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Development and validation of multiplex PCR assay for differentiating tunas and billfishes
김나예슬,박은지,이서현,문광호,양지영,김중범 한국식품과학회 2021 Food Science and Biotechnology Vol.30 No.4
Commercially available tunas and billfishes aregenerally processed as steaks, making it difficult to visuallydistinguish between the two. We developed and validatedspecies-specific primers to prevent the adulteration of tunasby billfishes. Tunas and billfishes primers were designed onthe cytochrome oxidase subunit I. Multiplex PCR bandsobtained were 579 bp, 291 bp and 114 bp for tunas, billfishesand internal control. Sensitivity was determined to be5 ng for tunas and billfishes. A total of 50 samples weremonitored: 49 for tunas and 1 for billfish. As a result of themonitoring, the fake tunas did not show due to the agreementbetween product name and the raw material of thewrapping paper. Our results indicate that the speciesspecificprimers developed in this study are suitable fordifferentiating tunas and billfishes. The newly developedmultiplex PCR assay is a time and cost effective techniquefor determining the authenticity of tunas and billfishes.
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김나예슬,김채린,김다운,정명인,오광교,김보은,류재기,정지은,전익성,류경열 한국식품위생안전성학회 2021 한국식품위생안전성학회지 Vol.36 No.6
본 연구는 로메인 상추에서 병원성미생물이 생존과 생육의 특성을 분석하여 안전관리 정보를 확보하고자 실시하였다. 로메인 상추에서 분무 접종한 E. coli O157:H7은 72시간 배양 후 초기균수 보다 2.0 log CFU/g 수준으로 증가하여 생존 및 증식이 가능한 것으로 판단되었다. 상추 잎의 상처 유무에 따른 E. coli O157:H7은 배양 72시 간 후 유의적 차이가 없었다. 상추 잎에 인위적인 상처에 내어 E. coli O157:H7을 접종하고 병원균의 분포를 조사한 결과 상처가 없는 상추는 표면이 매끄러워 균이 부착하지 못하거나 균수가 매우 낮았고, 상처가 있는 상추 잎은 거친 표면에 균이 밀집되어 상처를 통해 상추 내부로 침입하는 것으로 판단되었다. 병원성미생물의 상추 추출물 이용 여부는 10-100% 농도에서 배양 24시간 이후에 E. coli O157:H7 8.9 log CFU/mL, L. monocytogenes 8.6 log CFU/mL, P. carotovorum 8.8 log CFU/mL로 나타났다. 이는 병원성미생물과 식물병원균이 유사한 4 log CFU/g 이상의 증가율을 나 타내어 미생물이 상추 추출물을 영양원으로 사용할 수 있는 것으로 판단되었다. 상추 추출물 0.1%에서 초기 접종 농 도와 비교하여 E. coli O157:H7 2.7, L. monocytogenes 1.3, P. carotovorum 2.9 log CFU/mL 수준으로 증가하였다. 이에 따라 병원성 미생물의 최소생육농도는 0.1%보다 낮은 것으로 판단되었고, 상처를 통해 지속적으로 0.1% 수준의 상추 추출물이 병원성미생물에 제공되면 상추 내부에서도 생존 및 증식이 가능할 것으로 확인하였다. The purpose of this study was to provide safety management information by analyzing the survival and growth-related properties of foodborne pathogens from Romaine lettuce. After cultivating E. coli O157:H7 for 72 h on Romain lettuce via spray inoculation, the bacteria population increased by 2.0 log CFU/g from the initial population, confirming the possibility of survival and multiplication of the pathogen thereon. The study also revealed that there is no significant difference in the cultivation of E.coli O157:H7 after 72 h from inoculation on damaged and undamaged lettuce leaves. As a result of investigating distribution of E.coli O157:H7 on damaged lettuce leaves, it was found that the bacteria is unlikely to adhere on the smooth surface of undamaged leaves and, thus, results in a low population density, whereas the bacteria cluster on the rough surface of damaged leaves and easily enter through the damaged tissues. Furthermore, after 24 h of cultivation of the pathogenic microbe in the extract with concentrations of 10-100%, utilization of the lettuce extract by the pathogen was found to be 8.9 log CFU/mL E. coli O157:H7, 8.6 log CFU/mL L. monocytogenes, and 8.8 log CFU/mL P. carotovorum. The increase in the population of both the pathogenic microbe and foodborne pathogen reached over 4 log CFU/mL, implying the microbe can utilize the lettuce extract as a source of nutrition. Compared to the initial inoculation concentration in 0.1% lettuce extract, the final concentration has increased up to 2.7 log CFU/mL E. coli O157:H7, 1.3 log CFU/mL L. monocytogenes, and 2.9 log CFU/ mL P. carotovorum. Accordingly, the study confirms that the minimal growth concentration of the pathogenic microbe is lower than 0.1% and that the pathogen possibly survive and multiply inside the lettuce leaves given the lettuce extract with concentration of 0.1% is consistently supplied through the damaged tissues.
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김나예슬(Na-Ye-Seul Kim),양지영(Ji-Young Yang),김중범(Jung-Beom Kim) 한국식품과학회 2019 한국식품과학회지 Vol.51 No.3
본 연구에서는 형태학적으로 판별이 어려운 옥돔과 옥두어의 종 특이 primer를 개발, 검증한 후 모니터링을 통해 위변조 된 옥돔의 유통을 예방하고자 하였다. 옥돔과 옥두어 염기서열은 clustal omega 프로그램을 이용하여 정리하였고, primer3 프로그램을 이용하여 primer를 설계하였다. Multiplex PCR 결과는 옥돔과 옥두어에 대한 종 특이적 증폭이 확인되었고, PCR 반응을 확인하기 위한 공통 유전자에 대한 증폭이 확인되었다. 옥돔을 288 bp, 옥두어를 159 bp, 공통 유전자를 502 bp로 증폭되어 각각 PCR product 사이에 100 bp 이상 차이가 나타나 정확하게 판별이 가능하였다. Multiplex PCR 민감도 실험결과 옥돔 primer가 1 ng, 옥두어 primer가 1 ng, 공통 유전자 primer가 1 ng까지 밴드가 확인되었다. 모니터링 실험결과, 옥돔 38건, 옥두어 13건으로 판정되어 시료의 어종과 실험결과가 100% 일치함을 확인하였고, 위변조 사례는 나타나지 않았다. 본 실험에서 개발된 multiplex PCR 방법은 특이도와 민감도가 확보되었고 모니터링을 통해 유통, 판매되고 있는 옥돔과 옥두어의 판별에 적합함을 확인하였다. We developed and validated species-specific primers for Branchiostegus japonicus and Branchiostegus albus to prevent the sale of B. albus as B. japonicus. Primers for B. japonicus and B. albus were designed against the cytochrome b gene. Multiplex PCR showed a 288 bp amplicon for B. japonicus, a 159 bp amplicon for B. albus, and a 502 bp amplicon for the internal control. The PCR product bands for B. japonicus, B. albus, and the internal control were present at 1 ng each. The specificity and sensitivity of the primers developed in this study were validated by testing 38 B. japonicus strains and 13 B. albus strains. Using this monitoring method, fake fish did not appear due to the agreement between the experimental results and the species. Therefore, the developed multiplex PCR method was suitable for differentiating B. japonicus and B. albus.
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문광호,이한철,조아현,이서현,김나예슬,박은지,강주영,김중범,Mun, Kwang-Ho,Lee, Han-Cheol,Jo, Ah-Hyeon,Lee, Seo-Hyun,Kim, Na-Ye-Seul,Park, Eun-Ji,Kang, Ju-Yeong,Kim, Jung-Beom 한국식품영양학회 2019 韓國食品營養學會誌 Vol.32 No.3
The objective of this study was to evaluate the quality-based characteristics of Prunus mume fruit syrup, which is manufactured with various sugared sweeteners for suggestion of suitable alternative sweetener. Sweetener such as sucrose (MHP1), crystalline fructose (MHP2) and liquid fructo-oligosaccharide (MHP3) are used to manufacture Prunus mume fruit syrup. The sugar content of MHP1, MHP2 and MHP3 showed 53, 54 and $36^{\circ}Brix$, respectively. The total organic acid content of MHP1, MHP2 and MHP3 was 2.22, 3.07 and 3.71%. The total free sugar content of MHP1, MHP2 and MHP3 was 54.39, 47.52% and 31.62%, respectively. The appearance of MHP1 and MHP2 remained unchanged for the entire period but MHP3 had molded since the first week. This was as a result of the low total free sugar content in MHP3 sugared with liquid fructo-oligosaccharide compared to MHP1 and MHP2 sugared with solid sucrose and fructose. The sensory characteristics of MHP2 manufactured with crystalline fructose indicated an above average quality, indicating that it is difficult to manufacture Prunus mume fruit syrup using liquid sugar. It is suggested that crystalline fructose characterized solid form and lower glycemic index than sucrose be useful to manufacture Prunus mume fruit syrup as alternative sweetener.
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