[논문]Strain-specific Detection of Kimchi Starter Leuconostoc mesenteroides WiKim33 using Multiplex PCR

Lee, Moeun; Song, Jung Hee; Park, Ji Min; Chang, Ji Yoon

doi:10.7318/kjfc/2019.34.2.208

Strain-specific Detection of Kimchi Starter Leuconostoc mesenteroides WiKim33 using Multiplex PCR 원문보기

韓國食生活文化學會誌 = Journal of the Korean Society of Food Culture, v.34 no.2, 2019년, pp.208 - 216

Lee, Moeun (Advanced Process Technology and Fermentation Research Group, World Institute of Kimchi) , Song, Jung Hee (Advanced Process Technology and Fermentation Research Group, World Institute of Kimchi) , Park, Ji Min (Advanced Process Technology and Fermentation Research Group, World Institute of Kimchi) , Chang, Ji Yoon (Advanced Process Technology and Fermentation Research Group, World Institute of Kimchi)

Abstract ▼ AI-Helper

Leuconostoc spp. are generally utilized as kimchi starters, because these strains are expected to have beneficial effects on kimchi fermentation, including improvement of sensory characteristics. Here, we developed a detection method for verifying the presence of the kimchi starter Leuconostoc mesenteroides WiKim33, which is used for control of kimchi fermentation. A primer set for multiplex polymerase chain reaction was designed based on the nucleotide sequence of the plasmids in strain WiKim33, and their specificity was validated against 45 different strains of Leuconostoc spp. and 30 other strains. Furthermore, the starter strain consistently tested positive, regardless of the presence of other bacterial species in starter kimchi during the fermentation period. Our findings showed that application of a strain-specific primer set for strain WiKim33 presented a rapid, sensitive, and specific method for detection of this kimchi starter strain during natural kimchi fermentation.

주제어

표/그림 (9)

그림 Agarose gel electrophoresis of multiplex PCR products obtained from pure cultures of Leuconostoc mesenteroides WiKim33 in MRS broth. Lane M, size marker (100-bpladder); lane 1, primer 1; lane 2, primer 2; lane 3, primer 3; lane 4, primer 4; lane 5, integrated primers.
그림 Specificity test of the designed primer set. Lane M, size marker (100-bp ladder); lane 1, WiKim33; lanes 2-45, Leuconstoc spp.; lanes 46-76, other strains .
표 Summary^a of bacterial strains used in the PCR specificity test.
그림 Determination of the detection limit of the WiKim33-specific primer set. (A) Dilution with distilled water at the indicated concentrations. Lane M, size marker (100-bpladder); lanes 1-10, 1.8×10⁹ CFU/mL (277 ng of DNA) to 1.8×10⁰ CFU/mL (0.000277 pg of DNA). (B) Diluted with kimchi filtrate (10 ng of DNA for each sample). Lane M, size marker (100-bp ladder); lanes 1-10, kimchi filtrate with 1.8×10⁹ CFU/mL to 1.8×10⁰ CFU/mL of WiKim33.
표 Multiplex PCR primer sets used in this study
그림 Fermentation characteristics of starter kimchi containing Leuconostoc mesenteroides WiKim33. (A) pH profiles and titratable acidity. (B) Total viable bacteria and LAB counts of kimchi according to storage temperature.
그림 Application of kimchi to the WiKim33-specific detection system. (A) Analysis of the dominance ratio by measuring total viable bacteria and WiKim33 counts in kimchi. (B) Application of the multiplex PCR system to starter kimchi.
표 Bacterial strains used in the PCR specificity test
표 Bacterial strains used in the PCR specificity test (continued)

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제안 방법

A total of 189,111 reads (mean read length:11,305 bp) were obtained, and four contigs were formed (1 chromosomal DNA and 3 plasmid DNA). Four pairs of primer sets were designed based on the plasmid DNA sequence of strain WiKim33 using the National Center for Biotechnology Information Primer-Blast Tool (http://www. ncbi.nlm.nih.gov/tools/primer-blast/), and Macrogen, Ltd. (Seoul, Korea) synthesized all primers used in this study. The primer sequences are shown in Table 2 along with their corresponding GenBank accession numbers (CP021492- CP021494) and predicted product sizes.
To evaluate the possibility of applying the strain-specific detection system in the field, kimchi was manufactured and stored at different temperatures. Specific PCR was performed for kimchi, including the WiKim33 strain, and products were stored at 2, 0, 6, and 10℃ for 8 weeks. The physicochemical (pH and acidity) microbial enumeration (total viable bacteria and LAB) showed different fermentation aspects according to fermentation temperature [Supplementary Figure S2].

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