PCR patterns between cellular and lyophilized forms of probiotics. A, P1 (5'-ACGCAGGCA-3'); B, P2 (5'-ACGAGGCAC-3'); C, P3 (5'-ACGCGCCCT-3') primers. The figure is the representative from three independent experiments.

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