• 산업기술연구
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• 제29권B호
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• pp.115-119
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• 2009

Green fluorescent protein (GFPuv) was displayed on the surface of ethanol-producing bacteria Zymomonas mobilis using N-terminal domain of ice nucleation protein (INP) as an anchoring motif. To evaluate the ice nucleation protein as plausible anchor motif in Z. mobilis, GFPuv gene was subcloned into Zymomonas expression vector yielding pBBR1MCS-3/pPDC/INPN/GFPuv plasmid., INP-GFPuv fusion protein was expressed in Z. mobilis and its fluorescence was verified by confocal microscopy. The successful display of GFPuv on Zymomonas mobilis suggest that INP anchor motif could be used for future fusion partner in Z. mobilis strain improvement.

• 한국축산식품학회지
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• 제39권6호
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• pp.1008-1014
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• 2019

This study investigated the effects of pressure-mediated protein changes on the ice nucleation temperature of pork loins. To variate chemical state of meat proteins, pork loin was pressurized at varying pressure levels (100-500 MPa) for 3 min, and moisture content, expressible moisture (EM) and differential scanning calorimetry (DSC) were analyzed. Although, all treatments showed similar moisture content, EM and degree of protein unfolding of pork loin showed different features as of 300 MPa. At moderate pressure treatments (100-200 MPa), all protein fractions were detected in DSC experiments, and pork loin had lower EM than control (p<0.05). Meanwhile, myosin and actin of pork loin treated at greater than 300 MPa were completely unfolded, and the treatments showed high EM compared to control (p<0.05). Unfolding of meat proteins was a factor suppressing ice nucleation, and the ice nucleation temperature tended to decrease with increasing applied pressure level. The ice nucleation characteristics of pressurized pork loin exhibited a potential application in freezing storage of pressurized meat with less tissue damage comparing to freeze fresh meat, and further exploration regarding the quality change after freezing of fresh and pressurized meat was warranted.

• 한국잠사곤충학회지
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• 제36권1호
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• pp.62-68
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• 1994

뽕나무 추기벌채 후 피해가 큰 가지썩음 증상에서 빙핵활성세균의 분리, 생물검정 및 동정과 밀도를 조사하여 본 결과 1. 총 36개의 분리세균 중 -1$0^{\circ}C$ 이상 온도에서 동결이 되는 세균은 12개였으며, -5$^{\circ}C$ 이상의 온도에서는 2개균주였다. 2. 공시세균의 생물검정에 있어서 토마토와 옥수수에서는 -1$0^{\circ}C$ 이상의 동결균주에서는 일정한 경향을 볼 수 없었으나 뽕나무에서는 -5$^{\circ}C$ 이상에서 동결이 되는 세균에서만 육안으로 확실한 동상해피해를 볼 수 있었다. 3. 뽕나무, 옥수수, 토마토유묘에 피해를 주며 -5$^{\circ}C$ 이상에서 영핵활성이 있는 균주 SE9316, SE9338을 동정해 본 결과 gram음성, 간상형, 운동성이 있고 극모를 가지고 호기성균인 Pseudomonas syringae로 동정되었다. 4. 5cm, 10cm 이병뽕나무가지에서 Pseudomonas 속 영핵활성세균의 밀도는 2월과 4월이 가장 높았고, 5월에는 감소하는 경향이었으며 병반에서 멀어질수록 밀도는 감소하였다. 또한 그 밀도는 105cfu/g 이상으로서 동상해를 유발시키기에 충분한 밀도였다.

• 한국식품과학회지
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• 제29권2호
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• pp.326-336
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• 1997

INP를 포함한 빙핵활성세균을 식품 가공 단계에 직접 이용하기 위해 빙핵활성을 최대한 유지시키면서 완전 살균하는 방법을 찾기 위해 가열, 고압, 방사선으로 처리한 후 생균수 및 빙핵활성을 측정하였다. 균주는 Pseudomonas syringae, Xanthomonas campestris와 INP유전자를 포함한 plasmid가 재조합된 Escherichia coli JM109/pEIN229, Gluconobacter oxydans/pKIN230를 사용하였다. 빙핵활성을 T90 (drop의 90%가 어는 온도)으로 나타내었을 때 방사선 조사시 $0.3^{\circ}C{\sim}0.7^{\circ}C$, 초고압 처리시 $1^{\circ}C{\sim}2^{\circ}C$, 가열 처리시 $4^{\circ}C{\sim}7^{\circ}C$가 감소하여 방사선 조사에 의한 살균시 빙핵활성의 감소가 가장 적은 것으로 나타났다. 각 살균점에서의 cumulative ice nucleation activity spectra를 측정한 결과 A부류의 빙핵($-5^{\circ}C$ 이상에서 빙핵으로 작용)의 수는 방사선처리시 거의 감소가 없었으며 초고압처리시 90% 이상 감소하였으며 가열처리시 존재하지 않았다. 증류수에 방사선조사로 살균된 빙핵활성세균을 첨가한 후 DSC를 이용해 thermogram을 얻었다. 빙핵활성세균을 첨가하지 않은 증류수보다 $11^{\circ}C{\sim}15^{\circ}C$ 정도 높은 온도에서 발열 피크가 형성되기 시작했으나 흡열 피크는 큰 차이를 보이지 않았으며, 발열량 및 흡열량은 감소했다. 한편, DSC를 이용해 측정된 빙핵활성은 drop freezing method에 의해 측정된 값과 높은 연관성$(R^{2}>0.993,\;p<0.0001)$을 보여 간편하고 정확한 빙핵활성 측정법으로의 사용 가능성을 제시했다.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.234-238
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• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• The Plant Pathology Journal
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• 제38권4호
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• pp.403-409
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• 2022

In biological particles such as Fusarium species, ice nucleation activity (INA) has been observed. Fusarium strains isolated from apple declined trees in Korea were identified with a multilocus sequence analysis using the tef1 and rpb1 genes. Droplet-freezing and tube-freezing assays were used to determine the INA of the strains, using Pseudomonas syringae pv. syringae KACC 21200 as a positive control and resulting in seven INA+ fungal strains that were identified as F. tricinctum (KNUF-21-F17, KNUF-21-F18, KNUF-21-F29, KNUF-21-F32, KNUF-21-F38, KNUF-21-F43, and KNUF-21-F44). The effect of Fusarium INA+ KNUF-21-F29 was compared to that of INA- strains on Chrysanthemum morifolium cv. Shinma explants. A higher callus formation and no-shoot formation were observed, suggesting that fungal INA could play a role in cold injuries and be a factor to consider in rapid apple decline. To the best of our knowledge, this is the first report of INA fungal strains isolated in Korea.

• 한국균학회지
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• 제50권4호
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• pp.255-262
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• 2022

In this study, the fungal strain KNUF-21-F39 was isolated from a declined apple tree (Malus domestica) in the Chungcheongbuk province in Korea. The strain KNUF-21-F39 presented a slow growth rate and a variety of macroconidia shapes and sizes ranging from ovoid to fusoid and 1- to 5-septate, primarily showing 3- and 4-septate, with "S" -shaped macroconidia rarely observed. The strain was identified based on morphological characteristics along with phylogenetic analysis performed using the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-α (tef1), RNA polymerase largest subunit (rpb1), and calmodulin (cal) genes. The fungal strain KNUF-21-F39 was identified as Fusarium diversisporum, which has not been previously reported in Korea. The ice nucleation activity (INA) of the strain was also evaluated, identifying the strain as positive for INA. This is the first report characterizing F. diversisporum as an IN-active fungal species.

• Journal of Biosystems Engineering
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• 제39권4호
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• pp.330-335
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• 2014

Purpose: This study was conducted for enhancing the performance of a conventional quick freezer by introducing the supercooling state, using a low-temperature coolant. Methods: In the present investigation, the supercooling process was executed prior to quick freezing for reducing the time by which the temperature passes the zone of maximum ice crystal formation. Every food has different nucleation points and hence, we used silicone oil as the coolant for supercooling for easy modification of temperature. Additionally, for quick freezing, we used liquid nitrogen spray. Results: Using the heat exchanger-type precooler with silicone oil, the temperature of the chamber was easily changed for enabling supercooling. Particularly, the results of the freezing test with garlic indicated that this system improved the hardness of garlic after it was thawed, compared to the conventional freezing method. Conclusions: Before quick freezing, if the food item is subjected to the supercooling state, the time from nucleation to the temperature reaching the frozen state ($-5^{\circ}C$, which is the maximum ice crystal formation zone) will be shorter than that incurred using quick freezing alone. The combination of the heat exchanger-type supercooler and liquid nitrogen sprayer is expected to serve as a promising technology for improving the physicochemical qualities of frozen foods.

• 식물병과 농업
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• 제5권1호
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• pp.27-33
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• 1999

Frost injury of crops is closely related to the epiphytic population dynamics of ice nucleation-active (INA) bacteria, and the injury can be reduced by decreasing the INA bacterial population. In order to predict the epiphytic population of INA bacteria on crops, a rapid and accurate detection method has to be developed. In the previous report, we produced some antibodies against INA proteins purified from the outer membrane of INA bacteria. However it was difficult to produce the antibodies because the purification procedures of the INA proteins were complicated, and the final yield was too low. We designed a specific peptide from the N-terminal region of INA protein by computer analysis and synthesized the peptide in vitro in this experiment. The peptide sequence was Asp-Ser-Por-Leu-Ser-Leu-His-Ala-Asp, that is corresponding to the highly conserved region in several INA proteins, with predicted beta turn, coiling, and hydrophilic region. A polyclonal anti-INA peptide antiserum produced specifically recognized INA bacteria as few as 10 colony-forming units (CFU) in the ELISA reactions and did not respond to other non-INA bacteria. Serological specificity of the anti-INA peptide antiserum will facilitate the forecasting of the INA bacterial population dynamics on crops.

• 한국식물병리학회지
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• 제3권3호
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• pp.180-186
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• 1987

단감나무와 차나무의 표면에서 분리한 Pseudomonas syringae 2계통 PS8401, PS8402를 $2.5\%$ glycerol이 첨가된 nutrient agar에서 배양한 다음, 증류수로 세포현탁액($10^8$ colony forming unit/ml)을 조제하여 마이크로피펫법으로 동결온도를 측정하여 본 결과, 각각 -2.5와 $-3.8^{\circ}C$에서 동결하여 빙핵활성이 인정되었다. 한편 동일한 방법에 의한 증류수의 동결온도는 $-21.8^{\circ}C$였고 옥수수를 비롯한 8가지 작물즙액의 동결온도는 $-11.6^{\circ}C$ 이하였다. PS 8401의 nutrient broth 현탁액$(10^8\;cfu/ml)$을 살포한 옥수수유묘는 $-2^{\circ}C$에서부터 얼기 시작하여 $-4^{\circ}C$가 되면, 거의 전체 식물이 상해를 입었으나 nutrient broth만 살포한 대조구의 유묘는 온도가 $-9^{\circ}C$로 내려가기 전까지는 피해를 입지 않았다. PS8401 현탁액을 살포한 후 48시간에 측정한 유묘의 피해는 살포한 세균의 량이 증가함에 따라 심해지는 경향이었다. 상기 PS 8401의 빙핵활성은 현탁액내 세포의 수가 많아짐에 따라 증가하였으며 세균을 배양한 배지의 조성이 배양온도에 따라서도 빙핵활성이 변하였는데 $2.5\%$ glycerol 혹은 $2.5\%$ glucose를 첨가한 nutrient agar와 탄소원을 별도로 첨가하지 않은 nutrient agar에서 자란 세균현탁액$(10^2\;cfu/ml)$의 동결온도는 각각 -4.0, -4.4, $-7.2^{\circ}C$였고, 배지를 $2.5\%$ glycerol을 함유한 nutrient agar로 고정하고 온도를 달리하여 생육시킨 세균현탁액 $(10^2\;cfu/ml)$의 동결온도는 $-4.0^{\circ}C$(생육온도가 $15\~25^{\circ}C$인 경우)와 $-7.6^{\circ}C$(생육온도가 $30^{\circ}C$인 경우)였다.