• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.280-289
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• 2021

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

• Food Science of Animal Resources
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• v.23 no.3
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• pp.269-277
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• 2003

This study was carried out to evaluate the safety of Korean human milk. The microorganisms were identified from human milk of 149 healthy mothers by two collection methods, hand and pump expression. The means of total bacterial counts were 2.33x10$^4$ cfu/mL on the samples collected by the pump expression and 7.83xl0$^3$ cfu/mL on those collected by the hand expression. Therefore, the total bacterial counts of pump expression samples was 9.80xl0$^2$∼3.06x10$^4$ cfu/mL more than that of hand expression samples. The coliform counts of pump expression was 9.36xl0$^3$∼8.57xl0$^4$ cfu/mL more than that of hand expression. However, there was any significant differences of the lactic acid bacterial counts between the two samples collected by each methods. 100 strains of 5 patterns of total bacterial counts were isolated based on the morphology of colony in the standard plate count agar. 13 species were identified among the isolated strains. The dominant species in Korean human milk were Staphylococcus which 7 subspecies identified(81% in the rate of total bacteria, 1.07x10$^4$ cfu/mL). Other species identified were Micrococcus, Bacillus, Providencia, Pseudomonas, Yersinia and Acinetobacter. 36 strains of 6 patterns of lactic acid bacterial counts were isolated based on morphology of colony in the BCP agar. 7 species were identified among the isolated strains. The dominant species of lactic acid bacteria in Korean human milk were Lactobacillus brevis(50.9% in the rate of lactic acid bacteria, 4.72xl0$^4$ cfu/mL). Others species identified(49.1% lactic acid bacteria) were Lactobacillus curvatus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus acidophilus, Leuconostic lactis and Streptococcus salivarius subsp. thermophilus.