• International Journal of Oral Biology
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• 제46권3호
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• pp.146-153
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• 2021

Accurate identification of microbes facilitates the prediction, prevention, and treatment of human diseases. To increase the accuracy of microbiome data analysis, a long region of the 16S rRNA is commonly sequenced via paired-end sequencing. In paired-end sequencing, a sufficient length of overlapping region is required for effective joining of the reads, and high-quality sequencing reads are needed at the overlapping region. Trimming sequences at the reads distal to a point where sequencing quality drops below a specific threshold enhance the joining process. In this study, we examined the effect of trimming conditions on the number of reads that remained after quality control and chimera removal in the Illumina paired-end reads of the V3-V4 hypervariable region. We also examined the alpha diversity and taxa assigned by each trimming condition. Optimum quality trimming increased the number of good reads and assigned more number of operational taxonomy units. The pre-analysis trimming step has a great influence on further microbiome analysis, and optimized trimming conditions should be applied for Divisive Amplicon Denoising Algorithm 2 analysis in QIIME2 platform.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.856-867
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• 2017

Supplement of dietary fibers (DF) is regarded as one of the most effective way to prevent and relieve chronic diseases caused by long-term intake of a high-fat diet in the current society. The health benefits of soluble dietary fibers (SDF) have been widely researched and applied, whereas the insoluble dietary fibers (IDF), which represent a higher proportion in plant food, were mistakenly thought to have effects only in fecal bulking. In this article, we proved the anti-obesity and glucose homeostasis improvement effects of IDF from pear pomace at first, and then the mechanisms responsible for these effects were analyzed. The preliminary study by real-time PCR and ELISA showed that this kind of IDF caused more changes in the gut microbiota compared with in satiety hormone or in hepatic metabolism. Further analysis of the gut microbiota by high-throughput amplicon sequencing showed IDF from pear pomace obviously improved the structure of the gut microbiota. Specifically, it promoted the growth of Bacteroidetes and inhibited the growth of Firmicutes. These results are coincident with previous hypothesis that the ratio of Bacteroidetes/Firmicutes is negatively related with obesity. In conclusion, our results demonstrated IDF from pear pomace could prevent high-fat diet-induced obesity in rats mainly by improving the structure of the gut microbiota.

• 한국식품과학회지
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• 제50권4호
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• pp.400-406
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• 2018

본 연구는 국내 수도권, 충청, 강원 등지의 중소기업형 소규모 양조장에서 생산되는 탁주와 약주의 미생물 군집 분포를 차세대 염기서열 분석기법을 이용하여 조사하였다. 각 시료별로 미생물 분포에 따른 품질의 차이를 나타냈으며 특히 백미 또는 소맥분을 주로 사용한 탁주와 달리 약주는 찹쌀과 백미를 주원료로 사용하여 각각 독특한 주질과 미생물 분포에 영향을 주었다. 주요검출 미생물로 진균류는 S. cerevisiae가 대부분을 차지하였으며 세균의 경우 Firmicutes문에는 유산균인 Lactobacillus, Leuconostoc, Lactococcus, Weisella 속 등이 우점종으로 확인되었다. 곡물인 원재료에 의해 Cyanobacteria 문의 Chloroplast 속과 제조 환경에 의한 유입으로 추정되는 Cronobacter 속, Enterobacter 속 또한 검출되었다. 이러한 다양한 미생물의 분포는 제조 지역, 주원료, 그리고 제조 방법에 기인할 것으로 판단되며 명확한 상관관계는 확인할 수 없었으나 본 연구 결과 유통과정의 제품 안전성 확보를 위해 양조장별 미생물 관리가 필요할 것으로 판단된다.

• Journal of Animal Science and Technology
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• 제63권3호
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• pp.640-650
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• 2021

Particulate matter (PM) produced in pig houses may contain microbes which can spread by airborne transmission, and PM and microbes in PM adversely affect human and animal health. To investigate the microbiome in PM from pig houses, nine PM samples were collected in summer 2020 inside and outside of pig houses located in Jangseong-gun, Jeollanam-do Province, Korea, comprising three PM samples from within a nursery pig house (I-NPH), three samples from within a finishing pig house (I-FPH), and three samples from outside of the pig houses (O-PH). Microbiomes were analyzed using 16S rRNA gene amplicon sequencing. Firmicutes was the most dominant phylum and accounted for 64.8%-97.5% of total sequences in all the samples, followed by Proteobacteria (1.4%-21.8%) and Bacteroidetes (0.3%-13.7%). In total, 31 genera were represented by > 0.3% of all sequences, and only Lactobacillus, Turicibacter, and Aerococcus differed significantly among the three PM sample types. All three genera were more abundant in the I-FPH samples than in the O-PH samples. Alpha diversity indices did not differ significantly among the three PM types, and a principal coordinate analysis suggested that overall microbial communities were similar across PM types. The concentration of PM did not significantly differ among the three PM types, and no significant correlation of PM concentration with the abundance of any potential pathogen was observed. The present study demonstrates that microbial composition in PM inside and outside of pig houses is similar, indicating that most microbe-containing PM inside pig houses leaks to the outside from where it, along with microbe-containing PM on the outside, may re-enter the pig houses. Our results may provide useful insights regarding strategies to mitigate potential risk associated with pig farming PM and pathogens in PM.

• Journal of Animal Science and Technology
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• 제64권5호
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• pp.897-910
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• 2022

Bovine fecal microbiota is important for host health and its composition can be affected by various factors, such as diet, age, species, breed, regions, and environments. The objective of this study was to evaluate the impact of diet and gender on fecal microbiota in Korean native Hanwoo cattle. The 16S rRNA gene amplicon sequencing of fecal microbiota was conducted from 44 Hanwoo cattle divided into four groups: (1) 11 heifers fed an oat hay plus total mixed ration (TMR) diet for breeding (HOTB), (2) 11 heifers fed an early fattening TMR diet (HEFT), (3) 11 steers fed the early fattening TMR diet (SEFT), and (4) 11 steers fed the late fattening TMR diet (SLFT). Firmicutes and Bacteroidota were the first and second most dominant phyla in all the samples, respectively. The Firmicutes/Bacteroidota (F/B) ratio associated with feed efficiency was significantly greater in the SLFT group than in the other groups. At the genus level, Romboutsia, Paeniclostridium, and Turicibacter were the most abundant in the SLFT while Akkermansia, Bacteroides, and Monoglobus were the most abundant in the HOTB group. Although the same early fattening TMR diet was fed to Hanwoo heifers and steers, Marvinbryantia and Coprococcus were the most abundant in the HEFT group while Alistipes and Ruminococcus were the most abundant in the SEFT group. Shannon and Simpson diversity indices were significantly lower in the SLFT group than in the other groups. Distribution of fecal microbiota and functional genetic profiles were significantly different among the four treatment groups. The present study demonstrates that different diets and genders can affect fecal microbiota and the F/B ratio may be associated with feed efficiency in Hanwoo cattle. Our results may help develop strategies to improve gut health and productivity through manipulation of fecal microbiota using the appropriate diet considering Hanwoo cattle gender.

• 미생물학회지
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• 제39권1호
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• pp.45-50
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• 2003

분자 생물학적 방법 인 DGGE를 이용하여 저온에서 김치가 발효되는 동안 관여하는 미생물의 다양성과 변화양상을 분석하였다. 김치를 저온 ($4^{\circ}C$)에서 발효시키는 60 일 동안 5 일 마다 시료를 채취하였으며, 채취한 김치 시료에서 genomic DNA를 추출하여 실험을 수행하였다. 김치 시료 genomic DNA로부터 16S rDNA의 V3영역을 증폭하여 DGGE를 수행한 결과에서 관찰된 amplicon들의 염기서열을 분석한 결과 저온에서 김치가 발효되는 동안 젖산균들이 주요 미생물 군집으로 나타났으며, 그 중에서도 Weissella koreensis가 발효 전 과정 동안, Lactobacillus sakei의 경우는 발효 10 일째부터, 그리고 Leuconostoc gelidurn은 발효30 일째부터 amplicon들의 농도가 진하게 나타나 이들이 저온에서 김치 발효 과정 동안의 우점종 균주들 임을 알 수 있었다.

• 대한의생명과학회지
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• 제27권4호
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• pp.255-263
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• 2021

Human Astrovirus (HuAstV) is an important gastrointestinal pathogen that is frequently reported worldwide. Monitoring of contaminated groundwater has been suggested since HuAstV is transmitted through the fecal-oral route. This study developed a test method based on conventional reverse transcription (RT)-nested polymerase chain reaction (PCR) that involves SL^® non-specific reaction inhibitor for unknown non-specific amplification taking place in the groundwater environment. An optimal method for detecting HuAstV in groundwater sample through analysis and comparison against conventionally reported method was also suggested. The developed method enabled the production of nested PCR amplicon of 630 nt, which is a sufficient length for similarity analysis based on sequencing and genotyping. Amplicons suspected to be HuAstV were amplified in two out of the twenty groundwater samples collected in Korea, presenting 99.77% and 99.73% similarity against HuAstV 1 strain lhar/2011/kor (JN887820.1) in sequencing, respectively. These amplicons were identified as HuAstV 1.

• 한국축산식품학회지
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• 제44권2호
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• pp.390-407
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• 2024

Kuflu cheese, a popular variety of traditional Turkish mold-ripened cheeses, is characterized by its semi-hard texture and blue-green color. It is important to elucidate the microbiota of Kuflu cheese produced from raw milk to standardize and sustain its sensory properties. This study aimed to examine the bacteria, yeasts, and filamentous mold communities in Kuflu cheese using high-throughput amplicon sequencing based on 16S and ITS2 regions. Lactococcus, Streptococcus, and Staphylococcus were the most dominant bacterial genera while Bifidobacterium genus was found to be remarkably high in some Kuflu cheese samples. Penicillium genus dominated the filamentous mold biota while the yeasts with the highest relative abundances were detected as Debaryomyces, Pichia, and Candida. The genera Virgibacillus and Paraliobacillus, which were not previously reported for mold-ripened cheeses, were detected at high relative abundances in some Kuflu cheese samples. None of the genera that include important food pathogens like Salmonella, Campylobacter, Listeria were detected in the samples. This is the first experiment in which the microbiota of Kuflu cheeses were evaluated with a metagenomic approach. This study provided an opportunity to evaluate Kuflu cheese, which was previously examined for fungal composition, in terms of both pathogenic and beneficial bacteria.

• 생명과학회지
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• 제27권5호
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• pp.524-529
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• 2017

본 연구에서는 zebrafish에 인간의 KCNE1 유전자가 삽입된 형광단백질 vector를 microinjection하고, 그 발현 여부를 확인하고자 하였다. 먼저 양 말단에 제한효소(EcoRΙ, BamHΙ) site를 넣어 제작한 primer들로 genomic DNA에서 KCNE1 유전자를 분리하였다. 그 결과는 약 402 bp 크기의 DNA band였고 이 PCR 산물을 형광단백질 vector인 pPB-CMVp-EF1-GreenPuro 속에 클로닝하여 pPB-CMVp-hKCNE1-EF1-GreenPuro plasmid를 제작하였다. 이렇게 준비된 형광 vector를 zebrafish 수정란에 microinjection하였고, 부화된 치어에서 RT-PCR과 DNA sequencing을 통해 GFP 및 hKCNE1의 발현을 최종 확인하였다. 본 연구는 향후 QT 연장증후군(LQTs)에 대한 동물 모델로써 신경자극 전도, 유전자 치료, 유용 유전자 클로닝을 위한 기술 개발에 응용될 수 있을 것으로 기대된다.

• Journal of Korean Medical Science
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• 제33권52호
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• pp.337.1-337.14
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• 2018

Background: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. Methods: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. Results: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. Conclusion: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.