KR19980034989A - Insecticidal fungal medium using Chinese cabbage waste and its preparation method - Google Patents

Abstract

The present invention is a solid waste produced in kimchi factory, large fruit and vegetable market, damaged cabbage leaves, cabbage roots, radishes, etc. (more than 90% of the Chinese cabbage leaves) is crushed, compressed and separated into solid phase and liquid phase, respectively, and the appropriate process Through the pesticidal fungus Boberia bassiana ATCC 7159, Metalhizium anisopliae ATCC 20500 strains to prepare a cheap medium for the cultivation of industrially important fungal strains.

Description

Insecticidal fungal medium using Chinese cabbage waste and its preparation method

The present invention relates to a method for producing a cheap medium for the cultivation of useful microorganisms using cabbage waste produced in large quantities in a kimchi factory, a large fruit and vegetable market. At present, it is reported that organic wastes generated in Korea account for more than half of the total waste generation, and because of the high perishability, it is estimated that there is a high possibility of causing environmental problems such as odor generation and sewage leakage. Therefore, treatment / disposal technology for converting and reducing organic waste into stable materials has emerged as the biggest purpose of waste treatment. Waste treatment in Korea has been largely relied on landfill until now, but landfill treatment is not only a large amount of waste and takes a long time to stabilize, but also a secondary pollution by leachate, etc. Can't.

In particular, a large amount of organic wastes (mainly damaged cabbage leaves, cabbage roots, radishes, etc.) generated at the Kimchi factory and large fruit and vegetable market have been mostly reclaimed, even though they are easily recycled by composting because they have almost no impurities. have. However, in recent years, landfills are nearing their limits and the government's environmental policies are changing to advanced countries. However, there are many ways to prepare for such measures. However, effective resource-recycling technologies have not yet been established.

Chinese cabbage has been produced and shipped at an annual average of about 3 million tons for the past 10 years, of which about 300,000, or 900,000 tons, are disposed of as landfills every year, and if recycled, environmental protection and insecticidal fungus Boberia bassiana ATCC 7159 ) And mass production of useful microorganisms, such as metallium anisopliae ATCC 20500. Meanwhile, Boveria bassiana ATCC 7159 and Metalhizium anisopliae ATCC 20500 are insect pathogenic fungi that have the ability to infect various types of pests such as rice worms, aphids and moths and kill them. (Leger, RJ et al., 1987, J. Gen. Microbiol, 133, 1371-1382; Moorhouse, ER et al., 1993, Biocontrol Sci. Technol., 3, 63-72; Ziding, F , et al., 1988, Can. Entomol. 120, 133-144). Known insect pathogenic fungi are about 400 species, including Boveria bassiana, Metalhizium anisopliae, and Hisutella thonysonil (Mittler, TE et al). , 1987, Annual Review of Entomology.Vol. 32, pp. 228-229). In order to use such insecticidal fungi as a means of pest control, a process technology for mass production of cells and spores of these strains is required.

Therefore, in the present invention, in order to achieve the purpose of mass production of the above useful strains, solid and liquid cultures of Beauveria bassiana were performed using a medium using solid wastes of the Kimchi plant. As a result, when the solid culture using the solid content except the Chinese cabbage juice, conidia of 2.0 × 10 ⁸ cfu (colony forming units) / g was formed in two weeks. In liquid culture, 4.0 × 10 ⁷ blastosproes were generated per ml of culture medium (ml) at about 5 days of culture in Chinese cabbage broth. This medium had a much higher spore-forming ability than the medium for fungal growth such as PDB (potato dextrose broth), SDB (Sabouraud dextrose broth), and YPD (yeast extract peptone dextrose). The composition of these conventional media is as follows, respectively. PDB medium is prepared by dissolving 200 g of potato and 20 g of dextrose in distilled water so that the final volume is 1 liter. SDB medium is prepared by dissolving 10 g of neopeptone and 40 g of dextrose in distilled water to a final volume of 1 liter. The YDB medium was prepared by dissolving 10 g of yeast extract, 10 g of peptone, and 2 g of dextrose in distilled water so that the final volume was 1 liter. In the present invention, in order to test whether the spores produced by using the cabbage medium have insecticidal properties, the insecticidal effect on the insects of bee moths moth was tested, and also Boberia bassiana and metal beetle The optimal concentration, optimal incubation temperature, optimal incubation, pH, and dry weight of solid waste extracts for cultivating useful fungi, such as methium anisopliae, were determined.

Figure 1 is a photograph of the spore morphology under the microscope when the insecticidal fungus Boberia bassiana solid culture and liquid culture.

FIG. 2 is a photograph of spore morphology under sage name when solid and liquid cultures of the insecticidal fungus Metalhizium anisopliae.

Figure 3 is a comparison of the sporulation ability according to the concentration of the cabbage juice when the liquid culture of Boberia bassiana (Beauveria bassiana) in the Chinese cabbage liquid medium.

Figure 4 is a comparison of the sporulation ability according to the concentration of Chinese cabbage juice when the liquid culture of the metal herbium anisopliae (Metarhizium anisopliae) in the cabbage juice medium.

Figure 5 is a comparison of the sporulation ability according to the type of medium in the liquid culture of Boberia bassiana (Beauveria bassiana).

Figure 6 is a comparison of the sporulation ability according to the type of medium during the liquid culture of metallization anisopliae (Metarhizium anisopliae).

Figure 7 is a comparison of the cell weight (dry weight) according to the type of medium in the liquid culture of Boberia bassiana (Beauveria bassiana).

Figure 8 is a comparison of the cell weight (dry weight) according to the type of medium during the liquid culture of metallization anisopliae (Metarhizium anisopliae).

Figure 9 is a comparison of the sporulation ability according to the initial pH when the liquid culture of Boberia bassiana (Beauveria bassiana) in the cabbage juice medium (100%).

Figure 10 is a comparison of the sporulation ability according to the initial pH when the liquid culture of the metal herbium anisopliae (Metarhizium anisopliae) in the cabbage juice medium (100%).

Figure 11 is a comparison of the sporulation ability according to the culture temperature when the liquid culture of Boberia bassiana (Beauveria bassiana) in the cabbage juice medium (100%).

Figure 12 is a comparison of the sporulation ability according to the culture temperature when the liquid culture of the metal herbium anisopliae (Metarhizium anisopliae) in the cabbage juice medium (100%).

13 is a photograph showing the insecticidal effect on the larvae of honey bee moths according to spore infection in Boberia bassiana (Beauveria bassiana).

14 is a photograph showing the insecticidal effect on the larvae of the bee missing moth according to spore infection of the metal hydrium anisopliae (Metarhizium anisopliae).

Although the configuration and operation of the present invention will be described in detail with reference to the following examples, the scope of the present invention is not limited to the following examples.

Example 1: Preparation of Medium

The microbial growth medium was prepared after several steps of pretreatment using solid waste cabbage waste (1kg of Kimjang cabbage purchased from Daegu Chilsung Market as a raw material). The pretreatment process for the liquid medium includes (I) extracting cabbage juice using a pressurized green juice, (II) heating at 121 ° C. for 2 minutes using an autoclave, and (III) centrifuging at 12,000 rpm for 15 minutes. , (IV) The supernatant was taken and then filtered through filter paper (Whatman No. 1, USA). At this time, by adding water to the final produced cabbage juice to prepare a medium diluted to 100% (cabbage juice stock), 50%, 10% as a component of the basic cabbage medium of the present invention. In addition, carbon sources (glucose, sugar, lactose, potato starch, starch, gyros, molasses, etc.), nitrogen sources (peptone, tryptone, casamino acid, casein, yeast extract, soyton, corn gluten) Mill [CGM], corn steep liquor [CSL], soybean floor [SBF], etc.) can also be used as a general medium. The above pretreatment fixation was performed to easily quantify the growth of microorganisms in liquid culture by using a spectrophotometer by completely removing the crushed debris. After the pretreatment, the juice of the cabbage waste was sterilized for 15 minutes at 121 ℃ to prepare a liquid medium. In addition, when preparing a solid medium for the purpose of simply cultivating in a petri dish (petri dish) was prepared by adding agar 1.5-2.0% to the above liquid medium. The microscopic images of spores produced by solid and liquid cultures of the insecticidal fungus Boberia Bassiana and the Metal Hygidium Anisopli using the medium prepared by the above method are shown in FIGS. 1 and 2, respectively.

Example 2: Preparation of Solid Medium

The liquid cabbage juice medium of Example 1 was heat-dried at 80-100 ° C. for 12 hours or more to prepare a powder medium. The powder medium thus prepared is simply added with water to prepare the same medium as in Example 1. Therefore, depending on the application, it was used as a liquid medium or a solid medium.

Example 3: Preparation of Medium Using Crushed Waste

Solid except for the cabbage juice prepared in Example 1 was sterilized by adjusting the water content to 30-85%, preferably 50-70% and then insecticide fungus Boberia bassiana (Beauveria bassiana ATCC 7159), metal Soplihi (Metarhizium anisopliae ATCC 20500) was prepared as a medium for solid culture. As a result of inoculating 0.1 ml of 1.0 × 10 ⁵ / ml spore suspension with this medium to incubate Boberia bassiana cells, the number of conidia produced by the culture time was shown in Table 1.

TABLE 1

Number of colony forming units (cfu) per gram of Boberia bassiana cultured in medium using crushed ground

Example 4 Comparison of Spore Formation Capacity According to the Concentration of Chinese Cabbage Juice

10, 50, 100% of the cabbage juice medium prepared in Example 1 was placed in a 250 ml Erlenmeyer flask with 100 ml each. 0.5 ml of spore suspension at 1.0 × 10 ⁶ / ml concentration was inoculated into the culture medium, and the resultant agar spores were cultured at pH 6.0, 25 ° C, and 160 rpm. blastspores / ml) was the same as in Figs. As a result, the spore forming ability was highest in the cabbage juice medium of 50-100%. In addition, the cabbage juice medium of the present invention, in addition to the insecticidal fungus Boberia Bassiana and metallium aniseoply, penicillium notatum ATCC 7813, Cephalosporium acremonium (Cephalosporium acremonium ATCC 36225) , Aspergillus niger (Aspergillus niger ATCC 22343, ATCC 26550, etc.), Aspergillus oryzae ATCC 42149, Rhizopus oligosporus ATCC 22959, Saccharomyces cerevisiae (Saccharomyces cerevisiae ATCC 9763, 22244, etc.), Pleurotus ostreatus, Shiitake mushrooms (Lentinus edodes KCTC 6069) have been demonstrated to be effective. Therefore, the cabbage juice medium is used as a general medium for fungal culture.

Example 5 Comparison of Spore Formation Ability between Chinese Cabbage Juice Medium and Medium Medium]

100% cabbage juice medium prepared in Example 1 and PDB, SDB, and YPD medium, which are general mediums, were separately prepared and placed in 100 ml of 250 ml Erlenmeyer flasks. 0.5 ml of spore suspension at 1.0 × 10 ⁶ / ml concentration was inoculated into the culture medium, and the resultant agar spores were cultured at pH 6.0, 25 ° C, and 160 rpm. blastspores / ml) was the same as in Figs. As a result, it can be seen that the spore forming ability of the cabbage juice medium is much higher than that of the general medium.

[Example 6: Cell Production Amount According to the Type of Medium]

In order to find out the dry weight according to the medium of Boberia Bassiana and Metallgidium anisopli cultured in Examples 4 and 5, the culture medium was taken out one by one and centrifuged, and the precipitated cells were lyophilized. The weight was measured by drying at and the results are shown in FIGS. 7 and 8, respectively. As a result, 100% Chinese cabbage juice medium showed the highest cell production.

Example 7 Comparison of Spore Formation Capacity According to Initial pH

100% cabbage juice medium prepared in Example 1 was placed in a 250ml Erlenmeyer flask. 0.5 ml of spore suspension at 1.0 × 10 ⁶ / ml concentration was inoculated into the medium, and subcultured spores were produced by incubation at 25 ° C. and 160 rpm at various initial pHs. The number of (blastspores / ml) was as shown in FIGS. 9 and 10, respectively. As a result, Boberia bassiana showed the maximum sporulation ability at pH 4.5-5.5 and at pH 6.5-8.5 in Metalhizium Anisopli. Therefore, the pH of the maximum spore-forming ability of the fungus was found to vary depending on the strain.

Example 8 Comparison of Spore Formation Capacity According to Culture Temperature

100% cabbage juice medium prepared in Example 1 was placed in a 250ml Erlenmeyer flask. 0.5 ml of spore suspension at 1.0 × 10 ⁶ / ml concentration was inoculated into the medium, and subcultured spores were cultured at different culture temperatures at pH 6.0 and 160rpm. (blastspores / ml) numbers were the same as in FIGS. 11 and 12, respectively. As a result, Boberia Bassiana showed maximum sporulation at 20 ° C and at 25-30 ° C in Metallgidium Anisopli. Therefore, the maximum incubation temperature of the fungal spore forming ability was found to vary depending on the strain.

Example 9 Verification of Insecticidal Effect

In order to test whether the spores cultured in the cabbage juice medium of the above example had insecticidal properties, filter paper was placed in a 9 × 1.5 cm petri dish, and 8 larvae of honeybee moths were added at 1.0 × 10 ⁶ / ml concentration. 0.5 ml of Boberia Bassiana and Metallgiium Anisopli Spore Suspension were observed while storing at 25 ° C. for 4-7 days. As a result, as shown in FIGS. 123 and 14, the larvae were infected with the spores and were killed.

By completing the present invention by the above method it is possible to make a cabbage medium using a large amount of cabbage waste produced in Kimchi factory, large fruit and vegetable market. As a result, it can be used as an inexpensive medium for the mass cultivation of industrially useful fungi, including the insecticidal fungus Boberia Bassiana, Metallgidium anisopli, and the like. In addition, by recycling the cabbage waste as a fungal medium, environmental problems such as odor generation and sewage leakage caused by simple landfill have been solved.

Claims (10)

After extracting the cabbage juice using the pressurized juicer, the cabbage juice was extracted at 121 ℃ for 2 minutes in a autoclaved sterilizer and centrifuged at 12,000rpm for 15 minutes, followed by pretreatment to filter the supernatant, followed by sterilization at 121 ℃ for 15 minutes. Method of making a medium. The method of claim 1, wherein the agar is added to prepare a solid medium by adding 1.5-2.0%. A method for producing a medium from the crushed waste discarded in the manufacturing process according to claim 1. Chinese cabbage medium prepared according to claim 1. Chinese cabbage medium prepared according to claim 2. Chinese cabbage medium prepared according to claim 3. Dilution medium prepared by adding distilled water to the medium in any one of claim 4,5,6. A medium prepared by adding a carbon source and a nitrogen source in addition to the medium according to any one of claims 4, 5 and 6. The medium according to any one of claims 4,5,6,7,8 was selected from Beauveria bassiana ATCC 7159, Metalhizium anisopliae ATCC 20500, Penicillium chrysogenum (Penicillium chrysogenum ATCC 10238), Penicillium natatum ATCC 7813, Cephalosporium acremonium ATCC 36225, Aspergillus niger ATCC 22343, ATCC 26550, etc. Aspergillus oryzae ATCC 42149, Rhizopus oligosporus ATCC 22959, Saccharomyces cerevisiae ATCC 9763, 22244, etc., Pleurotus ostreatus, Shiitake Method for use as a culture medium for fungi, such as mushrooms (Lentinus edodes KCTC 6069). Method for using the medium according to any one of claims 4,5,6,7,8 as a fungal spore culture medium.