• Journal of Applied Biological Chemistry
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• v.61 no.3
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• pp.213-217
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• 2018

Tolaasin secreted by Pseudomonas tolaasii is a peptide toxin and causes brown blotch disease on the cultivated mushrooms by collapsing cellular and fruiting body structure. Toxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasin molecules form membrane pores on the red blood cells and destroy cell membrane structure. In the previous studies, we found that tolaasin cytotoxicity was suppressed by $Zn^{2+}$ and $Ni^{2+}$. $Ni^{2+}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its $K_i$ value was 1.8 mM. The hemolytic activity was completely inhibited at the concentration higher than 10 mM. The inhibitory effect of $Zn^{2+}$ on tolaasin-induced hemolysis was increased in alkaline pH, while that of $Ni^{2+}$was not much dependent on pH. When the pH of buffer solution was increased from pH 7 to pH 9, the time for 50% hemolysis ($T_{50}$) was increased greatly by $100{\mu}M$ $Zn^{2+}$; however, it was slightly increased by 1 mM $Ni^{2+}$ at all pH values. When the synergistic effect of $Zn^{2+}$ and $Ni^{2+}$ on tolaasin-induced hemolysis was measured, it was not dependent on the pH of buffer solution. Molecular elucidation of the difference in pH-dependence of these two metal ions may contribute to understand the mechanism of tolaasin pore formation and cytotoxicity.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.108-108
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• 2019

느타리버섯은 전남 장흥군에서 병재배로 재배된 '곤지 7호' 느타리버섯 품종을 $3^{\circ}C$에서 1일간 냉각시킨 후 실험에 사용하였다. 이산화탄소 처리는 10mm 두께의 아크릴로 제작한 밀폐 챔버($40{\times}70{\times}40cm$)를 이용하여 CO2가스를 주입하여 내부의 CO2 농도를 $30{\pm}1%$로 조정하여 3시간 동안 밀폐시켰으며 $3^{\circ}C$ 저온 저장고에서 수행하였다. 처리 후 포장필름은 3처리로 하여 PVC랩, 재배농가에서 이용하는 필름인 $20{\mu}m$ OPP필름, $30{\mu}m$ OPP필름을 재료로 하여 산소투과율을 $5,000cc/m2{\cdot}day{\cdot}atm$로 조정한 필름(OPP5)을 사용하였다. PVC랩 처리는 스티로폼 트레이에 옮긴 후 균일하게 한 겹으로 둘렀고, OPP와 OPP5는 봉지에 옮겨 봉지 입구 4cm 안쪽을 비닐접착기로 열접착하여 밀봉하였으며 $3^{\circ}C$에 저장하면서 필름내부의 기체조성, 경도, 색도, 이취, 종합선도 변화를 조사하였다. 포장된 느타리버섯의 포장재 내 이산화탄소 농도는 저장초기 0.03%에서 점차 증가하여 저장 3일 후 PVC랩은 3.5~3.9%, 나머지 처리는 18%이상으로 증가하였으며 OPP필름보다 OPP5필름의 경우 포장재 내 이산화탄소 농도가 더 낮게 유지되었다. 생체중 감소는 PVC랩에서 저장 26일 후 9.9% 이하로 육안으로 보이는 시들음이 관찰되었으며 나머지 처리들은 저장 26일 후까지 1.6% 이하의 중량감소율을 보였다. 느타리버섯 갓 경도는 유지되었으며 줄기표면의 황색도 $b^*$값은 PVC랩에서는 증가 경향을, OPP나 OPP5에서는 유지 경향을 보였다. 느타리버섯의 이취 및 전체적인 품질은 무처리 후 PVC랩 포장은 저장 6일 후에 이취가 상품성 한계로 발생하였고 OPP5 포장은 포장재 중 가장 이취발생이 늦게 발생하였으며 CO2 처리에 의해 지연되는 것을 알 수 있었다. 이런 결과를 종합하여 느타리버섯의 $3^{\circ}C$ 저장 중 품질유지기간은 각 PVC랩(6일) ${\rightarrow}$ 30% CO2+PVC랩(7일) ${\rightarrow}$ OPP(10일) ${\rightarrow}$ 30% CO2+OPP(17일) ${\rightarrow}$ OPP5(20일) ${\rightarrow}$ 30% CO2+OPP5(22일)로 조사되었다. CO2처리 후 산소투과율을 $5,000cc/m2{\cdot}day{\cdot}atm$으로 조정한 미세천공(OPP5)필름이 가장 높은 전체적 품질점수를 나타내었으며 상대적으로 낮은 이취발생, 갈변과 갓무름이 적어 높은 점수를 얻는데 영향을 준 것으로 보인다.

• Journal of Applied Biological Chemistry
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• v.62 no.3
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• pp.287-292
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• 2019

Pseudomonas tolaasii causes brown blotch disease on the oyster mushroom (Pleurotus ostreatus). Various pathogenic strains of P. tolaasii were isolated and divided into three subtypes, $P1{\alpha}$, $P1{\beta}$, and $P1{\gamma}$. For phage therapy, bacteriophages against to these subtype strains were applied to mushroom cultivation and very successful to prevent from the disease. In this study, bacteriophages were isolated against the representative strains of subtype pathogens and their polyclonal antibodies were synthesized to investigate structural relationship among capsid proteins of phages. Phage preparations over $10^{10}pfu/mL$ were injected to rabbit thigh muscle and polyclonal antibodies were obtained after three times of boost injection. Titers of the antibodies obtained were over $2{\times}10^7Ab/mL$ for the phage ${\phi}6264$, $1{\times}10^6Ab/mL$ for the phage ${\phi}HK2$, and $1{\times}10^7Ab/mL$ for the phage ${\phi}HK19$ and phage ${\phi}HK23$. High specific activities were observed between antibodies and the corresponding bacteriophages. Some cross-reactivities between the antibodies and non-corresponding bacteriophages were also measured. Antibody $Ab{\phi}6264$ inactivated all phages of $P1{\alpha}$ subtype and only phage ${\phi}HK16$ among $P1{\beta}$ subtype phages. Antibody $Ab{\phi}HK23$ of $P1{\gamma}$ subtype neutralized all phages of $P1{\beta}$ subtype as well as the phage ${\phi}HK23$, showing the widest phage-inactivation range. When the structural-similarity studies of phages were investigated by using phage antibodies, closeness obtained by phylogenetic analysis of 16S rRNA genes of pathogenic strains were quite different from that of polyclonal antibody-specific structural similarity of phage capsid proteins. In conclusion, there is weak correlation between the host strain specificity of bacteriophage and its capsid structural similarity measured by phage antibodies.

• Applied Biological Chemistry
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• v.21 no.3
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• pp.150-184
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• 1978

Nutritional characteristics and physio-chemical properties of mycelial growth and fruitbody formation of oyster mushroom(Pleurotus ostreatus)in synthetic media, the curtural condition for the commerical production in the rice straw and poplar sawdust media, and the changes of the chemical components of the media and mushroom during the cultivation were investigated. The results can be summarized as follows: 1. Among the carbon sources mannitol and sucrose gave rapid mycelial growth and rapid formation of fruit-body with higher yield, while lactose and rhamnose gave no mycelial growth. Also, citric acid, succinic acid, ethyl alcohol and glycerol gave poor fruit-body formation, and acetic acid, formic acid, fumaric acid, n-butyl alcohol, n-propyl alcohol and iso-butyl alcohol inhibited mycelial growth. 2. Among the nitrogen sources peptone gave rapid mycelial growth and rapid formation of fruit-body with higher yield, while D,L-alanine, asparatic acid, glycine and serine gave very poor fruit-body formation, and nitrite nitrogens, L-tryptophan and L-tyrosine inhibited mycelial growth. Inorganic nitrogens and amino acids added to peptone were effective for fruit-body growth, and thus addition of ammonium sulfate, ammonium tartarate, D,L-alanine and L-leucine resulted in about 10% increase fruit-body yield. L-asparic acid about 15%, L-arginine about 20%, L-glutamic acid, and L-lysine about 25%. 3. At C/N ratio of 15.23 fruit-body formation was fast, but the yield decreased, and at C/N ratio of 11.42 fruit-body formation was slow, but the yield increased. Also, at the same C/N ratio the higher the concentration of mannitol and petone, the higher yield was produced. Thus, from the view point of both yield of fruit-body and time required for fruiting the optimum C/N ratio would be 30. 46. 4. Thiamine, potassium dihydrogen phosphate and magnecium sulfate at the concentration of $50{\mu}g%$. 0.2% and 0.02-0.03%, respectively, gave excellent mycelial and fruit-body growth. Among the micronutrients ferrous sulfate, zinc sulfate and manganese sulfate showed synergetic growth promoting effect but lack of manganese resulted in a little reduction in mycelial and fruit-body growth. The optimum concentrati on of each these nutrients was 0.02mg%. 5. Cytosine and indole acetic acid at 0.2-1mg% and 0.01mg%, respectively, increased amount of mycelia, but had no effect on yield of fruit-body. The other purine and pyrimidine bases and plant hormones also had no effect on mycelial and fruit-belly yield. 6. Illumination inhibited mycelial growth, but illumination during the latter part of vegetative growth induced primordia formation. The optimum light intensity and exposure time was 100 to 500 lux and 6-12 hours per day, respectively. Higher intensity of light was injurous, and in darkness only vegetative growth without primordia formation was continued. 7. The optimum temperature for mycelial growth was $25^{\circ}C$ and for fruit-body formation 10 to $15^{\circi}C$. The optimum pH range was from 5.0 to 6.5. The most excellent fry it-body formation were produced from the mycelium grown for 7 to 10 days. The lesser the volume of media, the more rapid the formation of fruit-body; and the lower the yield of fruit-body; and the more the volume of media, the slower the formation of fruit-body, and the higher the yield of fruit-body. The primordia formation was inhibited by $CO_2$. 8. The optimum moisture content for mycelial growth was over 70% in the bottle media of rice straw and poplar sawdust. 10% addition of rice bran to the media exhibited excellent mycelial growth and fruit-body formation, and the addition of calciumcarbonate alone was effective, but the addition of calcium carbonate was ineffective in the presence of rice bran. 9. In the cultivation experiments the total yield of mushroom from the rice straw media was $14.99kg/m^2$, and from the sawdust media $6.52kg/m^2$, 90% of which was produced from the first and second cropping period. The total yield from the rice straw media was about 2.3 times as high as that from the sawdust media. 10. Among the chemical components of the media little change was observed in the content of ash on the dry weight basis, and organic matter content decreased as the cultivation progressed. Moisture content, which was about 79% at the time of spawning, decreased a little during the period of mycelial propagation, after which no change was observed. 11. During the period from spawning to the fourth cropping about 16.7% of the dry matter, about 19.3% of organic matter, and about 40% of nitrogen were lost from the rice straw media; about 7.5% of dry mallet, about 7.6% of organic matter, and about 20% of nitrogen were lost from the sawdust media. For the production of 1kg of mushroom about 232g of organic matter and about 7.0g of nitrogen were consumed from the rice straw media; about 235g of organic matter and about 6.8g of nitrogen were consumed from the sawdust media, 1㎏ of mushroom from either of media contains 82.4 and 82.3g of organic matter and 5.6 and 5.4g of nitrogen, respectively. 12. Total nitrogen content of the two media decreased gradually as the cultivation progressed, and total loss of insoluble nitrogen was greater than that of soluble nitrogen. Content of amino nitrogen continued to increase up to the third cropping time, after which it decreased. 13. In the rice straw media 28.0 and 13.8% of the total pentosan and ${\alpha}$-cellulose, respectively, lost during the whole cultivation period was lost during the period of mycelial growth; in the sawdust media 24.1 and 11.9% of the total pentosan and ${\alpha}$-cellulose, respectively, was lost during the period of mycelial growth. Lignin content in the media began to decrease slightly from the second cropping time, while the content of reduced sugar, trehalose and mannitol continued to increase. C/N ratio of the rice straw media decreased from 33.2 at spawining to 30.0 at ending; that of the sawdust media decreased from 61.3 to 60.0. 14. In both media phosphorus, potassium, manganese and zinc decreased, at magnesium, calcium and copper showed irregular changes, and iron had a tendency to be increased. 15. Enzyme activities are much higher in the rice straw media than in the sawdust media. CMC saccharifying and liquefying activity gradually increased from after mycelial propagation to the second cropping, after which it decreased in both media. Xylanase activity rapidly and greatly increased during the second cropping period rather than the first period. At the start of the third cropping period the activity decreased rapidly in the rice straw media, which was not observed in the sawdust media. Protease activity was highest after mycelial propagation, after which it gradually decreased. The pH of the rice straw media decreased from 6.3 at spawning to 5.0 after fourth cropping; that of the sawdust media decreased from 5.7 to 4.9. 16. The contents of all the components except crude fibre of the mushroom from the rice straw media were higher than those from the sawdust media. Little change was observed in the content of the components of mushroom cropped from the first to the third period, but slight decrease was noticed at the fourth cropping.