CN112779167B - Aureobasidin A high-yield strain and application thereof - Google Patents
Aureobasidin A high-yield strain and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of microbial pharmacy, and discloses an Aureobasidin A high-yield strain and application thereof. The strain is Aureobasidium pullulans (Aureobasidium pullulans), is obtained by carrying out compound mutagenesis and screening on an original strain separated from a soil sample in a Moganshan scenic area in Hangzhou, Zhejiang, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation numbers are as follows: CGMCC NO. 20887. The fermentation level of Aureobasidin A of the strain can reach more than 990 mug/ml, and the strain can be used as an Aureobasidin A industrial production strain, so that the large-scale production cost of Aureobasidin A is greatly reduced. In addition, the fermentation liquor, the bacterial suspension, the clarified liquid of the fermentation liquor and the concentrate of the fermentation liquor of the strain have strong antifungal activity and can be used for preparing antifungal agents.
Description
Technical Field
The invention relates to the technical field of microbial pharmacy, and particularly relates to an Aureobasidin A high-yield strain and application thereof.
Background
AureobasidinA is a cyclic lipopeptide antibiotic consisting of 9 amino acid molecules, which was first isolated from the culture broth of the black yeast Aureobasidium pullulans by the Kazutoh Takesako group of Japan in 1991. Subsequent researches prove that AureobasidinA has very strong antifungal capacity and can generate toxicity to yeast at a low concentration of 0.1-0.5 mu g/ml. The fungal species to which they are susceptible include: budding yeast (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Candida glabrata (Candida glabrata), Aspergillus nidulans (Aspergillus nidulans), and Aspergillus niger (Aspergillus niger). The mechanism of action of AureobasidinA is to inhibit the activity of Inositol Phosphamide (IPC) synthase, which is dependent on the growth of fungi, and interfere with sphingolipid synthesis, thereby further killing the strain. However, AureobasidinA does not disrupt DNA, RNA and protein synthesis.
Currently, there are few reports on the AureobasidinA production technology. In 1991, Aureobasidium pullulans strains were first isolated from leaves of the Martinus island by the KazutothTakesako team, and the yield of AureobasidinA after fermentation culture was about 140 mu g/ml, and no report of yield increase of AureobasidinA was found. Because of the strong antifungal activity of AureobasidinA, the application field is gradually expanded from potential human medication to biological pesticide, and therefore, the development of the high-level AureobasidinA production technology has important significance.
Disclosure of Invention
The invention provides an Aureobasidin A high-yield strain which is classified and named as Aureobasidium pullulans and is preserved in the common microorganism center of China microorganism culture preservation management committee (address: No. 3 of No. 1 Xilu Beijing Hopkins of the sunward district, China academy of sciences microbial research institute) within 10 months and 14 days in 2020, and the preservation number is as follows: CGMCC NO. 20887.
The invention also provides a preparation method of Aureobasidin A, which comprises the step of fermenting the strain.
Specifically, the fermentation temperature may be 15 to 27 ℃ (e.g., 15, 20, 21, 23, 24, 25, 26, 27 ℃), particularly 23 to 26 ℃.
In particular, the fermentation pH may be between 1.5 and 8.0 (e.g. 1.5, 2, 3, 4, 5, 6, 7, 8), in particular between 6 and 8.
Specifically, the fermentation time may be 1 to 15 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15 days), particularly 6 to 9 days.
Specifically, the strain inoculation amount for the above fermentation may be 0.5% to 10% (e.g., 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), particularly 0.5% to 5%.
Specifically, the medium for the above fermentation may comprise: glucose, yeast basic nitrogen source, polypeptone and ammonium sulfate; more specifically, the medium of the above fermentation may comprise: 40-80g/L (such as 40, 50, 55, 60, 65, 70, 80g/L) of glucose, 10-30g/L (such as 10, 15, 16, 18, 20, 22, 24, 25, 30g/L) of yeast basic nitrogen source, 20-40g/L (such as 20, 25, 26, 28, 30, 32, 34, 35, 40g/L) of polypeptone, 2-10g/L (such as 2, 3, 4, 5, 6, 7, 8, 9, 10g/L) of ammonium sulfate, and the balance of water.
Specifically, the preparation method may further comprise (before fermentation) a step of expanding culture of the strain.
Specifically, the temperature for the above-mentioned amplification culture may be 15 to 27 ℃ (for example, 15, 20, 21, 23, 24, 25, 26, 27 ℃), particularly 23 to 26 ℃.
Specifically, the above-mentioned pH for the scale-up culture may be 1.5 to 8.0 (e.g., 1.5, 2, 3, 4, 5, 6, 7, 8), particularly 6 to 8.
Specifically, the time for the above-mentioned amplification culture may be 6 to 60 hours (e.g., 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 hours), particularly 20 to 54 hours.
Specifically, the medium for the above-mentioned scale-up culture may comprise: glucose, yeast basic nitrogen source; more specifically, the medium for the above-mentioned scale-up culture may comprise: 10-30g/L (e.g., 10, 15, 16, 18, 20, 22, 24, 25, 30g/L) of glucose, 2-10g/L (e.g., 2, 3, 4, 5, 6, 6.7, 7, 8, 9, 10g/L) of yeast basic nitrogen source, and the balance of water.
Specifically, the above preparation method may further comprise a step of extracting the fermentation broth (after fermentation).
The invention also provides a fermentation liquor of the strain.
Specifically, the preparation method of the fermentation liquid comprises the step of fermenting the strain; more specifically, the fermentation parameters have the respective definitions of the invention described above.
In particular, the above method may further comprise an inactivation step, for example, inactivation under heating in a water bath at 70 ℃.
The present invention also provides a clarified liquid (e.g., supernatant, filtrate, etc., which does not contain solid matter (e.g., bacterial cells) in the fermentation broth) of the above-mentioned strain.
Specifically, the method for producing the above clarified liquid comprises a step of subjecting the above fermentation broth (for example, by centrifugation, filtration, sedimentation, or the like) to solid-liquid separation.
The invention also provides a concentrate of the above clarified liquid.
Specifically, the above concentrate may be a solid powder.
Specifically, the preparation method of the concentrate comprises the steps of extracting and concentrating clear liquid.
Specifically, the above extraction may be performed by solvent extraction, and the extraction solvent may be ethyl acetate.
The invention also provides a bacterial suspension of the bacterial strain.
Specifically, the method for producing the bacterial suspension includes a step of dispersing the cells of the strain (for example, obtained by solid-liquid separation of a fermentation liquid of the strain) in a solvent (for example, water).
Specifically, the cells in the bacterial suspension may be live cells or inactivated cells.
The invention also provides an antibacterial agent which comprises at least one of the fermentation liquor, the clear liquid, the concentrate and the bacterial suspension.
Specifically, the antibacterial agent may be at least antifungal (for example, budding yeast (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Candida glabrata (Candida glabrata), Aspergillus nidulans (Aspergillus nidulans), Aspergillus niger (Aspergillus niger) and the like).
Specifically, the antibacterial agent may be in the form of a drug, an agricultural chemical, a daily necessity, or the like.
The invention also provides the application of the strain and a fermentation product (such as fermentation liquor, supernatant of the fermentation liquor, concentrate of the supernatant, bacterial suspension and the like) thereof in the preparation of Aureobasidin A.
The invention also provides the application of the strain and fermentation products (such as fermentation liquor, clarified liquid of the fermentation liquor, concentrate of the clarified liquid, bacterial suspension and the like) thereof in the preparation of antibacterial agents (particularly antifungal agents).
The invention takes black yeast Aureobasidium pullulans separated from soil samples of Moganshan scenic spots in Hangzhou, Zhejiang as an original starting strain, obtains the Aureobasidin A high-yield strain through compound mutagenesis and screening, has the production capacity remarkably improved compared with that of the original strain, has the fermentation level of Aureobasidin A of more than 990 mu g/ml, is 13.75 times of that of the original strain under the same culture condition, has the remarkable improvement compared with other published report levels, can be used as an Aureobasidin A industrial production strain, and greatly reduces the large-scale production cost of Aureobasidin A. In addition, the fermentation liquor, the bacterial suspension, the clarified liquid of the fermentation liquor and the concentrate of the fermentation liquor of the strain have strong antifungal activity and can be used for preparing antifungal agents.
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FIG. 1 is a HPLC comparison chart of original strain fermentation liquid and a reference solution in the embodiment of the invention.
FIG. 2 shows a mass spectrum of positive ions of a control solution according to an embodiment of the present invention.
FIG. 3 shows a positive ion mass spectrum of the primary peak of the original strain fermentation broth in the embodiment of the present invention.
FIG. 4 shows the comparison of the yield of Aureobasidin A obtained from the high-producing strain prepared in the example of the present invention with that of the original strain.
FIG. 5 is a HPLC comparison chart of the original strain and the prepared high-yield strain fermentation broth in the embodiment of the present invention.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
In the present invention, the term "fermentation broth" refers to a mixture obtained by inoculating a liquid culture medium with a microorganism strain and culturing the mixture for a certain period of time, wherein the mixture contains microorganism cells and metabolites thereof, unused medium components (if possible), and the like.
In the present invention, "inoculum amount" means a ratio of a volume of the seed solution added to a volume of the culture solution after inoculation.
The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.
The present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.
In the following specific examples, unless otherwise specified, the reagents and apparatus used were those commonly used in the art and were obtained commercially; the methods used are conventional in the art, and those skilled in the art can understand how to implement the methods specifically according to the embodiment and achieve the corresponding results.
The media formulations used in the following examples consisted of:
PDA solid medium: 36g of finished culture medium is weighed for American BD company, and is dissolved in 1000ml of purified water before use, and the pH is natural.
Liquid seed culture medium: 20g/L glucose, 6.7g/L yeast basic nitrogen source and the balance of water, and the pH value is 6.5 before disinfection.
Fermentation medium: 60g/L glucose, 20g/L yeast basic nitrogen source, 30g/L polypeptone, 5g/L ammonium sulfate and the balance of water, wherein the pH value before disinfection is 6.5.
Example 1: acquisition of AureobasidinA original producer
The soil sample is taken from the lower layer of rotten leaves in the Mogan mountain scenic spot of Hangzhou city, Zhejiang, and 5g of the soil sample is taken, is diluted by sterile water in a gradient manner, is uniformly coated on a PDA culture medium added with 50 mu g/ml ampicillin sodium and 50 mu g/ml streptomycin sulfate at the same time, and is inversely cultured in a constant-temperature constant-humidity chamber with the relative humidity of 60 percent at the temperature of 25 ℃ for 3 days. Taking a plate with independent single colony distribution, respectively dibbling the cream colony on a fresh PDA culture medium added with 80 mug/ml ampicillin sodium and 80 mug/ml streptomycin sulfate by using a sterile toothpick, inversely culturing for 9 days at 25 ℃ in a constant temperature and humidity chamber with relative humidity of 60 percent, and observing every day. Colonies which were creamy white in the early stage but blackened in the later stage were numbered, individually prepared in a clean bench, and examined under a microscope. Selecting the colony with the corresponding number and the yeast shape in microscopic examination, inoculating the colony to a fermentation culture medium, and performing shake culture in a constant temperature and humidity shaking table at 25 ℃ and 250rpm for 6 days to obtain fermentation liquor. The fermentation liquor takes candida albicans as a verification bacterium, and screening and determination are carried out by adopting a filter paper sheet method, so as to finally obtain a black yeast Aureobasidium pullulans with obvious resistance to the candida albicans. The strain is purified for 5 successive rounds and then deposited in the strain preservation management center of the applicant, and the internal number of the strain is A.pullulans HDCC 101-R13.
Morphological growth characteristics:
inoculating the separated and purified A.pullulans HDCC101-R13 bacterial strain on a PDA solid culture medium, performing inverted culture in a constant-temperature constant-humidity chamber with the relative humidity of 60% at the temperature of 25 ℃, and after 3 days, visually observing cream-colored bacterial colonies with smooth surfaces; the colony portion turned into dark green starting at 7 days; filamentous hyphae can be seen on the surface and the periphery of the colony after 12 days, the color is dark brown, and no free spores can be seen in the examination of a cover glass tabletting microscope.
Inoculating the separated and purified A.pullulans HDCC101-R13 bacterial strain on a PDA solid culture medium, respectively placing the bacterial strain in constant-temperature constant-humidity chambers with relative humidity of 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 27 ℃, 30 ℃, 32 ℃, 37 ℃ and 60 percent, inverting and culturing, observing the size of bacterial colonies, and finding that the suitable growth temperature range is 15-27 ℃ and the optimal growth temperature is 23-25 ℃.
The separated and purified A.pullulans HDCC101-R13 strain is inoculated in liquid seed culture media with different pH values, and the strain can grow well within the pH range of 1.5-8.0.
Example 2: validation of AureobasidinA production ability of pullulans HDCC101-R13 Strain
The separated and purified A.pullulans HDCC101-R13 strain is inoculated in a PDA solid culture medium and is cultivated for 7 days in a constant temperature and humidity chamber with the relative humidity of 60 percent at the temperature of 25 ℃. Inoculating proper amount of lawn to 100ml sterile liquid seed culture medium, and culturing at 25 deg.C and 220rpm on shaking table for 48 hr. After microscopic examination, 0.4ml of seed liquid is sucked by a pipette and inoculated into a 250ml triangular flask containing 40ml of fermentation medium, 5 bottles of fermentation medium are inoculated in total, and the mixture is cultured on a shaking table at the temperature of 25 ℃ and the rpm of 220 for 8 days. And after the fermentation culture is finished, taking 4ml of fermentation liquor, uniformly mixing with 1 volume of absolute ethyl alcohol, ultrasonically soaking for 20min, uniformly mixing again, centrifuging for 15min at 3000rpm, filtering supernate with a 0.45-micrometer membrane, respectively sending the filtered supernate to HPLC (high performance liquid chromatography) and LC-MS (liquid chromatography-mass spectrometry) for detection, and comparing and confirming the filtered supernate with an Aureobasidin A reference substance. The fermentation broth was found to have a distinct peak at RT5.152min, consistent with RT5.149 of AureobasidinA control (as shown in FIG. 1). Further LC-MS analysis determined that the peak had a major relative molecular weight of 1100.7 (as shown in fig. 2 and 3), consistent with AureobasidinA. According to the identification content of Aureobasidin A reference substances, the titers of Aureobasidin A in 5 bottles of fermentation liquor are respectively calculated to be 63.3 mu g/ml, 69.2 mu g/ml, 76.4 mu g/ml, 72.1 mu g/ml and 71.6 mu g/ml, and the average value is 70.5 mu g/ml.
Example 3: AureobasidinA high-yield strain preparation and verification
1) Taking HDCC101-R13 as an original starting strain, inoculating the strain to a PDA solid culture medium, and inversely culturing for 5 days in a constant-temperature constant-humidity chamber with the relative humidity of 60% at the temperature of 25 ℃ to obtain a fresh seed source.
2) Taking a well-grown solid culture lawn 1 dish, scraping off the lawn by using a sterile steel stick, transferring into a triangular flask added with glass beads and 15ml of sterile normal saline, bundling, placing on a shaking table, and vibrating and scattering for 30min to obtain a well-dispersed bacterial suspension.
3) The prepared bacterial suspension is evenly coated on a PDA solid culture medium added with a specific resistance reagent after being subjected to mutagenesis treatment, is subjected to inverted culture for 4 days in a constant-temperature constant-humidity chamber with the relative humidity of 25 ℃ and 60 percent, is then dibbled on a fresh PDA solid culture medium added with the specific resistance reagent, and is subjected to inverted culture for 7 days in a constant-temperature constant-humidity chamber with the relative humidity of 25 ℃ and 60 percent.
4) Taking cultured dibbling bacterial colonies, scraping each bacterial colony by a steel stick separately, inoculating into a triangular flask containing 25ml of liquid seed culture medium, fully grinding the bacterial colony on the flask wall, washing into the culture medium, binding, and placing on a shaker at 25 ℃, 220rpm for culturing for 48 h. Then 0.4ml of the seed solution is respectively sucked into a 250ml triangular flask containing 40ml of the fermentation medium by a liquid suction device for each bottle of seeds, and a 424 bottle (10 bottles of a control) of the fermentation bottles is co-inoculated. All fermentation flasks were incubated on a shaker at 25 ℃ and 220rpm for 8 days.
7) After the fermentation culture is finished, 1ml of fermentation liquor is taken, is uniformly mixed with 4 times of volume of absolute ethyl alcohol, is ultrasonically soaked for 20min, is uniformly mixed again, is centrifuged at 14000rpm for 10min, and is taken as supernatant to be sent to HPLC for detection. Finally, a high-yield strain is obtained, and the titer of AureobasidinA is 990.6 mu g/ml. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 10 months and 14 days in 2020, and the preservation number is as follows: CGMCC NO. 20887.
Example 4: AureobasidinA high-yield strain re-screening verification
1) Using the screened AureobasidinA high-producing strain as a test strain (F)0Generation), inoculating to PDA solid culture medium, culturing in a constant temperature and humidity chamber at 25 deg.C and 60% relative humidity for 4 days to obtain single colony (F)1Generation).
2) Taking cultured single colony, respectively dibbling to PDA solid plate containing specific resistance reagent with toothpick, culturing in a constant temperature and humidity chamber at 25 deg.C and 60% relative humidity for 4 days to obtain dibbled single colony (F)2Generation). The seeds are continuously dibbled and passed for 5 rounds to obtain F5And (5) seeding single colonies.
3) Taking cultured F5And (3) inoculating single colonies, scraping each colony by using a steel stick independently, inoculating the colony into a triangular flask containing 25ml of liquid seed culture medium, fully grinding the colony on the flask wall, washing the colony into the culture medium, binding, and culturing on a shaking table at 25 ℃ and 220rpm for 48 hours. Then 0.4ml of the seed solution was pipetted into a 250ml Erlenmeyer flask containing 40ml of the fermentation medium for each individual bottle of seeds.The mixture was inoculated into 40 flasks and cultured on a shaker at 25 ℃ and 220rpm for 8 days.
4) After the fermentation culture is finished, 1ml of fermentation liquor is taken, is uniformly mixed with 4 times of volume of absolute ethyl alcohol, is ultrasonically soaked for 20min, is uniformly mixed again, is centrifuged at 14000rpm for 10min, and is taken as supernatant to be sent to HPLC for detection. As a result, it was found that the minimum titer of 40 bottles of fermentation samples was 878.8. mu.g/ml, the maximum titer was 1087.0. mu.g/ml, and the average titer was 993.2. mu.g/ml.
The embodiment shows that the AureobasidinA high-yield strain provided by the invention has the advantages of strong production capacity and good genetic stability, and can be used as an industrial production strain of AureobasidinA.
Example 5: initial evaluation of antifungal activity of Aureobasidin A high-yield strain fermentation broth
1) The Aureobasidin A high-yield strain is inoculated in a PDA solid culture medium, and is inversely cultured for 7 days in a constant-temperature constant-humidity chamber with the relative humidity of 60 percent at the temperature of 25 ℃. Inoculating proper amount of lawn to 100ml sterile liquid seed culture medium, and culturing at 25 deg.C and 220rpm on shaking table for 48 hr. Sucking 0.4ml of the seed solution by a pipette, inoculating the seed solution into a 250ml triangular flask containing 40ml of fermentation medium, placing the flask on a shaker at 25 ℃ and 220rpm for culturing for 8 days to obtain fermentation liquor, and combining all the fermentation liquor, heating in 70 ℃ water bath for 1h for inactivation for later use.
2) And transferring the inactivated fermentation liquor to a sterile centrifuge cup, centrifuging for 15min at 14000rpm, and respectively collecting thalli and supernatant. The thalli is washed twice by sterile water and then resuspended by 9 times of the weight of the sterile water to obtain bacterial suspension. Soaking the supernatant with 3 times volume of ethyl acetate, extracting, rotary evaporating, concentrating, and removing ethyl acetate to obtain solid powder (concentrate).
3) Candida albicans (Candida albicans) was inoculated onto a tyrose agar slant, cultured at 30 ℃ for 4 days, and the lawn was washed with a sterile physiological saline solution and broken up with glass beads to give a suspension for assay.
4) Taking a tyrose agar culture medium which is sterilized and cooled to 50 ℃, respectively adding the inactivated fermentation liquor, the bacterial suspension, the supernatant and the concentrate according to the proportion of 0.01% (w/v), 0.02% (w/v), 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.5% (w/v), 1% (w/v), 2% (w/v) and 5% (w/v), adding sterile physiological saline into a control group according to the corresponding proportion, fully shaking, dissolving/uniformly mixing, pouring a flat plate, and solidifying for later use.
5) And (3) taking the solidified plates, inoculating 50 mu L of candida albicans detection bacterial suspension on each plate, uniformly coating, culturing in a constant-temperature constant-humidity chamber with the temperature of 30 ℃ and the relative humidity of 60 percent for 7-14 days, and observing and counting the growth condition of bacterial colonies. 5 replicates were made for each test group and the statistics of Candida albicans growth for each test group are shown in the following table.
TABLE 1 initial evaluation results of antifungal Activity of Aureobasidin A high-yielding strain fermentation broth
Remarking: the bacterial lawn is fully paved on the plate, which shows that the verification bacteria grow vigorously;
"+ +" is a sparse colony on the plate (< 100 cfu/plate-indicating that the test bacteria can grow;
"+" is very few microcolonies (< 3 cfu/dish) on the plate, indicating that the growth of the test bacteria is significantly inhibited;
"-" is the growth of a sterile colony on the plate, indicating that the growth of the test bacterium was completely inhibited.
The results of the example show that the Aureobasidin A high-yield strain inactivated fermentation broth, the supernatant, the bacterial suspension and the concentrate have different degrees of inhibition on the growth of Candida albicans, wherein the inhibition of the concentrate is most obvious, and the fermentation broth and the supernatant are second. The result shows that the Aureobasidin A high-yield strain inactivated fermentation liquor, the supernatant, the bacterial suspension and the concentrate have better development prospects in the field of antifungal agents.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by changing and modifying the embodiments described herein or by using the equivalent structures or equivalent processes of the present invention, and are included in the scope of the present invention.
The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.
Claims (8)
1. A kind ofAureobasidium pullulansThe strain is preserved in China general microbiological culture Collection center (CGMCC) in 2020, 10 months and 14 days, and the preservation number is as follows: CGMCC NO. 20887.
2. A method for preparing Aureobasidin a, comprising the step of fermenting the strain of claim 1.
3. The method of claim 2, wherein the fermentation temperature is 15-27 ℃;
the fermentation pH is 1.5-8.0;
the medium of the fermentation comprises: glucose, yeast basic nitrogen source, polypeptone and ammonium sulfate.
4. The method of claim 2, wherein the fermentation temperature is 23-26 ℃.
5. The method of claim 2, wherein the fermentation pH is 6-8.
6. The method of claim 2, wherein the fermentation medium comprises: 40-80g/L of glucose, 10-30g/L of yeast basic nitrogen source, 20-40g/L of polypeptone, 2-10g/L of ammonium sulfate and the balance of water.
7. Use of the strain of claim 1 in the preparation of Aureobasidin a.
8. Use of the strain of claim 1 in the preparation of an antibacterial agent.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03220199A (en) * | 1990-01-23 | 1991-09-27 | Takara Shuzo Co Ltd | Novel r106 compounds |
| JPH0539204A (en) * | 1991-02-19 | 1993-02-19 | Takara Shuzo Co Ltd | Agricultural/horticultural fungicide |
| EP0547484A1 (en) * | 1991-12-19 | 1993-06-23 | Nippon Kayaku Kabushiki Kaisha | Process for producing antibiotic R106-I |
| US5260214A (en) * | 1988-07-19 | 1993-11-09 | Takara Shuzo Co. | Aureobasidium pullulans which produces antibiotic R106 |
| CA1334180C (en) * | 1988-07-19 | 1995-01-31 | Kazutoh Takesako | Antibiotics r106 |
-
2021
- 2021-01-11 CN CN202110032299.9A patent/CN112779167B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5260214A (en) * | 1988-07-19 | 1993-11-09 | Takara Shuzo Co. | Aureobasidium pullulans which produces antibiotic R106 |
| CA1334180C (en) * | 1988-07-19 | 1995-01-31 | Kazutoh Takesako | Antibiotics r106 |
| JPH03220199A (en) * | 1990-01-23 | 1991-09-27 | Takara Shuzo Co Ltd | Novel r106 compounds |
| JPH0539204A (en) * | 1991-02-19 | 1993-02-19 | Takara Shuzo Co Ltd | Agricultural/horticultural fungicide |
| EP0547484A1 (en) * | 1991-12-19 | 1993-06-23 | Nippon Kayaku Kabushiki Kaisha | Process for producing antibiotic R106-I |
Non-Patent Citations (2)
| Title |
|---|
| A study on Candida biofilm growth characteristics and its susceptibility to aureobasidin A;Munusamy, Komathy;《REVISTA IBEROAMERICANA DE MICOLOGIA》;20180312;第35卷(第2期);第68-72页 * |
| AUREOBASIDINS，NEW ANTIFUNGAL ANTIBIOTICS TAXONOMY，FERMENTATION，ISOLATION，AND PROPERTIES;Kazutoh Tkaesako;《TheJournal Of Antibiotics》;19910130;第44卷(第9期);第919-924页 * |
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