WO2012070891A2 - Medium composition for culturing the simplicillium lamellicola bcp strain, and culturing method using the composition - Google Patents
Medium composition for culturing the simplicillium lamellicola bcp strain, and culturing method using the composition Download PDFInfo
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- WO2012070891A2 WO2012070891A2 PCT/KR2011/009028 KR2011009028W WO2012070891A2 WO 2012070891 A2 WO2012070891 A2 WO 2012070891A2 KR 2011009028 W KR2011009028 W KR 2011009028W WO 2012070891 A2 WO2012070891 A2 WO 2012070891A2
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- the present invention relates to a medium composition for culturing Simplici Ilium lamellicola BCP strains and a method of culturing the same.
- Mycelial growth or mycelial shape may vary depending on which medium is used for strain culture.
- the strains When culturing strains, the strains may show changes in mycelial growth or irregularities in the tip of the bacteria, which are often caused by changes in the composition or culture conditions of the medium. In some cases, depending on the type of medium, there may be difficulty in mass-proliferating bacteria.
- PDB potato dextrose broth
- the present invention provides an optimal medium composition for the cultivation of Simplici Ilium lamellicola BCP strain and a method for culturing the Simplicilium lamellicola BCP strain using the same.
- At least one carbon source selected from the group consisting of fructose and dextrin;
- a medium composition for simplicity of culture of Pilisimpli Ilium lamellicola BCP comprising at least one nitrogen source medium selected from the group consisting of soybean meal and tryptone, and simple using the composition It is an object to provide a method for culturing Lysillium lamelicola BCP.
- the present invention provides a medium composition containing an optimal carbon source and a nitrogen source, and by setting the optimum culture degree, the initial culture pH, and the incubation time, Simplici Ilium lamellicola BCP (Simplici Ilium lamellicola BCP) )
- the culture medium composition and culture conditions for strain culture were optimized.
- the present invention provides at least one carbon source selected from the group consisting of sucrose, fructose and dextrin;
- a medium composition for Simplici Ilium lamellicola BCP strain culture comprising at least one nitrogen source selected from the group consisting of soybean meal and tryptone as a medium component.
- the carbon source may include 1.0 to 5.0 w / v%, preferably 2 to 4 w / v%, more preferably 3 to 3.5 w / v%. Outside the above range, the yield of spore production may be lowered.
- the medium composition of the present invention may contain 0.5 to 4.0 w / v%, preferably 2 to 4.0 w / v%, more preferably 2.5 to 3.0 w / v%. Outside the above range, the yield of spores may be lowered.
- the medium composition of the present invention may further include an inorganic source.
- an inorganic source In one embodiment of the invention, but not limited to, magnesium sulfate, potassium chloride, magnesium chloride, sodium chloride, ferrous sulfate, calcium chloride, dipot ass him phosphate, At least one selected from the group consisting of copper sulfate, zinc sulfate, potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, potassium iodide and sodium molybdate is preferred.
- the present invention particularly provides a medium composition for the culture of simple Lysillium lamelicola BCP strain comprising fructose, soy flour, and magnesium sulfate as a medium component.
- the medium is more preferably 1.0 to 5.0 w / v% fructose; 0.5 to 4.0 soy flour; By including 0.1 to 1 magnesium sulfate, it is possible to increase the yield of spores of the Simpliclium la meli cola BCP strain.
- the medium may further include an inorganic source such as potassium chloride, potassium diphosphate, and iron sulfate, and in the medium, 0.1 to 1 / ⁇ ⁇ ) potassium chloride; 0.05 to 0.5 l ⁇ potassium diphosphate; And 0.0001 to 0.01 / ⁇ iron sulfate.
- an inorganic source such as potassium chloride, potassium diphosphate, and iron sulfate, and in the medium, 0.1 to 1 / ⁇ ⁇ ) potassium chloride; 0.05 to 0.5 l ⁇ potassium diphosphate; And 0.0001 to 0.01 / ⁇ iron sulfate.
- the medium prepared according to the medium composition is inoculated with an initial concentration of 0.1 to 5 w / v3 ⁇ 4, more specifically 0.5 to 2 w / v% of the Simlyillium lamelicola BCP strain seed. Outside the concentration range, there is a fear that growth is somewhat lower.
- the culture temperature may be 15 to 37 ° C., preferably 15 to 33 ° C., more preferably 20 to 25 ° C.
- the culture temperature is 15 ° C or less, the growth rate is increased, but the growth rate is slow and the spore formation yield may drop significantly, and when the temperature is above 37 ° C, contamination occurs easily and the amount of the final culture product may be reduced.
- the pH is suitably adjusted to 3 to 8, preferably pH 3.5 to 6, more preferably pH 4 to 5. If the pH is less than 3, it is almost impossible to grow, the yield of spore production may be low, and if the pH is exceeded 8, the amount of the final culture may be reduced.
- the pH refers to the pH of the initial culture
- the initial culture refers to the sterilization of the nutrient medium and immediately after the inoculation of the bacteria in the state before the inoculation of the simple bacilli. pH adjustment is carried out before sterilization, but final pH measurement is made immediately after sterilization, so the pH at the time of inoculation and growth of the first bacteria is taken as the initial pH.
- the culture period may be 20 hours or more, specifically 30 hours to 7 days, more specifically 50 hours to 6 days, based on 500 to 5000 L incubator. If the incubation period is less than 20 hours, there is a fear that the yield is lower when drying more spores than spores, and if the incubation period exceeds 7 days, the material produced during the culturing process may kill the microorganisms and lower the yield.
- the stirring speed and the air input rate may be appropriately set according to the growth of microorganisms, and may vary depending on the culture conditions and conditions.
- bubbles generated during the culture may cause problems such as contamination, it is preferable to remove them.
- defoaming may be performed using a device such as KONION GE-304.
- the initial input ratio may be 0.1 to 1 w / v% level, and can be defoamed every time bubbles are generated at the level of 0.01 to 0.1 / ⁇ during the culture.
- freeze-drying may be performed by freezing at a temperature of -90 to -10 ° C, specifically -80 to -20 ° C, more specifically -70 to -30 ° C, but the amount of the culture medium is high.
- the final pH can be adjusted to less than 5 to suppress contamination and the growth of microorganisms.
- the present invention can obtain a homogeneous medium by establishing the optimum medium composition and culture conditions of the strains of Simpliclium lamellicola BCKSimplici Ilium lamellicola BCP), more than double the yield of spores of the Simpliclium lamellicola BCP strain It can be increased, and the spore stability is greatly improved.
- Figure 1 shows the results of measuring the spores production yield of Simplicillium lamelicola BCKSimplici Ilium lamellicola BCP) according to the culture temperature.
- Figure 2 shows the results of measuring the spores production yield of the Simplisium lamelicola BCP according to the initial culture P H.
- Figure 3 is a graph of the spore production of the simplicity lamella cola BCP over time incubation.
- Figure 4 is a graph of spore production of Simplisium lamelicola BCP according to the initial inoculation concentration.
- Figure 5 shows the results of measuring the spores production yield of simplicity lamellicola BCP according to the fructose concentration (fructose).
- Figure 6 shows the results of measuring the spores production yield of Simpliclium lamella cola BCP according to the soybean meal concentration.
- Figure 7 shows the results of the main effect analysis of the media components on the yield of spores of the simplicity la melicola BCP strain according to Plackett-Burman Design (PBD).
- Figure 8 is a graph showing the change in culture yield of Simplisium lamelicola BCP according to the type of medium.
- Figure 9 is a graph showing the changes over time stability of Simplisium lamelicola BCP according to the type of medium.
- Growth medium was prepared according to the composition described in Table 1 below:
- Simplicium lamelicola BCP strain was distributed from Korea Research Institute of Chemical Technology and used [Accession Number: KCTC0639BP].
- the strain 1 H medium was inoculated to carry out the experiment as follows.
- CDB (Czapek-Dox Broth: sucrose 30 g / L, sodium nitrate 3.0 g / L, dipotassium phosphate 1.0 g / L, magnesium sulfuate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L , pH 7.3).
- PDA was inoculated with 5 x 5raf sections of the bacterium grown for 7-10 days.
- the number of spores in the culture was counted using a homocytometer under a 400x microscope.
- the physical properties of the cultures in the culture were largely homogeneous, but a large number of small cell masses were formed as a whole, and the microscopic observation showed that many spores were collected in the mass. So, after incubation, grinding with a homogenizer for 10 seconds to homogenize the spores and fill with a vortex mixer. After suspension, the spore count was counted. The test was repeated three times.
- the initial P H was 7.3 when the medium was prepared, and the culture silver was cultured at 120 rpm for 5 days at 20, 22, 25, 27, and 30 ° C., and then the number of spores in the culture medium was counted according to the method described above.
- Simplisium lamelicola BCP showed a slight drop in sporulation yield at a low temperature below 20 ° C., 22 ° C. was the best silver, and a decrease in sporulation yield after 25 ° C. [FIG. 1] .
- the culture temperature was fixed at 22 ° C.
- the pH of the medium was adjusted to 4, 5, 6, 7, 8 using HC1 and NaOH, respectively, and cultured for 5 days (120 rpm), and the number of spores in the culture was counted. .
- the optimum silver is 22 ° C and optimally to the initial pH 4.5 condition three times in 1L flask (working volume 200 ml) 9 ilgan culture (150 rpm), while the culture medium was collected for each 5 ml sample in a 24-hour interval My spore count was counted.
- sucrose which is used as a carbon source of CDB medium
- sucrose which is used as a carbon source of CDB medium
- 1.5% (w / v) of different carbon sources Carbon sources tested in this experiment were soluble starch, dextrin, maltose, lactose, mannitol, galactose, fructose and glucose. , CMC.
- Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. The number of spores in the final culture was counted and the properties of the culture were also investigated. The test was repeated three times.
- Fructose was less homogeneous than basal medium CDB, but spore count increased by approximately 47% and little mycelial mass was seen. No mold growth occurred at Mannier and CMC. This was thought to be due to the fact that the carbon source of this type was not available to the Simlyillium lamelicola BCP.
- the medium was prepared by removing the sucrose used as a carbon source from the CDB basal medium and replacing the fructose with an increase in the fructose content from 1.0 w / v% to 5.0 w / v% in units of 1.0 w / v%.
- Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. Spore count was counted in the final culture. The test was repeated three times.
- the optimum concentration of fructose was investigated according to the above carbon source test results. Tests from 1.0 w / v% to 5.0 w / v% at 1.0 w / v% intervals showed high yields of spore production from 3.0 to 5.0 w / v%, with decreasing trend before then (Fig. 5). Considering the variation in carbon source concentration of fructose, the optimal condition was set at 3.0%.
- the yield of spore production according to the concentration was investigated for the soybean meal having the highest yield of spores and excellent physical properties of the culture solution.
- a carbon source was used for 3 h of fructose and a nitrogen source was prepared by increasing soy flour 1.0 hVA from 0.5 w / v% to 4.0 w / v%.
- Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. Spore count was counted in the final culture. The test was repeated three times.
- concentration test was performed from 0.5 w / v% to 4.0 w / v% in units of 1.0 w / v% from 3.0 to 4.0 w / v3 ⁇ 4.
- the maximum spore yield was shown (FIG. 6).
- the optimum condition was 3.0 in consideration of the variation of nitrogen source concentration of soy flour and raw material cost.
- PBD Plackett-Burman Design
- composition of liquid culture medium of S. simillium lamelicola BCP strain Composition of liquid culture medium of S. simillium lamelicola BCP strain
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Abstract
The present invention relates to a medium composition for culturing the Simplicillium lamellicola BCP strain and to a culturing method using the composition. More particularly, the present invention relates to a medium composition for culturing the Simplicillium lamellicola BCP strain, wherein the medium composition comprises a specific carbon source and a specific nitrogen source, and to a method for culturing the Simplicillium lamellicola strain using the composition.
Description
1 09028 1 09028
【명세서】 【Specification】
【발명의 명칭】 [Name of invention]
심플리실리움 라멜리콜라 BC P 균주 배양용 배지 조성물 및 이의 배양방법 Media composition for cultivation of Simplicrium lamelicola BC P strain and its culture method
【기술분야】 Technical Field
본 발명은 심플리실리움 라멜리콜라 BCP(Simplici Ilium lamellicola BCP) 균주 배양용 배지 조성물 및 이의 배양방법에 관한 것이다. 【배경기술】 The present invention relates to a medium composition for culturing Simplici Ilium lamellicola BCP strains and a method of culturing the same. Background Art
균주 배양 시 어떠한 배지를 사용하느냐에 따라 균사 생장력이나 균총 모양 이 달라질 수 있다. 균주를 배양하다 보면 균주들의 균사 생장력이 떨어지거나 균 총의 선단부분이 불규칙하게 변하는 등의 변화를 나타내기도 하는데, 이는 배지의 조성이나 배양 조건이 달라진 데에 따른 경우가 많다. 경우에 따라서, 배지의 종류 에 따라균을 대량 증식시키는 데에도 어려움이 있을 수 있다. Mycelial growth or mycelial shape may vary depending on which medium is used for strain culture. When culturing strains, the strains may show changes in mycelial growth or irregularities in the tip of the bacteria, which are often caused by changes in the composition or culture conditions of the medium. In some cases, depending on the type of medium, there may be difficulty in mass-proliferating bacteria.
따라서, 기존의 PDB (포테이토 덱스트로스 브로스, Potato dextrose broth)를 대체 가능하면서도 심플리실리움 라멜리콜라 BCP균주의 배양을 최적화할 수 있는 배지 선정이 중요하다. 심플리실리움 라멜리콜라 BCP 균주의 배양에는 PDB가 주로 사용되어 왔으나, PDB는 복합배지로서 정확한 성분을 알 수 없는 문제점이 있다. Therefore, it is important to select a medium that can replace the existing PDB (potato dextrose broth), while optimizing the culture of the Simplisium lamelicola BCP strain. PDB has been mainly used for the cultivation of S. simillium lamelicola BCP strains, but PDB is a complex medium has a problem that does not know the exact components.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】 [Technical problem]
본 발명은 심플리실리움 라멜리콜라 BCP(Simplici Ilium lamellicola BCP) 균주의 배양을 위한 최적의 배지 조성물 및 이를 이용한 심플리실리움 라멜리콜라 BCP 균주의 배양방법을 제공하며, 구체적으로 본 발명은 수크로오스, 과당 및 덱스트린으로 이루어진 그룹으로부터 선택되는 하나 이상의 탄소원; 및
대두분 (soybean meal) 및 트립톤 (tryptone)으로 이루어진 그룹으로부터 선택되는 하나 이상의 질소원올 배지 성분으로 포함하는 심플리실리움 라멜리콜라 PiSimplici Ilium lamellicola BCP) 균주 배양용 배지 조성물 및 상기 조성물을 이용하여 심플리실리움 라멜리콜라 BCP을 배양하는 방법을 제공하는데 목적이 있다. The present invention provides an optimal medium composition for the cultivation of Simplici Ilium lamellicola BCP strain and a method for culturing the Simplicilium lamellicola BCP strain using the same. At least one carbon source selected from the group consisting of fructose and dextrin; And A medium composition for simplicity of culture of Pilisimpli Ilium lamellicola BCP) comprising at least one nitrogen source medium selected from the group consisting of soybean meal and tryptone, and simple using the composition It is an object to provide a method for culturing Lysillium lamelicola BCP.
【기술적 해결방법】 Technical Solution
상기 문제점을 해결하기 위해, 본 발명에서는 최적 탄소원 및 질소원을 포함하는 배지 조성을 제공하고, 최적의 배양은도, 배양초기 pH, 배양시간을 설정함으로써, 심플리실리움 라멜리콜라 BCP(Simplici Ilium lamellicola BCP) 균주 배양용 배지 조성물 및 배양 조건을 최적화하였다. 이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다. 본 발명은 수크로오스, 과당 및 덱스트린으로 이루어진 그룹으로부터 선택 되는 하나 이상의 탄소원; 및 대두분 (soybean meal) 및 트립톤 (tryptone)으로 이루 어진 그룹으로부터 선택되는 하나 이상의 질소원을 배지성분으로 포함하는 심플리 실리움 라멜리콜라 Simplici Ilium lamellicola BCP) 균주 배양용 배지 조성물 을포함한다. In order to solve the above problems, the present invention provides a medium composition containing an optimal carbon source and a nitrogen source, and by setting the optimum culture degree, the initial culture pH, and the incubation time, Simplici Ilium lamellicola BCP (Simplici Ilium lamellicola BCP) ) The culture medium composition and culture conditions for strain culture were optimized. Referring to the present invention in more detail as follows. The present invention provides at least one carbon source selected from the group consisting of sucrose, fructose and dextrin; And a medium composition for Simplici Ilium lamellicola BCP strain culture comprising at least one nitrogen source selected from the group consisting of soybean meal and tryptone as a medium component.
본 발명의 배지 조성물 중에는 탄소원이 1.0 내지 5.0 w/v%, 바람직하게 2 내지 4 w/v%, 보다 바람직하게 3 내지 3.5 w/v%포함될 수 있다. 상기 범위 밖에서 는 포자 생산 수율이 낮아질 우려가 있다. In the medium composition of the present invention, the carbon source may include 1.0 to 5.0 w / v%, preferably 2 to 4 w / v%, more preferably 3 to 3.5 w / v%. Outside the above range, the yield of spore production may be lowered.
본 발명의 배지 조성물 중에는 질소원이 0.5 내지 4.0 w/v%, 바람직하게 2 내지 4.0 w/v%, 보다 바람직하게 2.5 내지 3.0 w/v%포함될 수 있다. 상기 범위 밖 에서는 포자 생산 수율이 낮아질 우려가 있다. The medium composition of the present invention may contain 0.5 to 4.0 w / v%, preferably 2 to 4.0 w / v%, more preferably 2.5 to 3.0 w / v%. Outside the above range, the yield of spores may be lowered.
본 발명의 배지 조성물은, 무기물원을 추가로 포함할 수 있다. 본 발명의 일 예에서, 이로 제한되지는 않으나, 황산마그네슘, 염화칼륨, 염화마그네슘, 염화 나트륨, 황산철 (ferrous sulfate), 염화칼슘, 이인산칼륨 (di pot ass him phosphate),
황산구리 , 황산아연, 질산칼륨, 질산암모늄, 인산이수소칼륨, 요오드화칼륨 및 몰 리브덴산나트륨으로 이루어진 그룹으로부터 선택되는 하나 이상이 바람직하다. 본 발명은 특히 과당, 대두분 , 황산마그네슴을 배지성분으로 포함하는 심플 리실리움 라멜리콜라 BCP균주 배양용 배지 조성물을 제공한다. The medium composition of the present invention may further include an inorganic source. In one embodiment of the invention, but not limited to, magnesium sulfate, potassium chloride, magnesium chloride, sodium chloride, ferrous sulfate, calcium chloride, dipot ass him phosphate, At least one selected from the group consisting of copper sulfate, zinc sulfate, potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, potassium iodide and sodium molybdate is preferred. The present invention particularly provides a medium composition for the culture of simple Lysillium lamelicola BCP strain comprising fructose, soy flour, and magnesium sulfate as a medium component.
상기 배지는 보다 바람직하게 1.0 내지 5.0 w/v%의 과당; 0.5 내지 4.0 대두분; 0.1 내지 1 황산마그네슴을 포함함으로써, 심플리실리움 라 멜리콜라 BCP 균주의 포자 생산수율을 높일 수 있다. The medium is more preferably 1.0 to 5.0 w / v% fructose; 0.5 to 4.0 soy flour; By including 0.1 to 1 magnesium sulfate, it is possible to increase the yield of spores of the Simpliclium la meli cola BCP strain.
또한, 상기 배지에는 염화칼륨, 이인산칼륨 및 황산철과 같은 무기물원이 추가로 포함될 수 있으며, 상기 배지 중에는 0.1 내지 1 /\^)의 염화칼륨; 0.05 내 지 0.5 l \ 이인산칼륨; 및 0.0001 내지 0.01 /^의 황산철이 포함될 수 있다. 본 발명은 상기 언급된 배지 조성물 중에서 심플리실리움 라멜리콜라 BCP 균주를 배양하는 방법을 제공한다. In addition, the medium may further include an inorganic source such as potassium chloride, potassium diphosphate, and iron sulfate, and in the medium, 0.1 to 1 / \ ^) potassium chloride; 0.05 to 0.5 l \ potassium diphosphate; And 0.0001 to 0.01 / ^ iron sulfate. The present invention provides a method of culturing the Simpliclium lamella cola BCP strain in the above-mentioned medium composition.
상기 배지 조성에 따라 제조한 배지에 초기 농도 0.1 내지 5 w/v¾, 보다 구 체적으로 0.5 내지 2 w/v% 수준의 심플리실리움 라멜리콜라 BCP 균주 씨드를 접종 한다. 상기 농도범위 밖에서는 생육이 다소 낮아질 우려가 있다. The medium prepared according to the medium composition is inoculated with an initial concentration of 0.1 to 5 w / v¾, more specifically 0.5 to 2 w / v% of the Simlyillium lamelicola BCP strain seed. Outside the concentration range, there is a fear that growth is somewhat lower.
배양 시 , 배양 온도는 15 내지 37 °C, 바람직하게 15 내지 33 °C, 보다 바람 직하게 20 내지 25°C로 할 수 있다. 배양 온도가 15°C 이하인 경우, 생육 정도는 증가하나 생육 속도가 늦고 포자형성 수율이 크게 떨어질 우려가 있으며, 37°C 이 상인 경우 오염이 쉽게 일어나고 최종 배양산물의 양도 감소할 우려가 있다. In the culture, the culture temperature may be 15 to 37 ° C., preferably 15 to 33 ° C., more preferably 20 to 25 ° C. When the culture temperature is 15 ° C or less, the growth rate is increased, but the growth rate is slow and the spore formation yield may drop significantly, and when the temperature is above 37 ° C, contamination occurs easily and the amount of the final culture product may be reduced.
배양 시, pH는 3 내지 8로 조절하는 것이 적합하며, 바람직하게 pH 3.5 내 지 6, 보다 바람직하게 pH 4 내지 5일 수 있다. pH 3 미만에서는 생육이 거의 불가 능하고, 포자 생산 수율이 낮아질 우려가 있고, pH 8을 초과하는 경우 최종 배양산 물의 양이 감소할 수 있다. 상기 pH는 배양 초기의 pH를 말하며, 배양 초기란 영양 배지를 멸균하고 심플리실리움 균을 접종하기 전 상태에서 균을 접종한 직후까지를 말한다. pH 조정은 멸균 이전에 이투어지나 최종적인 pH 측정은 멸균 직후에 이루 어지므로 최초로 균을 접종하여 생장하기 시작하는 시기의 pH를 배양초기 pH로 잡 는다.
배양시, 배양 기간은 500 내지 5000 L배양기 기준으로, 20시간 이상, 구체 적으로 30시간 내지 7일, 보다 구체적으로 50시간 내지 6일일 수 있다. 배양 기간 이 20시간 미만인 경우, 포자 보다 균사가 많아 건조 시 수율이 낮아질 우려가 있 고, 배양 기간이 7일을 초과하는 경우 배양 과정에서 생긴 물질이 미생물을사멸시 켜 수율이 낮아질 우려가 있다. In culture, the pH is suitably adjusted to 3 to 8, preferably pH 3.5 to 6, more preferably pH 4 to 5. If the pH is less than 3, it is almost impossible to grow, the yield of spore production may be low, and if the pH is exceeded 8, the amount of the final culture may be reduced. The pH refers to the pH of the initial culture, the initial culture refers to the sterilization of the nutrient medium and immediately after the inoculation of the bacteria in the state before the inoculation of the simple bacilli. pH adjustment is carried out before sterilization, but final pH measurement is made immediately after sterilization, so the pH at the time of inoculation and growth of the first bacteria is taken as the initial pH. In the culture, the culture period may be 20 hours or more, specifically 30 hours to 7 days, more specifically 50 hours to 6 days, based on 500 to 5000 L incubator. If the incubation period is less than 20 hours, there is a fear that the yield is lower when drying more spores than spores, and if the incubation period exceeds 7 days, the material produced during the culturing process may kill the microorganisms and lower the yield.
배양 시, 교반속도 및 공기 투입율은 미생물의 생육에 맞추어 적절히 설정 할 수 있으며, 배양 조건 및 상태에 따라 변동될 수 있다. 또한, 배양 시 생성되는 기포는 오염 등의 문제를 일으킬 수 있기 때문에 제거하는 것이 바람직하며, 예컨 대 KONION GE-304등과 같은 장치를 이용하여 소포를 실시할 수 있다. 초기 투입비 율은 0.1내지 1 w/v%수준으로 할 수 있으며 , 배양 중에 0.01 내지 0.1 /^의 수 준으로 기포가 생길 때마다 투입하여 소포할 수 있다. During the cultivation, the stirring speed and the air input rate may be appropriately set according to the growth of microorganisms, and may vary depending on the culture conditions and conditions. In addition, since bubbles generated during the culture may cause problems such as contamination, it is preferable to remove them. For example, defoaming may be performed using a device such as KONION GE-304. The initial input ratio may be 0.1 to 1 w / v% level, and can be defoamed every time bubbles are generated at the level of 0.01 to 0.1 / ^ during the culture.
최초 배양 직후 -90 내지 -10°C, 구체적으로 -80 내지 -20 °C, 보다 구체적 으로 -70 내지 -30 °C의 은도로 동결하여 동결건조를 실시할 수 있으나, 배양액의 양이 많아 한 번에 동결을 시키기 어려운 경우에는 최종 pH를 5미만으로조정함으 로써 오염과 미생물의 번식을 억제할 수 있다. Immediately after the initial incubation, freeze-drying may be performed by freezing at a temperature of -90 to -10 ° C, specifically -80 to -20 ° C, more specifically -70 to -30 ° C, but the amount of the culture medium is high. In cases where it is difficult to freeze at a time, the final pH can be adjusted to less than 5 to suppress contamination and the growth of microorganisms.
【유리한 효과】 Advantageous Effects
본 발명은 심플리실리움 라멜리콜라 BCKSimplici Ilium lamellicola BCP) 균주의 최적 배지 조성 및 배양 조건을 확립함으로써, 균질한 배지를 얻을 수 있고, 심플리실리움 라멜리콜라 BCP 균주의 포자 생산 수율을 2배 이상 높일 수 있으며, 포자 안정성을 크게 향상시킨다는 장점이 있다. The present invention can obtain a homogeneous medium by establishing the optimum medium composition and culture conditions of the strains of Simpliclium lamellicola BCKSimplici Ilium lamellicola BCP), more than double the yield of spores of the Simpliclium lamellicola BCP strain It can be increased, and the spore stability is greatly improved.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 배양 온도에 따른 심플리실리움 라멜리콜라 BCKSimplici Ilium lamellicola BCP)의 포자 생산수율 측정 결과를 도시한 것이다. Figure 1 shows the results of measuring the spores production yield of Simplicillium lamelicola BCKSimplici Ilium lamellicola BCP) according to the culture temperature.
도 2는 배양 초기 PH에 따른 심플리실리움 라멜리콜라 BCP의 포자 생산 수 율 측정 결과를 도시한 것이다.
도 3은 배양 시간에 따른 심플리실리움 라멜리콜라 BCP의 포자 생산 그래프 이다. Figure 2 shows the results of measuring the spores production yield of the Simplisium lamelicola BCP according to the initial culture P H. Figure 3 is a graph of the spore production of the simplicity lamella cola BCP over time incubation.
도 4는 초기 접종 농도에 따른 심플리실리움 라멜리콜라 BCP의 포자 생산 그래프이다. Figure 4 is a graph of spore production of Simplisium lamelicola BCP according to the initial inoculation concentration.
도 5는 과당 (fructose) 농도에 따른 심플리실리움 라멜리콜라 BCP의 포자 생산수율 측정 결과를 도시한 것이다. Figure 5 shows the results of measuring the spores production yield of simplicity lamellicola BCP according to the fructose concentration (fructose).
도 6은 대두분 (Soybean meal) 농도에 따른 심플리실리움 라멜리콜라 BCP의 포자 생산 수율 측정 결과를 도시한 것이다. Figure 6 shows the results of measuring the spores production yield of Simpliclium lamella cola BCP according to the soybean meal concentration.
도 7은 PBD(Plackett-Burman Design)에 따라 배지 성분이 심플리실리움 라 멜리콜라 BCP균주의 포자 생산 수율에 미치는 주요인 효과 분석 결과를 도시한 것 이다. Figure 7 shows the results of the main effect analysis of the media components on the yield of spores of the simplicity la melicola BCP strain according to Plackett-Burman Design (PBD).
도 8은 배지 종류에 따른 심플리실리움 라멜리콜라 BCP의 배양 수율 변화를 나타낸 그래프이다. Figure 8 is a graph showing the change in culture yield of Simplisium lamelicola BCP according to the type of medium.
도 9는 배지 종류에 따른 심플리실리움 라멜리콜라 BCP의 경시 안정성 변화를 나타낸 그래프이다. Figure 9 is a graph showing the changes over time stability of Simplisium lamelicola BCP according to the type of medium.
【발명의 실시를 위한 최선의 형태】 [Best form for implementation of the invention]
이하, 본 발명에 따르는 실시예 및 본 발명에 따르지 않는 비교예를 통하여 본 발명을 보다 상세히 설명하나, 본 발명의 범위가 하기 제시된 실시예에 의해 제 한되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited by the examples given below.
<실시예 1> <Example 1>
하기 표 1에 기재된 조성에 따라 생장 배지를 제조하였다: Growth medium was prepared according to the composition described in Table 1 below:
【표 1】 Table 1
배지 조성
Badge composition
<실험예 > Experimental Example
1. 균주 및 기본 배지 선정 1. Selection of strains and basal medium
심플리실리움 라멜리콜라 BCP 균주를 한국화학연구원으로부터 분양받아 사 용하였다 [기탁번호: KCTC0639BP]. 상기 균주 1 H배지에 접종하여 하기와 같 은 실험을 수행하였다. Simplicium lamelicola BCP strain was distributed from Korea Research Institute of Chemical Technology and used [Accession Number: KCTC0639BP]. The strain 1 H medium was inoculated to carry out the experiment as follows.
기본 배지로서 CDB(Czapek-Dox Broth: sucrose 30 g/L, sodium nitrate 3.0 g/L, dipotassium phosphate 1.0 g/L, magnesium sulfuate 0.5 g/L, potassium chloride 0.5 g/L, ferrous sulfate 0.01 g/L, pH 7.3)를 선정하였다. CDB (Czapek-Dox Broth: sucrose 30 g / L, sodium nitrate 3.0 g / L, dipotassium phosphate 1.0 g / L, magnesium sulfuate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate 0.01 g / L , pH 7.3).
2. 물리적, 화학적 배양조건 검토 2. Review of physical and chemical culture conditions
CDB 배지 (50ml, 250ml-삼각플라스크)에서 배양 온도, 배양 초기 pH, 배양 시간과 같은 물리적, 및 화학적 배양 조건이 심플리실리움 라멜리콜라 BCP 균주의 포자 생산량에 미치는 영향을 조사하였다. The effects of physical and chemical culture conditions such as culture temperature, initial pH and culture time on CDB medium (50ml, 250ml-triangular flasks) on the spore production of S. aureus spp.
PDA에서 7 ~ 10일간자란 균총의 5x5raf 절편 5개를 접종하였다. 포자 생산 수율을 조사하기 위해서, 배양액 내 포자의 수를 400배 현미경 하에서 호모사이토 미터를 이용하여 계수하였다. 배양액 내의 배양액의 물성은 대체로 균질적이었으나 전체적으로 작은 균체 덩어리가 다수 형성되었으며 현미경으로 관찰한 결과 이 덩 어리에 많은 포자가 뭉쳐있는 것이 관찰되었다. 그래서 배양이 끝난 후 균질기로 10초간 마쇄하여 포자를 균질하게 풀어 준 후에 보르텍스 믹서 (vortex mixer)로 충
분히 현탁시킨 다음 포자 수를 계수하였다. 시험은 3회 반복 시험하였다. PDA was inoculated with 5 x 5raf sections of the bacterium grown for 7-10 days. To examine the yield of spore production, the number of spores in the culture was counted using a homocytometer under a 400x microscope. The physical properties of the cultures in the culture were largely homogeneous, but a large number of small cell masses were formed as a whole, and the microscopic observation showed that many spores were collected in the mass. So, after incubation, grinding with a homogenizer for 10 seconds to homogenize the spores and fill with a vortex mixer. After suspension, the spore count was counted. The test was repeated three times.
(1) 배양은도에 따른 영향측정 (1) Influence measurement according to culture
배지 제조 시 초기 PH는 7.3이었고, 배양 은도는 20, 22, 25, 27, 30°C로 달리하여 5일간 배양 (120 rpm)한 다음, 위에 설명한 방법에 따라 배양액 내 포자 수를 계수하였다. The initial P H was 7.3 when the medium was prepared, and the culture silver was cultured at 120 rpm for 5 days at 20, 22, 25, 27, and 30 ° C., and then the number of spores in the culture medium was counted according to the method described above.
심플리실리움 라멜리콜라 BCP은 20°C 이하의 저온에서는 포자 형성 수율이 다소 떨어졌고, 22°C가 최적 은도였으며, 25°C 이후부터 포자 형성 수율이 떨어지 는 경향을 나타내었다 [도 1]. Simplisium lamelicola BCP showed a slight drop in sporulation yield at a low temperature below 20 ° C., 22 ° C. was the best silver, and a decrease in sporulation yield after 25 ° C. [FIG. 1] .
(2) 배양초기 PH에 따른 영향 측정 (2) Measurement of influence according to initial culture P H
배양 온도는 22°C로 고정하였고, 배지의 pH는 HC1과 NaOH를 사용하여 각각 4, 5, 6, 7, 8로 조정하여 5일간 배양 (120 rpm)한 다음, 배양액 내 포자 수를 계수 하였다. The culture temperature was fixed at 22 ° C. The pH of the medium was adjusted to 4, 5, 6, 7, 8 using HC1 and NaOH, respectively, and cultured for 5 days (120 rpm), and the number of spores in the culture was counted. .
배지의 초기 pH가 심플리실리움 라멜리콜라 BCP의 포자 생산 수율에 미치는 영향을 검토한 결과 7.0 이상의 염기성 pH에서는 포자 생산 수율이 낮았고 pH 4.0 ~ 5.0에서 우수하였으며 6,0 이상에서는 낮아졌다. 최적 pH는 4.5로 판단되었다 [도 2]. (3) 배양 시간에 따른 영향 As a result of examining the effect of the initial pH of the medium on the spore production yield of Simpliclium lamelicola BCP, the spore production yield was low at basic pH of 7.0 or higher, excellent at pH 4.0-5.0, and lower than 6,0. The optimum pH was determined to be 4.5 [FIG. 2]. (3) Effect of incubation time
상기 두 실험에서 결정된 최적 은도 22°C와 최적 초기 pH 4.5 조건으로 1L 플라스크 (working volume 200 ml)에서 3회 반복으로 9일간 배양 (150 rpm)하면서 24 시간 간격으로 5 ml씩 시료를 채취하여 배양액 내 포자 수를 계수하였다. Determined in two experiments the optimum silver is 22 ° C and optimally to the initial pH 4.5 condition three times in 1L flask (working volume 200 ml) 9 ilgan culture (150 rpm), while the culture medium was collected for each 5 ml sample in a 24-hour interval My spore count was counted.
배양 시간에 따른 포자 생산 추이를 조사한 결과 3일째까지는 포자가 거의 형성되지 않다가 3일 이후부터 급속히 형성되기 시작하여 5일째 거의 최대 수준에 도달하였으며, 8일까지 일정 수준을 유지하였다 (도 3).
3. 배지 최적화조건 실험 As a result of investigating the trend of spore production according to the incubation time, almost no spores were formed until day 3, but rapidly formed after 3 days, reaching the maximum level on day 5, and maintaining a constant level until day 8 (Fig. 3). . 3. Medium optimization condition experiment
(1) 탄소원에 따른 영향 측정 (1) measuring the impact of carbon sources
탄소원이 포자 생산 수율이 미치는 영향을 조사하기 위하여 CDB 배지의 탄 소원으로 쓰이고 있는 수크로오스 (sucrose)를 각각 다른 탄소원 1.5%(w/v)로 대체 첨가하여 제조하였다. 이 실험에서 시험한 탄소원은 가용성 전분 (soluble starch), 덱스트린 (dextrin), 말토오스 (maltose), 락토오스 (lactose) , 만니를 (mannitol), 갈 락토오스 (galactose), 과당 (fructose) , 글루코오스 (glucose) , CMC였다. 배양조건은 온도 22°C, 초기 pH 4.5, 120 rpm으로 고정하여 5일간 배양하였다. 최종 배양액에 서 포자 수를 계수하였고 배양액의 물성도 조사하였다. 시험은 3회 반복 시험하였 다. In order to investigate the effect of spore production yield on the carbon source, sucrose, which is used as a carbon source of CDB medium, was prepared by substituting 1.5% (w / v) of different carbon sources. Carbon sources tested in this experiment were soluble starch, dextrin, maltose, lactose, mannitol, galactose, fructose and glucose. , CMC. Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. The number of spores in the final culture was counted and the properties of the culture were also investigated. The test was repeated three times.
탄소원이 포자 생산 수율에 미치는 영향을 조사한 결과 기본 배지인 CDB의 수크로오스 보다 우수한 효과별로 보인 탄소원은 과당이었고, 나머지 탄소원들은 수크로오스 보다 포자 생산 수율이 낮았을 뿐만 아니라 배양액의 물리적인 특성도 불리하였다 (표 2). 가용성 전분은 균사덩어리가 매우 크게 형성되었고 말토오스, 락토오스, 갈락토오스도 비교적 큰 균사덩어리가 매우 많이 형성되었던 반면, 포자 수는 낮았다. 글루코오스는 균사덩어리가 비교적 적은 편이었지만 포자수가낮았다. 덱스트린은 배양액이 덩어리가 거의 없이 매우 균질적이었으나, 포자수가 비교적 낮은 편이었다. 과당은 기본 배지 CDB보다는 덜 균질적이었지만, 포자수가 CDB 약 47%증가하였고, 균사 덩어리도 거의 볼 수 없었다. 만니를과 CMC에서는 곰팡이 생 장이 일어나지 않았다. 이것은 이 형태의 탄소원을 심플리실리움 라멜리콜라 BCP이 이용하지 못하기 때문으로 생각되었다. As a result of investigating the effect of carbon source on the spore production yield, the carbon source showed better effect than sucrose of CDB, the basal medium, and the other carbon sources showed lower spore production yield than sucrose as well as the physical characteristics of the culture (Table 2). Soluble starch was very large in mycelial mass and maltose, lactose and galactose were also relatively large in mycelial mass, while the spore count was low. Glucose had a relatively low mycelial mass but low spore count. Dextrins were very homogenous with little lumps, but relatively low spore count. Fructose was less homogeneous than basal medium CDB, but spore count increased by approximately 47% and little mycelial mass was seen. No mold growth occurred at Mannier and CMC. This was thought to be due to the fact that the carbon source of this type was not available to the Simlyillium lamelicola BCP.
【표 2】 Table 2
심플리실리움 라멜리콜라 BCP의 포자 생산 수율과 배양 특성에 미치는 탄소원의 영향
탄소원 포자 밀도 Effects of Carbon Sources on Spore Production Yield and Culture Characteristics of Simpliclium Lamelicola BCP Carbon Source Spore Density
배양액의 특성 Characteristics of culture
(1.5 w/v%) (conidia/ml ) (1.5 w / v%) (conidia / ml)
가용성 전분 (Soluble starch) 3.2E+6 균사덩어리 형성 (+++) 덱스트린 (Dextrin) 4.2E+7 균질적 (+++) 말토오스 (Mal tose) 6.3E+6 균사명어리 형성 (++) 락토오스 (Lactose) 1.9E+7 균사명어리 형성 (++) 만니톨 (Manitol ) 생장하지 않음 - 갈락토오스 (Galactose) 7.5E+6 균사명어리 형성 (++) Soluble starch 3.2E + 6 mycelial formation (+++) Dextrin 4.2E + 7 Homogeneous (+++) Mal tose 6.3E + 6 Mycelial formation (++) Lactose 1.9E + 7 Mycelial Formation (++) Manitol does not grow-Galactose 7.5E + 6 Mycelial Formation (++)
과당 (Fructose) 2.7E+8 균질적 (+) 글루코오스 (Glucose) 3.4E+6 균사덩어리 형성 (+) Fructose 2.7E + 8 Homogeneous Glucose 3.4E + 6 Mycelial formation (+)
CMC 생장하지 않음 - 수크로오스 (Czapek-Dox broth, 대조) 1.7E+8 균질적 (++) CMC Not Grown-Sucrose (Czapek-Dox broth, Control) 1.7E + 8 Homogeneous (++)
(2) 과당 (Fructose) 농도에 따른 영향 측정 (2) measuring the effect of fructose concentration
상기 탄소원 실험에서 포자 생산 수율이 제일 높고 배양액의 물성도 우수했 던 과당에 대하여 농도에 따른 포자 생산 수율을 조사하였다 . CDB 기본 배지에서 탄소원으로 쓰이고 있는 수크로오스를 빼고 과당으로 대체하여 1.0 w/v%부터 1.0 w/v% 단위로 5.0 w/v%까지 과당 함량을 높여가며 배지를 제조하였다. 배양조건은 온도 22°C , 초기 pH 4.5, 120 rpm으로 고정하여 5일간 배양하였다 . 최종 배양액에 서 포자 수를 계수하였다 . 시험은 3회 반복 시험하였다 . In the carbon source experiment, the yield of spore production according to the concentration of fructose, which had the highest yield of spore production and excellent physical properties, was investigated. The medium was prepared by removing the sucrose used as a carbon source from the CDB basal medium and replacing the fructose with an increase in the fructose content from 1.0 w / v% to 5.0 w / v% in units of 1.0 w / v%. Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. Spore count was counted in the final culture. The test was repeated three times.
위의 탄소원 시험 결과에 따라 과당의 최 적 농도를 조사하였다 . 1.0 w/v%부 터 1.0 w/v% 간격으로 5.0 w/v%까지 시험한 결과, 3.0 ~ 5.0 w/v%까지 높은 포자 생산 수율을 보여주었고 그 이 전에는 감소하는 경 향을 보였다 (도 5) . 과당의 탄소 원 농도 변이를 고려하여 최 적 조건은 3.0%로 정하였다 . The optimum concentration of fructose was investigated according to the above carbon source test results. Tests from 1.0 w / v% to 5.0 w / v% at 1.0 w / v% intervals showed high yields of spore production from 3.0 to 5.0 w / v%, with decreasing trend before then (Fig. 5). Considering the variation in carbon source concentration of fructose, the optimal condition was set at 3.0%.
(3) 질소원에 따른 영향 측정 (3) measuring the effect of nitrogen source
질소원이 포자 생산 수율이 미 치는 영 향을 조사하기 위하여 CDB 배지의 질
소원으로 쓰이고 있는 NaN03를 3 /^를 없애고 각각 다른 질소원을 1.0 w/v% 첨가 하여 제조하였다. 탄소원은 앞의 실험에서 결정된 과당 3 ΦΛ로 수크로오스 3 h 대체하였다. 이 실험에서 시험한 질소원은 대두분, 이스트 추출물, 트립톤 (tryptone), 펩톤 (peptone), 비프 추출물 (beef extract), (NH4)2S04, NH4C1, H2C02, N¾N03였다. 배양조건은 온도 22 °C, 초기 pH 4.5, 120 rpm으로 고정하여 5일간 배 양하였다. 최종 배양액에서 포자 수를 계수하였다. Quality of CDB medium to investigate the effect of nitrogen source on spore production yield NaN0 3 used as a wish was prepared by removing 3 / ^ and adding another nitrogen source 1.0 w / v%. The carbon source was replaced with sucrose 3 h by fructose 3 ΦΛ, determined in the previous experiment. The nitrogen sources tested in this experiment were soybean meal, yeast extract, tryptone, peptone, beep extract, (NH4) 2 S0 4 , NH 4 C1, H 2 C0 2 , and N¾N0 3 . Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. Spore count was counted in the final culture.
질소원이 포자 생산 수율에 미치는 영향을 조사한 결과, 기본 배지인 CDB의 질산나트륨보다 우수한 효과별로 보인 질소원은 대두분 (soybean meal) 및 트립톤이 었다 (표 3). 전반적으로 암모니아 형태의 질소원은 심플리실리움 라멜리콜라 BCP이 잘 이용하지 못하는 것으로 나타났다. As a result of investigating the effect of nitrogen source on the spore production yield, the soybean meal and trypton were shown to be superior to sodium nitrate of CDB, the basal medium (Table 3). Overall, the nitrogen source in the form of ammonia was found to be poorly used by the Simlyillium lamelicola BCP.
【표 3】 Table 3
심플리실리움 라멜리콜라 BCP의 포자 생산 수율과 배양 특성에 미치는 질소원의 영향 Influence of Nitrogen Sources on Spore Production Yield and Culture Characteristics of Simplisium Lamelicola BCP
상기 질소원 실험에서 포자 생산수율이 제일 높았고 배양액의 물성도 우수 했던 대두분에 대하여 농도에 따른 포자 생산수율을 조사하였다. 기본 배지 CDB에 서 탄소원은 과당 3 h 사용하였고 질소원은 대두분을 0.5 w/v%부터 4.0 w/v% 까지 1.0 hVA 증가시켜 배지를 제조하였다. 배양조건은 온도 22°C, 초기 pH 4.5, 120 rpm으로 고정하여 5일간 배양하였다. 최종 배양액에서 포자 수를 계수하였다. 시험은 3회 반복 시험하였다. In the nitrogen source experiment, the yield of spore production according to the concentration was investigated for the soybean meal having the highest yield of spores and excellent physical properties of the culture solution. In the basal medium CDB, a carbon source was used for 3 h of fructose and a nitrogen source was prepared by increasing soy flour 1.0 hVA from 0.5 w / v% to 4.0 w / v%. Culture conditions were fixed at a temperature of 22 ° C, initial pH 4.5, 120 rpm and incubated for 5 days. Spore count was counted in the final culture. The test was repeated three times.
위 질소원 실험에서 우수한 질소원으로 선발된 대두분의 최적 농도를 알아 보기 위하여 0.5 w/v%부터 1.0 w/v%단위로 4.0 w/v%까지 농도 시험을 시행한 결과 3.0 ~ 4.0 w/v¾에서 최대 포자 생산수율을 보였다 (도 6). 그러나, 2.0 w/v%이하 에서는 급격히 포자 생산 수율이 감소하는 것으로 나타났다. 대두분의 질소원 농도 변이 및 원료비용 등을 고려하여 최적 조건은 3.0 정하였다. In order to find out the optimum concentration of soy flour selected as an excellent nitrogen source in the above nitrogen source experiment, concentration test was performed from 0.5 w / v% to 4.0 w / v% in units of 1.0 w / v% from 3.0 to 4.0 w / v¾. The maximum spore yield was shown (FIG. 6). However, below 2.0 w / v%, the yield of spores decreased rapidly. The optimum condition was 3.0 in consideration of the variation of nitrogen source concentration of soy flour and raw material cost.
(5) 무기물원에 따른 영향 (5) Influence by mineral source
CDB에서 무기물원으로 함유되어 있는 이인산칼륨 (Dipotassiura phosphate), 황산마그네슘 (magnesium sulfuate), 염화칼륨 (potassium chloride), 황산철 Dipotassiura phosphate, magnesium sulfate, potassium chloride and iron sulfate, which are contained as mineral sources in CDB
(ferrous sulfate)을 ¾배고 9가지 무기물 (magnesium sulfate, sodium chloride, potassium chloride, ferrous sulfate, calcium chloride, cupric sulfate, magnesium chloride, Zinc sulfate, barium chloride)을 한 가지씩 0.1 w/v% 첨가 하여 포자 생산 수율에 미치는 영향을 시험하였다. 탄소원은 과당 3.0 w/v%, 질소 원은 대두분 3.0 h 첨가하였다. 배양조건은 배양온도 25°C, 초기 pH 4.5, 120 rpm으로 하여 5일간 배양하였다. 최종 배양액에서 포자 수를 계수하였고 시험은 3 회 반복하였다. Spore production by adding ¾ ferrous sulfate and 0.1 w / v% of 9 minerals (magnesium sulfate, sodium chloride, potassium chloride, ferrous sulfate, calcium chloride, cupric sulfate, magnesium chloride, Zinc sulfate, barium chloride) The effect on yield was tested. The carbon source was added 3.0 w / v% of fructose and the nitrogen source 3.0 h. Culture conditions were incubated for 5 days at a culture temperature of 25 ° C, initial pH 4.5, 120 rpm. Spore count in the final culture was counted and the test was repeated three times.
각종 무기물원이 포자 생산 수율에 미치는 영향을 조사한 결과, 단일 무기물이 포자 생산 수율을 증가시키는 효과는 나타나지 않았다 (표 4). 이것은 여러 무기물이 동시에 일정양이 필요하거나 어느 한 가지가 절대적으로 필요하지는 않다는 것을 의미할 수 있다.
1 As a result of investigating the effect of various mineral sources on the yield of spores, there was no effect of increasing the yield of spores from a single mineral (Table 4). This may mean that several minerals need a certain amount at the same time, or that neither is absolutely necessary. One
【표 4】 Table 4
심플리실리움 라멜리콜라 BCP의 포자 생산 수율과 배양 특성에 미치는 무기물원의 영향 Influence of Mineral Sources on Spore Production Yield and Culture Characteristics of Simplisium Lamelicola BCP
이 실험으로 필요한 무기물원을 결정하기 어 렵기 때문에 CDB에서 주로 사용 되는 무기물원을 중심으로 주요인 효과를 찾아내는 PBD 기술을 적용하여 주로 필요 한 무기물원을 선정하고자 하였다. Since it is difficult to determine the mineral source required by this experiment, we tried to select the mineral source that is mainly needed by applying the PBD technique that finds the main effects centered on the mineral source mainly used in the CDB.
(6) PBDCPlackett-Burman Design) (6) PBDCPlackett-Burman Design)
상기 실험에서 탄소원으로 선발된 과당과 질소원으로 선발된 대두분을 포함 하여 기존의 질소원 질산나트륨과 기본 배지 CDB의 무기물 4가지 (magnesium sul fate, dipotassium phosphate, potassium chloride, ferrous sul fate) 증에서 포자 생산 수율에 크게 영향을 미치는 요인을 찾아내기 위하여 PBD(Plackett- Burman Design)에 따라 시험하였다. PBD란 사용된 배지성분이 생장에 어떤 영향을 주는지를 알 수 있는 통계적 처리 방법 이며, 이 것은 배지 성분 중 주효과를 나타내 는 성분들을 찾아낼 수 있다 .
PBD는 하기 표 5와 같이 12개의 실험군과 3개의 기준군을 사용하고 12개의 실험군에는 +run 은 200% 의 배지 성분을 , -run 은 20¾)의 배지 성분으로 하여 12개 run을 각각 배지별로 +6개 , -6개씩 무작위화 하여 만든 배지와 중심 값인 0값으로 이뤄진 배지 (기준군)를 3개 만들어 그 값을 기준으로 한다 . 1~12번까지는 +와 - 의 무작위화한 배지 조성에 의한 것이고 , 1315번은 중심값에 의 한 배지 조성 이다. 이 각 run에 대하여 얻어진 포자 밀도값에 대하여 표준 편차 의해 그 실행치의 증가가 그 값에 어떤 효과를 보여주는지를 알 수 있게 된다. 주효과는 각 성분에 대한 +run의 표준편차 값에서 -run 값을 뺀 것으로 나타내는데 +표준편차 값이 높은 것일수록 주요 효과를 나타내는 배지 성분이 되며, 이 성분의 증가가 세포의 성장에 절대적 인 도움이 된다는 것을 의미 한다 . Production of spores from four kinds of minerals of the existing nitrogen source sodium nitrate and basic medium CDB (magnesium sul fate, dipotassium phosphate, potassium chloride, ferrous sul fate), including fructose selected as carbon source and soy flour selected as nitrogen source in the experiment. In order to find a factor that greatly affects the yield, it was tested according to Plackett-Burman Design (PBD). PBD is a statistical method of knowing how the media component used affects the growth, and it is possible to find out the components of the media component that have the main effect. PBD uses 12 test groups and 3 reference groups as shown in Table 5 below. In 12 test groups, + run is a medium component of 200% and -run is a medium component of -20¾). Create three randomized badges of 6 and -6 pieces each, and create three badges (reference group) consisting of a central value of zero. Numbers 1-12 are due to the randomized media composition of + and-, and number 1315 is the media composition by the median. With respect to the spore density values obtained for each of these runs, the standard deviation shows how the increase in the run value affects the values. The main effect is indicated by subtracting the -run value from the standard deviation value of + run for each component.The higher the standard deviation value is, the more important the media component is.The increase in this component is an absolute help for cell growth. It means.
【표 5】 Table 5
이 연구에 적용한 Plackett-Burman Design(PBD) Plackett-Burman Design (PBD) applied to this study
Soybean Magnesium Sodium Dipotassium Potassium FerrousSoybean Magnesium Sodium Dipotassium Potassium Ferrous
Run Fructose Run fructose
meal sul fate nitrate phosphate chlor ide sul fate meal sul fate nitrate phosphate chlor ide sul fate
1 +(30) -(3.0) +(5.0) -(0.3) -(0.1) -(0.5) +(0.01)1 + (30)-(3.0) + (5.0)-(0.3)-(0.1)-(0.5) + (0.01)
2 +(30) +(30) -(0.5) +(3.0) -(0.1) -(0.5) -(0.001)2 + (30) + (30)-(0.5) + (3.0)-(0.1)-(0.5)-(0.001)
3 -(3.0) +(30) +(5.0) -(0.3) +(1.0) -(0.5) -(0.001)3-(3.0) + (30) + (5.0)-(0.3) + (1.0)-(0.5)-(0.001)
4 +(30) -(3.0) +(5.0) +(3.0) -(0.1) +(5.0) -(0.001)4 + (30)-(3.0) + (5.0) + (3.0)-(0.1) + (5.0)-(0.001)
5 +(30) +(30) -(0.5) +(3.0) +(1.0) -(0.5) +(0.01)5 + (30) + (30)-(0.5) + (3.0) + (1.0)-(0.5) + (0.01)
6 +(30) +(30) +(5.0) -(0.3) +(1.0) +(5.0) -(0.001)6 + (30) + (30) + (5.0)-(0.3) + (1.0) + (5.0)-(0.001)
7 -(3.0) +(30) +(5.0) +(3.0) -(0.1) +(5.0) +(0.01)7-(3.0) + (30) + (5.0) + (3.0)-(0.1) + (5.0) + (0.01)
8 -(3.0) -(3.0) +(5.0) +(3.0) +(1.0) -(0.5) +(0.01)8-(3.0)-(3.0) + (5.0) + (3.0) + (1.0)-(0.5) + (0.01)
9 -(3.0) -(3.0) -(0.5) +(3.0) +(1.0) +(5.0) -(0.001)9-(3.0)-(3.0)-(0.5) + (3.0) + (1.0) + (5.0)-(0.001)
10 +(30) -(3.0) -(0.5) -(0.3) +(1.0) +(5.0) +(0.01)10 + (30)-(3.0)-(0.5)-(0.3) + (1.0) + (5.0) + (0.01)
11 -(3.0) +(30) -(0.5) -(0.3) -(0.1) +(5.0) +(0.01)11-(3.0) + (30)-(0.5)-(0.3)-(0.1) + (5.0) + (0.01)
12 -(3.0) -(3.0) -(0.5) -(0.3) -(0.1) -(0.5) -(0.001)
13 0(16.5) 0(16.5) 0(2.75) 0(1.75) 0(0.55) 0(2.75) 0(0.0055)12-(3.0)-(3.0)-(0.5)-(0.3)-(0.1)-(0.5)-(0.001) 13 0 (16.5) 0 (16.5) 0 (2.75) 0 (1.75) 0 (0.55) 0 (2.75) 0 (0.0055)
14 0(16.5) 0(16.5) 0(2.75) 0(1.75) 0(0.55) 0(2.75) 0(0.0055)14 0 (16.5) 0 (16.5) 0 (2.75) 0 (1.75) 0 (0.55) 0 (2.75) 0 (0.0055)
15 0(16.5) 0(16.5) 0(2.75) 0(1.75) 0(0.55) 0(2.75) 0(0.0055)15 0 (16.5) 0 (16.5) 0 (2.75) 0 (1.75) 0 (0.55) 0 (2.75) 0 (0.0055)
* 괄호안의 값은 각 성분의 함량 (g/L) * The values in parentheses indicate the content of each component (g / L)
최적 탄소원 과당과 최적 질소원 대두분 외에 어느 무기물원이 심폴리실리움 라멜리콜라 BCP의 포자 생산 수율을 향상시키는데 필요한지를 알아보기 위하여 PBD 기술을 적용하였다. 그 결과, 과당, 대두분, 황산마그네슴, 염화칼륨의 함량은 더 높여주는 것이 포자 생산 수율에 유리한 것으로 나타났고, 반대로 질산나트륨의 함량은 줄이는 것이 유리한 것으로 나타났다 (도 7). 이인산칼륨, 황산철은 포자 생산 수율에 큰 영향을 미치지는 않는 것으로 나타났다. In addition to the optimal carbon source fructose and the optimum nitrogen source soy flour, the PBD technique was applied to determine which mineral source is needed to improve the spore production yield of the Simpolysilium lamelicola BCP. As a result, it was found that increasing the content of fructose, soy flour, magnesium sulfate, and potassium chloride is advantageous for the yield of spores, and conversely, it is advantageous to reduce the content of sodium nitrate (FIG. 7). Potassium diphosphate and iron sulfate did not appear to have a significant effect on the yield of spore production.
【표 6】 Table 6
심플리실리움 라멜리콜라 BCP균주의 액체배양 배지 조성 Composition of liquid culture medium of S. simillium lamelicola BCP strain
상기 실험 결과를 종합하여 하기 표 7과 같은 최적 배지 조성을 정하였다. PBD 결과에서 양을 늘리는 것이 유리하다고 판단되었던 황산마그네슘 (magnesium sulfate)과 염화칼륨 (potassium chloride)의 양은 CDB의 함량 기준 0.05 w/v¾보다 높게 0.5 정하였다. 대신 함량올 줄이는 것이 유리하다고 판단되었던 질산나트륨 (sodium nitrate)은 배지 조성에서 제거하였다. PBD에서 주효과가 크게 나타나지 않았던 이인산칼륨 (dipotassium phosphate), 황산철 (ferrous sulfate)은 CDB와 동일한 함량으로 정하였다. 과당과 대두분의 함량은 PBD 결과에 따르면
함량을 늘리는 것이 포자 생산 수율을 올리는데 유리한 것으로 나타났지만 농도별 결과에 따라 농도를 을릴 경우 억제 효과가 나타날 우려가 있었으므로 앞서 실험에서 결정된 최적 농도로 정하였다. 【표 7】 By combining the results of the experiment to determine the optimum medium composition as shown in Table 7. The amount of magnesium sulfate and potassium chloride, which was considered to be beneficial in PBD results, was set to 0.5 higher than 0.05 w / v¾ based on the CDB content. Instead, sodium nitrate, which was found to be beneficial in reducing its content, was removed from the media composition. Potassium diphosphate (dipotassium phosphate) and ferrous sulfate (ferrous sulfate), which did not show major effects in PBD, were set to the same content as CDB. The content of fructose and soy flour is based on PBD results. Increasing the content was found to be advantageous to increase the yield of spores, but according to the results of each concentration, the concentration was reduced, there was a risk of suppression effect, so it was determined as the optimum concentration determined in the previous experiment. Table 7
최적 배지 조성 Optimal medium composition
상기 표 7의 최적 배지 조성과 PDB (포테이토 덱스트로스 배지)로 심플리실리움 라멜리콜라 BCP를 배양한 결과를 도 8 및 도 9에 나타내었다.
8 and 9 show the results of culturing the Simplisilium lamelicola BCP with the optimal medium composition and PDB (potato dextrose medium) shown in Table 7 above.
Claims
【청구항 1】 [Claim 1]
과당, 덱스트린 및 수크로오스로 이루어진 그룹으로부터 선택되는 하나 이 상의 탄소원; 및 At least one carbon source selected from the group consisting of fructose, dextrin and sucrose; And
대두분 (soybean meal) 및 트립톤 (tryptone)으로 이루어진 그룹으로부터 선 택되는 하나 이상의 질소원 One or more nitrogen sources selected from the group consisting of soybean meal and tryptone
을 배지성분으로 포함하는 것을 특징으로 하는 심플리실리움 라멜리콜라 Simplici Ilium lamellicola BCP) 균주 배양용 배지 조성물. Simplici Ilium lamellicola BCP) strain culture medium composition comprising a as a medium component.
【청구항 2】 [Claim 2]
제 1 항에 있어서, The method of claim 1,
황산마그네슘, 염화칼륨, 염화마그네슘, 염화나트륨, 황산철, 염화칼슴, 이 인산칼륨, 황산구리, 황산아연, 질산칼륨, 질산암모늄, 인산이수소칼륨, 요오드화 칼륨 및 몰리브덴산나트륨으로 이루어진 그룹으로부터 선택되는 하나 이상의 무기 물원을 추가로 포함하는 것을 특징으로 하는 심플리실리움 라멜리콜라 BCP균주 배 양용 배지 조성물. At least one selected from the group consisting of magnesium sulfate, potassium chloride, magnesium chloride, sodium chloride, iron sulfate, calcium chloride, potassium phosphate, copper sulfate, zinc sulfate, potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, potassium iodide and sodium molybdate A culture medium composition for simplicity lamella cola BCP strain culture further comprising an inorganic water source.
【청구항 3] [Claim 3]
제 1 항에 있어서, The method of claim 1,
상기 배지에 1.0 내지 5.0 ΦΛ^\ 탄소원 및 0.5 내지 4.0 /^의 질소원이 함유되는 것을 특징으로 하는 심플리실리움 라멜리콜라 BCP균주 배양용 배지 조성 물. A culture medium composition for simplicity lamella cola BCP strain culture, characterized in that the medium contains 1.0 to 5.0 ΦΛ ^ \ carbon source and 0.5 to 4.0 / ^ nitrogen source.
【청구항 4】 [Claim 4]
제 1 항에 있어서, The method of claim 1,
상기 배지는 1.0 내지 5.0 \ 과당; 0.5 내지 4.0 대두분; 0.1 내지 1 황산마그네슘을 필수 배지성분으로 포함하는 것을 특징으로 하는 심
플리실리움 라멜리콜라 BCP 균주 배양용 배지 조성물 . The medium comprises 1.0 to 5.0 \ fructose; 0.5 to 4.0 soy flour; Shim, characterized in that containing 0.1 to 1 magnesium sulfate as an essential medium component Medium composition for cultivation of plysilium lamelicola BCP strains.
【청구항 5] [Claim 5]
제 4 항에 있어서 , The method of claim 4,
0.1 내지 1 ΦΛ^ 염화칼륨 ; 0.05 내지 0.5 \ 이인산칼륨 ; 및 0.0001 내지 0.01 /^의 황산철을 추가로 포함하는 것을 특징으로 하는 심플리실리움 라 멜리콜라 BCP 균주 배양용 배지 조성물 . 0.1 to 1 ΦΛ ^ potassium chloride; 0.05 to 0.5 \ potassium diphosphate; And 0.0001 to 0.01 / ^ of the culture medium composition for simplicity la meli cola BCP strain further comprising iron sulfate.
【청구항 6] [Claim 6]
제 1 항 내지 제 5 항 증 어느 한 항의 배지 조성물에서 심플리실리움 라멜 리콜라 BCP 균주를 배양하는 방법 . A method of culturing a Simpliclium lamella coli BCP strain in the medium composition of any one of claims 1 to 5.
【청구항 7】 [Claim 7]
제 6 항에 있어서, The method of claim 6,
상기 배지에 0.1 내지 5 심플리실리움 라멜리콜라 BCP를 접종하는 것을 특징으로 하는 심플리실리움 라멜리콜라 BCP 균주를 배양하는 방법 . A method for culturing the S. similisium lamelicola BCP strain, characterized in that the medium is inoculated with 0.1 to 5 S. similisium lamelicola BCP.
【청구항 8] [Claim 8]
제 6 항에 있어서, The method of claim 6,
배양 온도가 15 내지 37°C인 것을 특징으로 하는 심플리실리움 라멜리콜라Simplisium lamelicola, characterized in that the culture temperature is 15 to 37 ° C.
BCP 균주를 배양하는 방법 . How to Cultivate BCP Strains.
【청구항 9] [Claim 9]
제 6 항에 있어서, The method of claim 6,
배양 PH가 3 내지 8인 것을 특징으로 하는 심플리실리움 라멜리콜라 BCP 균 주를 배양하는 방법 .
【청구항 10] A method for culturing a S. similirium lamelicola BCP strain, characterized in that the culture P H is 3 to 8. [Claim 10]
제 6 항에 있어서 , The method of claim 6,
배양 기간이 20시간 이상인 것을 특징으로 하는 심플리실리움 라멜리콜라 BCP 균주를 배양하는 방법 .
A method of culturing the Simpliclium lamella cola BCP strain, characterized in that the culture period is 20 hours or more.
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CN117801964A (en) * | 2024-02-29 | 2024-04-02 | 中国食品发酵工业研究院有限公司 | Culture medium for rapid enrichment culture of fungi, culture method and application |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20020034755A (en) * | 2000-11-03 | 2002-05-09 | 고두모 | Medium for Production 0f Cephalosporin C with High Concentration and Producing Method thereof |
JP2005073583A (en) * | 2003-09-01 | 2005-03-24 | Shizuoka Prefecture | New simplicillium microorganism having ability to control citrus disease injury and microbe-controlling method using the same |
KR100576576B1 (en) * | 1997-02-20 | 2006-05-04 | 디에스엠 아이피 어셋츠 비.브이. | Fermentative production of valuable compounds on an industrial scale using chemically defined media |
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KR20010062501A (en) * | 1999-12-21 | 2001-07-07 | 복성해 | A new fungal strain Acremoniem sp. MT70646(KCTC 8973P), novel compounds produced by this strain and their use |
KR20020034755A (en) * | 2000-11-03 | 2002-05-09 | 고두모 | Medium for Production 0f Cephalosporin C with High Concentration and Producing Method thereof |
JP2005073583A (en) * | 2003-09-01 | 2005-03-24 | Shizuoka Prefecture | New simplicillium microorganism having ability to control citrus disease injury and microbe-controlling method using the same |
Cited By (4)
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CN111304100A (en) * | 2020-03-19 | 2020-06-19 | 贵州医科大学 | Entomogenous fungus strain capable of protecting human umbilical vein endothelial cell damage and application thereof |
CN111304100B (en) * | 2020-03-19 | 2022-03-04 | 贵州医科大学 | Entomogenous fungus strain capable of protecting human umbilical vein endothelial cell damage and application thereof |
CN117801964A (en) * | 2024-02-29 | 2024-04-02 | 中国食品发酵工业研究院有限公司 | Culture medium for rapid enrichment culture of fungi, culture method and application |
CN117801964B (en) * | 2024-02-29 | 2024-05-28 | 中国食品发酵工业研究院有限公司 | Culture medium for rapid enrichment culture of fungi, culture method and application |
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