KR100187754B1 - A medium for bacteria with cabbage waste and producing method for the medium - Google Patents

Abstract

The present invention separates and recovers damaged solid cabbage leaves, cabbage roots, radishes (more than 90% of them are cabbage leaves), which are solid waste generated in the kimchi factory and large-scale fruit and vegetable market, into solid and liquid phases after crushing and compression treatment, There is an excellent effect of providing an inexpensive bacterial culture medium using industrial wastes for cultivation of industrially important bacteria including Bacillus subtilis ATCC 6633 and a preparation method thereof.

Description

Bacterium culture medium using Chinese cabbage waste and manufacturing method thereof

The present invention relates to an inexpensive bacterial culture medium for culturing beneficial microorganisms using a large amount of Chinese cabbage waste produced in a kimchi factory, a large-sized fruit and vegetable market, and a method for producing the same. It is reported that organic wastes generated in Korea account for more than half of total wastes, and it is highly likely that environmental problems such as odor generation and sewage leak are caused due to the large perishability. Therefore, a treatment / disposal technique for converting organic waste into a stable material and reducing the amount of organic waste is the most important purpose of the waste treatment. The method of disposal of wastes in Korea has been largely dependent on landfill so far. The landfill treatment method requires a lot of waste and takes a long time to stabilize, and secondary pollution by leachate is inevitable. I can not.

Especially, a large amount of organic wastes (mainly damaged Chinese cabbage leaves, Chinese cabbage roots, radishes, etc.), which are generated in kimchi factories and large fruits and vegetables market, are almost free from impure solid matter, It is coming. However, recently landfill sites have reached the limit, and the government's environmental policy is changing to the advanced country type, and various methods are being sought in order to prepare a countermeasure against them. However, the effective reuse technology has not yet been established.

About 3 million tons of Chinese cabbage is produced and shipped annually in the last 10 years. Approximately 30% of the plant is landfilled as waste every year. About 90% of the cabbage is recycled, and therefore, environmental protection effects such as Bacillus subtilis ATTC 6633 Mass production of microorganisms becomes possible. On the other hand, Bacillus sp. Strains such as Bacillus subtilis produce and secrete proteases, amylase, and antibiotics. Among them, proteolytic enzymes are the most industrially produced and used enzymes for food processing, leather processing, brewing industry, enzyme detergents, pharmaceutical processing, and tools for amino acid sequence analysis of proteins (Ward, OP, 1986. Proteolytic Enzymes: Compreshensive Biotechnology. Vol III. Pp. 789-818). In particular, subtilisin produced by Bacillus subtilis has been used for over 30 years (Ward, OP, 1986. Proteolytic Enzymes: Comprehensive Biotechnology, Vol III, pp, 789 -818). In order to industrially utilize such useful microorganisms, a process technology for mass production of cells and enzymes of these strains is needed.

Therefore, in the present invention, in order to achieve the object of mass production of the useful strains described above, cultivation of Bacillus subtilis was carried out using a medium using solid waste of a Kimchi factory. As a result, the absorbance (OD ₆₀₀ ) at ₆₀₀ nm reached maximum 9.0 at 6 days when the liquid culture was performed in the Chinese cabbage liquid medium. This medium was much higher in growth of bacteria than in the medium used for bacterial growth such as LB medium (Luria-Bertani medium). The medium composition of the LB medium was prepared by dissolving 10 g of tryptone, 5 g of yeast extract and 10 g of salt (NaCl) in distilled water to a final volume of 1 liter. In addition, in the present invention, the optimum concentration, optimal culture temperature, optimum culture pH, etc. of the solid waste extract for culturing the useful microorganisms including Bacillus subtilis were determined.

Figure 1 is an optical micrograph (100X) of Bacillus subtilis after Gram stain.

2 is an optical micrograph (100X) of Bacillus subtilis after spore staining.

FIG. 3 is a growth curve of the bacillus subtilis according to the concentration of the cabbage juice when the Bacillus subtilis is liquid cultured in the Chinese cabbage liquid medium.

FIG. 4 is a growth curve of the bacillus subtilis according to the incubation temperature when liquid culture of Bacillus subtilis is carried out in the cabbage liquid medium (100%).

FIG. 5 is the growth curve of the bacillus subtilis according to the initial pH when liquid culture of Bacillus subtilis is performed in the Chinese cabbage liquid medium (100%).

FIG. 6 is the result of high-pressure liquid chromatography (HPLC) analysis of culture broth when liquid culture of Bacillus subtilis was performed in a Chinese cabbage liquid medium (100%).

The structure and function of the present invention will be specifically described with reference to the following examples, but the scope of the present invention is not limited to the following examples.

[Example 1]

Preparation of medium

Microbial growth medium was prepared after several pretreatment steps using the solid waste, Chinese cabbage waste (1 kg of kimchi cabbage purchased from Chilgong Market, Daegu) as a raw material. The pretreatment process for liquid medium was (I) extraction of chinese cabbage juice using a pressurized green juice, (II) heating at 121 ° C for 2 minutes using autoclave, (III) centrifugation at 12,000 rpm for 15 minutes , And (IV) supernatant, followed by filtration through a filter paper (Whatman No. 1, USA). At this time, a culture medium is prepared using the final stock of Chinese cabbage juice, which is used as a component of the basic Chinese cabbage culture medium of the present invention. Also, the amount of the carbon source (glucose, sugar, lactose, potato starch, starch, xylose, molasses and the like) required for the general medium, nitrogen source (peptone, tryptone, casein, yeast extract, Wheat [CGM], corn steep liquor [CSL], soybean flour [SBF], etc.) are added and used as a general medium. The pretreatment process was carried out to easily quantify the growth of microorganisms in a liquid culture by using a spectrophotometer by completely removing the crushed residue. After the pretreatment, the juice of the cabbage waste was sterilized at 121 ° C for 15 minutes to prepare a liquid medium. Also, when preparing a solid medium for the purpose of culturing on a petri dish, 1.5-2.0% of agar was added to the above liquid medium. Bacillus subtilis was cultivated using the medium prepared above, and the resulting cells and spores were observed under a microscope to show in FIGS. 1 and 2, respectively.

[Example 2]

Comparison of cell growth curves according to concentration of cabbage juice

The Chinese cabbage liquid medium prepared in Example 1 was diluted with distilled water to make a cabbage liquid medium of 1, 10, 40, 70, 100% (Chinese cabbage stock solution), and 100 ml of the cabbage liquid medium was filled in a 250 ml Erlenmeyer flask. 2.5 ml of Bacillus subtilis pre-cultured in a 20 ml test tube was inoculated on this medium for 2 days, and cultured at pH 6.0, 37 ° C, and 200 rpm with shaking. The growth curve of the cells was as shown in FIG. 3 . As a result, it was found that the cell growth was the most active in the 100% Chinese cabbage liquid medium. In addition, the Chinese cabbage liquid culture medium of the present invention may contain Bacillus amyloliquefaciens ATCC 23350, Bacillus brevis ATCC 8186, Bacillus cereus ATCC 23350, 9634), Bacillus circulans ATCC 9500, Bacillus polymyxa ATCC 12321, Bacillus thuringiensis ATCC 10792, 35646, etc., Cellulomonas cellulans ATCC 12830, Corynebacterium glutamicum ATCC 13032, Streptomyces argenteolus ATCC 5 11009, Streptomyces fungicidicus ATCC 21013, Streptomyces tuberidicus, (Streptomyces tubercidicus ATCC 25502), E. coli (Escherichia coil ATCC 9637, 10798, etc.) It was. Therefore, the Chinese cabbage liquid medium was useful as a general medium for culturing bacteria.

[Example 3]

Comparison of cell growth between cabbage liquid medium and normal medium

The 100% Chinese cabbage broth prepared in Example 1 and the LB broth, which is a general medium, were separately prepared, and 100 ml of each broth was placed in a 250 ml Erlenmeyer flask. 0.5 ml of Bacillus subtilis preincubated in a 20 ml test tube for 2 days was inoculated into each of these mediums and shake cultured at pH 6.0, 37 ° C, and 200 rpm to determine the degree of cell growth The absorbance at ₆₀₀ nm (OD ₆₀₀ ) was measured. The results are shown in Table 1 below.

As shown in the results of Table 1, the growth of Bacillus subtilis was somewhat better in the LB medium than in the cabbage liquid medium until the third day of culture, but was much higher in the cabbage liquid medium thereafter.

[Example 4]

Comparison of cell growth by incubation temperature

100 ml of the 100% cabbage liquid medium prepared in Example 1 was placed in a 250 ml Erlenmeyer flask. 0.5 ml of Bacillus subtilis pre-cultured in a 20-ml test tube was inoculated on this medium for 2 days. After shaking culture at pH 6.0 and 200 rpm at various temperatures, the growth curve of the cells was determined according to the incubation time. It's like hell. The results showed that the growth of the cells was the most active at 30-37 ° C.

[Example 5]

Comparison of cell growth by initial pH

100 ml of the 100% cabbage liquid medium prepared in Example 1 was placed in a 250 ml Erlenmeyer flask. 0.5 ml of Bacillus subtilis pre-cultured in a 20 ml test tube was inoculated on this medium for 2 days. After shaking culture at 37 ° C and 200 rpm at various initial pHs, the growth curve of the cells was determined according to the incubation time. 5 is the same. The results showed that the cell growth was the most active at pH 6.0-7.0.

[Example 6]

Sugar analysis of the culture using high pressure liquid chromatography (HPLC)

When Bacillus subtilis is cultured by using the Chinese cabbage liquid medium, it can be seen that there is a common two-stage growth curve as in FIGS. Ie, a small growth curve at the beginning of culture and a large growth curve of the latter period. For more accurate analysis, saccharides were analyzed by high pressure liquid chromatography (Varian, USA) equipped with an Aminex HPX-42C column (300 mm × 7.8 mm, Bio-rad, USA) Fig. According to the results of analysis at the important points in the culture time, the initial small growth curve shows that the glucose present in the Chinese cabbage liquid (the substance which flows out in the 14th minute of the sixth stage), the fructose (in the 16th minute of the sixth stage, Materials) and microorganisms (microorganisms). Because the sixth degree. The monosaccharides present at the beginning of A are shown in Fig. B at 66 hours of incubation. When the monosaccharide is completely consumed, the microbial growth is reduced. After that, the microbial growth is continuously increased again. This strain induces enzymes that decompose oligosaccharides and polysaccharides (cellulose, xylene, pectin, etc.) It can be decomposed and used as a carbon source. This is the sixth degree. B showed that the oligosaccharides and polysaccharides of 13 minutes or less were incubated for 149 hours. In C, it can be seen through disappearance or reduction.

As apparent from the above examples, the present invention provides an inexpensive bacterial culture medium using a large amount of Chinese cabbage waste produced in a Kimchi factory, a large-scale fruit and vegetable market, and the like. The present invention is not only effective in providing a method for producing an excellent medium for culturing industrially useful bacterium containing Bacillus subtilis in large quantity, It is an extremely useful invention in the bio-industry and the environment industry because it has the effect of solving environmental problems such as odor generation and sewage leakage caused by landfilling.

Claims (5)

The Chinese cabbage leaves were squeezed out, juice was extracted from the juice, and the mixture was treated in a pressure sterilizer at 121 ° C for 2 minutes, centrifuged at 12,000 rpm for 15 minutes, filtered, and sterilized at 121 ° C for 15 minutes Cabbage liquid medium for bacterial culture. The Chinese cabbage solid medium according to claim 1, wherein 1.5-2.0% of agar is added to the liquid medium. The culture medium according to claim 1 or 2, wherein the culture medium is prepared by additionally adding a carbon source and a nitrogen source to the culture medium. The method of claim 1, wherein the bacterium is selected from the group consisting of Bacillus subtilis ATCC 6633, Bacillus amyloliquefaciens ATCC 23350, Bacillus brevis ATCC 8186, Bacillus cereus ATCC 9634, , Bacillus circulans ATCC 9500, Bacillus polymyxa ATCC 12321, Bacillus thuringiensis ATCC 10792, 35646, etc., Cellulomonas cellulans ATCC 12830, Corynebacterium glutamicum ATCC 13032, Streptomyces argenteolus ATCC 11009, Streptomyces fungicidicus ATCC 21013, Streptomyces tubercidicus ATCC 11409, 25502), or Escherichia coli (ATCC 9637, 10798, etc.). . The Chinese cabbage leaf was squeezed, and the juice was extracted from the juice. The juice was treated in a pressure sterilizer at 121 ° C for 2 minutes, centrifuged at 12,000 rpm for 15 minutes, filtered, and sterilized at 121 ° C for 15 minutes A method for producing a Chinese cabbage liquid medium for culturing bacteria.