KR20040053906A - Method for formation of synnemata from mycelium of beauveria bassiana - Google Patents

Abstract

PURPOSE: A method for formation of synnemata from mycelium of Beauveria bassiana is provided, thereby forming synnemata of Beauveria bassiana which has bio-stimulating activity, so that it can be used in studies on pharmacological effects of synnemata of Beauveria bassiana. CONSTITUTION: The method for formation of synnemata from mycelium of Beauveria bassiana comprises the steps of: (1) liquid culturing conidiospore of Beauveria bassiana to form mycelium of Beauveria bassiana; and (2) inoculating the mycelium of Beauveria bassiana to a specific medium for forming synnemata containing 30 to 35 wt.% of unpolished rice, 1 to 7 wt.% of pupa powder, 1 to 3 wt.% of yolk protein, 1 to 3 wt.% of soybean powder and 140 ml of sterilized water and culturing it at 25 to 27 deg. C for 5 to 10 days and at 18 to 22 deg. C for 20 to 30 days under the light of 1,000 to 4,000 lux, maintaining water content of the medium at 55 to 65%.

Description

Method for formation of conidia in the mycelia of Bacillus {Method for formation of synnemata from mycelium of Beauveria bassiana}

The present invention relates to a method for forming the conidia congenital from the mycelium of Bacillus Bacillus. More specifically, the present invention relates to a method for forming a conidia conidia using a mycelium formed from the conidia of spores of Beauveria bassiana , a raw fungus of Baekja, known to be excellent in promoting various physiological activities. The present invention relates to a method for forming a conidia of Bacillus pneumoniae by culturing the liquid and inoculating the mycelium formed therefrom into a special medium for forming conidia in accordance with the present invention.

Insect pathogenic fungi, including Cordyceps sinensis, are known to have about 100 genera and about 750 species worldwide. Among them, entomopathogenic fungi, which form perennials, that are fruiting bodies and belong to Ascomycetes, are Cordyceps sp., Shimizumyces sp., Torrubiella sp. The third genus is known, but the typical genus can be called Cordyceps genus is Cordyceps genus.

Insect pathogenic fungi belonging to the genus Cody sepseu is known to currently 300 species distributed throughout the world, baekganggyun (Beauveria bassiana), silkworm Cordyceps (Paecilomyces tenuipes) and most of the insect pathogenic fungi Cody sepseu belonging to incomplete gyungang (Deuteromycetes) as It is known as the incomplete generation of the genus. Since Paecilomyces tenuipes are imperfect fungi, they do not form fruiting bodies, but they form a conidia well in artificial media and are currently commercialized under the name of silkworm cordyceps. However, the Bacillus Bacillus, which induces a white nap, does not form a fruiting body or conidia, but grows only in the form of mycelium and conidia on the host surface, and thus has not commercialized the conidia as in silkworm cordyceps.

Some Cordyceps cordyceps and incomplete fungus Baekgangjam, which form fruiting bodies, have been used as an excellent herbal medicine since ancient times in China, Korea, and Japan. Cordyceps mainly take fruiting bodies, but leuksae take silkworm larvae infected with mycelium. Representative Cordyceps species forming fruiting bodies include Cordyceps sinensis (C. sinensis) Based on the larvae of bat moths, they form fruiting bodies and are found only in China. In addition, Codycept Militaris (C. militaris), Cordyceps Mathias (C. martialis), Cordyceps opioglosoidis (C. ophioglossoides), Cordyceps Soborifera (C. soborifera), Cordyceps Hokesh (C. hawkesii), Tolipocladium (Tolypocladiumsp.), Boberia Bassiana (B. bassiana) Is used for medical purposes.

Recently, research into the development of biotechnology has been conducted to search for useful bioactive substances from these Cordyceps sinensis or herbal medicines. Through some studies, cordycepin, a nucleoside derivative that exhibits various physiological activities such as anticancer effects, has been studied. Cordycepin (3'-deoxyadenosin)] has been characterized. In addition, D-mannitol, steric acid, mycose, ergosterol, uracil, adenine from C. sinensis Adenosine was identified and galactomannan, a polysaccharide that inhibits the growth of sarcoma 180, was isolated from C. cicadae . In addition, polysaccharides with anticancer activity were also isolated from C. ophioglossoides .

Since Baekgang was first discovered by Bassi in 1835 among insect pathogenic fungi including Cordyceps sinensis, much research has been conducted worldwide. As a result, the antibiotics oposporin and bastiatin have anticoagulant effects. However, research on leukemia has been limited to mycelium because the conidia of Leukococcus are not naturally formed in nature but could not be formed artificially. As a result, there is no research on the formation of conidia coliforms and the biologically active effects or bioactive substances of conidia coli.

Therefore, for Korea, which has a relatively narrow land area and poor resources, the development of new resources that can actively respond to the global opening trend is the best way to maximize national competitiveness. The importance of developing technology to explore, discover, and resource resources is emphasized. In particular, it is urgently needed to increase the added value of domestic microbial resources by forming the conidia bottle of Bacillus bacillus, which has not been studied at home and abroad, and separating, purifying, characterizing and mass producing useful materials.

Accordingly, the present inventors have made a thorough study on the method of forming the conidia congenital pathology of Bacillus Bacillus in order to use the Bacillus bacterium more efficiently, the mycelium formed by liquid culture of the conidia of Bacillus ( Beauveria bassiana ) according to the present invention After inoculating into a special medium for forming and searching for the optimal culture conditions for the formation of the conidia congener, and found under the cultivation under these conditions, it was found that the conidia of the Bacillus bacterium can be formed and completed the present invention.

Accordingly, it is an object of the present invention to provide a method for forming conidia congenital from the mycelium of Bacillus fungus, an insect pathogenic fungus, as an incomplete generation of Cordyceps sinensis.

Another object of the present invention is a special medium for forming conidia congenital disease, including brown rice, chrysalis, egg yolk protein, soy flour and sterile water, and the optimal culture conditions that can form the conidia of Bacillus bacterium using the same. To provide.

1 is a photograph of conidia of the Bacillus.

Figure 2 is a photograph showing the conidia congenital formation of Bacillus bacterium according to the present invention.

The present invention relates to a method for forming a conidia by using a mycelium formed from conidia of coliform ( Beauveria bassiana ), which is a raw mold of Bacillus japonicus. It characterized by comprising the step of inoculating into a special medium for forming the conidia of the leukemia to form the conidia of the leukemia.

Hereinafter, the present invention will be described in more detail.

Dried worms of silkworm moths infected with leukemia have been used as a medicine for the treatment of cramps, epilepsy, strokes and numb necks in children. It has been used for the treatment of headache, laryngitis, tonsillitis, adenosis, itching of the skin, solitary, and diabetes.

Bacillus bassiana is known to have the broadest host range among insect pathogenic fungi that can infect about 700 species of insects, including the sky cow. Leukococcus forms conidia on the surface of the host, conidia form conidia on mycelia, then form a round flask-shaped pialide on top of it, forming a long neck at the top of the pyramid. To form. At the end of these nodes, one conidia develop in a zigzag shape and have a similar shape to grape clusters. The color is colorless. Because large amounts of spores are produced on the medium, it looks like wheat flour with the naked eye.

In the present invention, the bacterium isolated from all kinds of host insects can be used as the bacterium, for example, the bacterium isolated from silkworms can be used. Bacillus cultivars cultured under special culture conditions as in the present invention form an orange-yellow conidia, the length of which is about 10 cm or more and splits like a branch at the end. After a certain incubation period, white mycelium forms at the end and forms conidia. This bacterium enters all stages of growth in almost all insect groups. As described above, the leukococcus, which induces leukemia, grows only in the form of mycelium and conidia on the surface of the host, and until now, commercializes the conidia as well as silkworm cordyceps. Although it did not, when culturing leukemia by using a special medium for forming conidia bottle prepared by using brown rice, chrysalis, egg yolk protein, soy flour and sterile water according to the present invention, the conidia of the leukemia, that is, fruiting body You can shape it.

The method of forming conidia of the Bacillus bacterium according to the present invention is as follows.

(1) liquid culture of the conidia with the Bacillus as a seed; And

(2) inoculating a mycelium formed from the conidia of spores of the liquid culture Bacillus in a special medium for forming conidia congeners to form a conidia of Bacillus Bacillus;

It includes.

The step (1) is a step for obtaining the mycelium from the conidia of the Bacillus sp., And can be arbitrarily selected by appropriately selecting a known method for culturing Bacillus. For example, the conidia of Bacillus spp. Cultured in potato agar medium (potato dextrose agar) were inoculated into a 250 ml Erlenmeyer flask containing potato medium (potato dextrose broth), and then stirred at a rotation speed of 150 rpm at a temperature of 25 ° C. Incubate for about 5 days.

Inoculating 10 to 15% of the mycelium of Bacillus Bacillus liquid cultured in the step (1) for each medium and incubating for 5 to 10 days at a temperature of 25 to 27 ° C., and then incubating at a temperature of 18 to 22 ° C. for 20 to 30 days. desirable.

In the method for forming the conidia of the Bacillus mycelium according to the present invention, in order to form the conidia of the Bacillus mycelium from the mycelium, the water content in the medium is 55 to 65%; The lid of the culture bottle is closed so that the air inflow amount is not large; The temperature is 18 to 22 ° C .; It is preferable to incubate under the conditions of 1000-4,000 lux of irradiation amount.

In the step of inducing the formation of the conidia in the step (2), the special medium for forming the conidia conidia, for example, 30 to 35% by weight brown rice, 1 to 7% by weight of chrysalis, egg yolk in an 850 ml mushroom culture bottle A mixed medium prepared by mixing 1% to 3% by weight of protein, 1% to 3% by weight of soy flour, and 140 ml of sterile water may be used, but is not limited thereto. This would be possible.

When white Leukococcus is cultured according to the method for forming the conidia of the Bacillus bacterium according to the present invention, a pale white mycelium starts to form on the surface of the medium after 3 days of inoculation, and after 7 days, the mycelium of Bacillus is formed on the whole medium. After 20-30 days under the condition of 18-22 ℃, the conidia bottle grows completely. At this time, the dry weight of harvested conidia bottle is about 5.5-6.5g per cultivation bottle.

Leprosy conidia congenital disease according to the present invention is characterized by the first artificial artificial formation of the conidia congenital bacteria of Bacillus, which is not naturally formed in nature. This conidia bottle has a feature that can be usefully used to develop a functional food, a new drug or a medicinal product of various efficacy by separating and purifying the useful material from itself or from it.

Hereinafter, the conidia bottle forming method of the present invention will be described in more detail through the following examples and experimental examples. However, these are only embodiments of the present invention and do not limit the scope of the present invention, and ordinary modifications and applications in the art may also be included in the scope of the present invention.

EXAMPLES Formation of Bacillus Bacillus Conidia

Beauveria bassiana isolated from silkworms was incubated in potato agar medium, and then the conidia separated from the silkworm were inoculated into a 250 ml Erlenmeyer flask containing potato medium and agitated at 150 rpm for 5 rpm. Incubated daily.

Special medium for forming conidia bottle was prepared by putting 72g brown rice, chrysalis powder 8g, egg yolk protein 4g, 4g soy flour and 140ml sterilized water in 850ml mushroom culture bottle and sterilized at 121 ℃ for 50 minutes. Mycelia of the liquid cultured Bacillus bacteria were inoculated by 10% per the special medium and incubated at 26 ° C. for 7 days, and then at 20 ° C. for 20-30 days.

In the above process, the medium and culture conditions to be applied in the formation of the leukemia congenital disease bottle, whether or not the congenital disease bottle is formed from the mycelia of the bacterium according to each condition as in the following experimental example The freeze dryer was selected by examining the dry weight.

Experimental Example 1 Searching for Optimal Aerobic Conditions for the Formation of Conidia B.

The optimal aerobic conditions for the formation of conidia bottle of Bacillus bacterium were investigated by controlling the air inflow by using the mushroom culture bottle cap.

As a result, as shown in Table 1, when the air inflow was increased by opening the lid, the condensation disease was not formed due to the decrease of water content in the medium. It was found that the genus was formed.

Incubated with the lid open When incubated with the lid closed Conidia bottle not formed Produced in Conidia

Experimental Example 2 Investigation of Optimal Temperature Conditions for Formation of Conidia Bottles of Bacillus Bacillus

As a result of culturing by varying the temperature of the conidia bottle culture medium, as shown in Table 2, the conidia bottle was produced only at 20 ℃ culture, the conidia bottle was not produced at any temperature below 15 ℃ or more than 25 ℃, only mycelium Produced.

Incubation temperature (℃) Formation of Conidia Bottles 10 Not formed 15 Not formed 20 Formed 25 Not formed 30 Not formed

Experimental Example 3 Investigation of Optimum Water Content Condition for Conidia Formation of Bacillus Bacillus

The water content of the conidia bottle culture medium was adjusted to 50% to 90%, and then cultured. The conidia bottle was separated and lyophilized to measure the weight of each.

As a result, as shown in Table 3, when the water content was 60%, most conidia were produced. At 50%, 70%, and 80%, fruiting bodies were produced less, and at 90%, conidia were not formed. .

Moisture content (%) Repeat number [conidia bottle weight (g)] Average 1 repetition 2 repetitions 3 repetitions 50 6.920 4.508 5.236 5.555 60 26.006 25.575 25.004 25.528 70 8.084 7.636 6.185 7.302 80 5.864 5.959 6.678 6.167 90 Conidia bottle not formed

Experimental Example 4 Investigation of Optimal Irradiation Conditions for Conidia Formation of Bacillus Bacillus

In order to find the optimal irradiation conditions, 3000 lux, 6000 lux and 9000 lux light were irradiated to mushroom culture bottles inoculated with mycelium mycelium, and then the cultured conidia bottles were isolated, lyophilized and weighed. It was.

As a result, as shown in Table 4, the highest concentration of conidia was produced when the dose was 3,000 lux, and less than that under the stronger irradiation conditions. In addition, when the light was not irradiated, that is, 0 lux, no conidia bottle was formed.

Dose (lux) Repeat number [conidia bottle weight (g)] Average 1 repetition 2 repetitions 3 repetitions 9,000 2.8 3.0 3.2 3.000 6,000 4.1 4.8 4.7 4.553 3,000 26.006 25.575 25.004 25.528 0 Conidia bottle not formed

As can be seen from the above Experimental Examples 1 to 4, the optimum conditions for forming the conidia congenital pathology of Bacillus Bacillus is about 55 to 65% of the water content in the medium before and after 60%, so that the inflow of the culture bottle It turns out that a lid is closed, the temperature is about 18-22 degreeC about 20 degreeC, and the irradiation amount is about 1,000 to 4,000 degree around 3,000 lux.

As described above, the present invention relates to a method for forming a conidia bottle from the mycelium of Bacillus Bacillus using a mycelium formed from the conidia of the Bacillus Bacillus ( Beauveria bassiana ), a raw fungus of Baekja sleep known to be excellent in promoting various biological activities It provides an effective way to form a conidia bottle. The conidia of the Bacillus bacterium formed by the method of the present invention is artificially formed that is not naturally formed in the natural world, and more active research can be conducted with respect to the pharmacological efficacy of the Bacillus bacterium congenital disease, and useful materials therefrom. It can be usefully developed by separating and purifying functional foods, new drugs or medicinal products with various efficacy, and when it is mass-produced and distributed, it can be expected to improve the income of domestic farmers, improve national health and improve physical fitness using them. It is a very useful invention.

Claims (7)

In the method of forming the conidia of the Bacillus ( Beauveria bassiana ), (1) liquid culture of the conidia with the Bacillus as a seed; And (2) inoculating a mycelium formed from the conidia of spores of the liquid culture Bacillus in a special medium for forming conidia congeners to form a conidia of Bacillus Bacillus; Conidia bottle formation method of Bacillus bacterium comprising a. The method of claim 1, wherein the special medium for forming conidia bottle is 30 to 35% by weight brown rice, 1 to 7% by weight pupa powder, 1 to 3% by weight egg yolk protein, 1 to 3% by weight soy flour and 140 ml of sterilized water. Conidia bottle formation method of Bacillus Bacillus, characterized in that it was prepared. The method of claim 1, wherein the method for forming the conidia of the Bacillus bacterium is inoculated by 5 to 15% of the mycelium formed from the conidia of the liquid cultured Bacillus sp. Incubated for 5 to 10 days at 20 to 30 days at a temperature of 18 to 22 ℃ for the conidia bottle forming method, characterized in that the incubation. The method of claim 1, wherein the method for forming conidia congeners of Bacillus fungi has a water content of 55 to 65%; Close the lid of the culture bottle so that the air inflow is not high; The temperature is 18 to 22 ° C; A method of forming conidia congenital disease of Bacillus Bacillus, characterized in that the dose is cultured under the conditions of 1,000 to 4,000 lux. The conidia of the Bacillus bacterium, characterized in that formed by the method of any one of claims 1 to 4. Special medium for forming conidia of Bacillus bacillus, comprising 30 to 35 wt% brown rice, 1 to 7 wt% pupa powder, 1 to 3 wt% egg yolk protein, 1 to 3 wt% soy flour, and 140 ml of sterile water . The conidia of the Bacillus bacterium, characterized in that formed using a special medium for forming the conidia of the Bacillus bacterium according to claim 6.