KR20000063185A - Control of aflatoxin using inhibition microorganism CP220 and bean fermentation food and animal feed applied to such microorganism - Google Patents

Abstract

PURPOSE: An antagonistic microorganism Bacillus subtilis CP220 is provided, which or 0.1-50% cultured broth of which inhibits growth of aflatoxin-producing fungi, or decomposes aflatoxin. Fermented soybean food and animal feed-stuff containing the Bacillus subtilis CP220 are also provided. CONSTITUTION: A method of isolating and identifying antagonistic microorganism, Bacillus subtilis CP220, from soil reagent or customary fermented soybeans comprises the steps of: mixing the reagent with tris-HCl buffer solution(pH 7.5); inoculating the mixture into a nutrient broth, and culturing at 20-30°C for 20-40minutes; taking some of the cultured solution, and diluting with saline(0.85% NaCl) step by step; smearing each diluted solution on a nutrient agar, culturing at 20-30°C for 3days, and isolating colony; inoculating the Aspergillus flavus and the Aspergillus parasiticus on potato dextrose agar and culturing at 20-30°C for a day; inoculating the antagonistic microorganism at four positions around the culture medium and culturing continuously; selecting the bacillus that form inhibition zone after culturing for 7days; selecting the most superior antagonistic microorganism and identifying it as the Bacillus subtilis CP220.

Description

Control method of aflatoxin using inhibition microorganism CP220 and bean fermentation food and animal feed applied to such microorganism}

The present invention relates to a method for inhibiting the growth of aflatoxin-producing mold using the antagonistic Bacillus subtilis CP220, and a fermented soybean food and livestock feed fermented with the antagonist.

Various fungi that produce fungal toxins are known to be widely contaminated with food or various agricultural products, and a number of studies have been reported on the occurrence of fungal toxin poisoning in humans and livestock ingesting these toxins.

Aflatoxin B ₁ , the most lethal among fungal toxins, is a secondary metabolite produced by Aspergillus flavus, Aspergillus parasiticus, Penicillium puberulum, etc. As well as reduced mortality or fertility in livestock, and carcinogenicity, teratogenicity and mutagenicity have been reported in humans.

Aflatoxins are found mainly in cereals, peanuts, and corn throughout the world, and humans are exposed not only to direct intake through contaminated produce, but also to indirect intake through secondary products of livestock fed contaminated feed, such as aflatoxin M in milk. It is a situation.

In Korea, 200 to 300 ppb of aflatoxin was detected in peanuts imported for livestock feed in the late 70's. Especially, it was exposed to contaminated foods in our diets common to fermented foods using molds such as traditional jang. The possibilities are also very high. In fact, 84 ppb of aflatoxin was detected in some doenjang samples from Busan, which was 2.8 times higher than the WHO standard. Therefore, it is very important to suppress contamination of aflatoxins in food or to inactivate already produced aflatoxins.

Research trends for inactivation of aflatoxins include: ammonia treatment of contaminated cotton thread, volatile organic solvents such as acetic acid and propionic acid, treatment of 1 ppm barium, methods using pesticides or herbicides, plants A method of extracting and treating essential oil components of the plant has been reported.

However, most of the antifungal substances listed above have not been put to practical use because they are not suitable for processing in foods or feeds and there is also a possibility of secondary pollution to the environment.

Therefore, the technical problem of the present invention is that aflatoxin B ₁ produced by some fungi is the most potent toxic substance discovered to date, and inactivates or inhibits the production of aflatoxin, which is known to be carcinogenic, teratogenic and mutagenic in humans. Instead of various chemical or physical methods in order to invent a biological inhibition method by antagonistic microorganisms, in particular antagonistic CP220 (KCTC 8831P) is isolated from various samples in nature.

Such biological inhibition using antagonistic microorganisms present in nature can be said to be an ideal method with almost no side effects. In the present invention, antagonistic microorganisms have been separated by antagonistic test through replacement culture, and aflatoxin in the early stage of mold growth. Inhibition of fungal growth and aflatoxin degradation of isolated antagonists were investigated. In addition, the expression of antagonism was confirmed in real doenjang and meju, and the taste and flavor components of doenjang were analyzed.

1 is a photograph showing the inhibition of growth of Aspergillus playbus and Aspergillus paraciticus by the antagonist CP220.

Figure 2 is a photograph showing the growth inhibition of Aspergillus placebus and Aspergillus paraciticus when treated with the culture filtrate of the antagonist CP220.

Figure 3 is a photograph showing the growth inhibition of Aspergillus placebus and Aspergillus paraciticus on a solid medium containing the culture medium of the antagonist CP220.

Figure 4 is a photograph showing the growth inhibition of Aspergillus placebus and Aspergillus paraciticus in the liquid medium containing the culture filtrate of the antagonist CP220.

Accordingly, an object of the present invention is to provide a method for inhibiting aflatoxin-producing mold growth or degrading aflatoxin through the antagonist Bacillus subtilis CP220 (Accession No. KCTC-8831P) or 0.1 to 50% culture thereof.

At this time, the aflatoxin-producing fungus is characterized by Aspergillus flavus and Aspergillus paraciticus, while the production of aflatoxin fermented with the antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P) is suppressed and taste and flavor Providing fermented foods containing improved soybean, soy sauce, cheonggukjang, ssamjang, red pepper paste, chunjang, and aflatoxin-producing livestock feed containing antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P).

Hereinafter, the present invention will be described.

Antagonist CP220 (KCTC 8831P) used in the present invention was isolated from soil samples or meju prepared by traditional methods. The isolation method of the strain is well mixed with tris-HCl buffer (pH 7.5), inoculated in nutrient broth (Nutrient Broth) and incubated for 20 to 40 minutes at 20-30 ℃, and then taken part of the culture solution saline solution Diluted stepwise in (0.85% NaCl), apply on a proper agar medium (Nutrient Agar) at an appropriate concentration, and incubate at 20-20 ° C for 3 days to separate the bacterial flora. Inoculate Aspergillus Flavor and Aspergillus paraciticus in the center of potato glucose agar medium (Potato Dextrose Agar) and incubate at 20-30 ℃ for 1 day, and continue inoculating each of the four antagonists at the surrounding spots. . After 7 days of incubation, strains that form an inhibition zone are selected as antagonists. As a result, the best antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P) was selected.

Treatment of antagonist CP220 culture filtrate resulted in superior growth inhibition of Aspergillus Placebus and Aspergillus paraciticus. Antagonist activity was 98% for Aspergillus Placebus and Aspergillus para. 93% of Cyticus cases.

When fungi were grown in solid medium of the culture filtrate of antagonist CP220, aspergillus placebo and aspergillus paraciticus showed excellent growth inhibition, and antagonism was 95% for aspergillus placebo. 82% for Aspergillus paraciticus.

In addition, the antagonistic bacterium of the present invention has the ability to decompose aflatoxin B1. Looking at the effect, the culture medium and the antagonistic bacterium in which aflatoxin B ₁ was produced by liquid culturing Aspergillus playbus and Aspergillus paraciticus for 4-5 days CP220 culture solution was mixed 1: 1 and incubated at 30 ° C. for 5 days, and the amount of aflatoxin B ₁ in the culture solution was analyzed and quantified by TLC method. When the antagonist CP220 was incubated for 5 days in a fungal culture containing aflatoxin B ₁ , the Aspergillus playverse culture showed a degradation rate of about 95% of aflatoxin B _{1, and} the aflatoxin in the Aspergillus paraciticus culture. The decomposition rate of 87% of B ₁ is shown.

Isolation of the antagonist was produced by culturing the antagonist CP220 (KCTC 8831P) in nutrient agar medium at 30 ℃ for 3 days and centrifuged to freeze-dried the supernatant. The separated antagonists were purified by solvent extraction, separation by silica gel adsorption chromatography, separation by Sephadex LH-20 chromatography, and the antagonistic effects thereof were measured. Aspergillus Placebus and Aspergillus were measured. It showed strong growth inhibition and antagonistic effects on paraciticus.

In addition, as a result of preparing a doenjang by the conventional method by adding the antagonist of the present invention, no aflatoxin was detected in the doenjang added with the antagonist, and the aflatoxin content of the doenjang prepared by treating the isolated antagonist and aflatoxin-producing bacterium Aflatoxin B ₁ was reduced by 96.9% by treatment with antagonists at 35.9 ppb when inoculated by bus, and aflatoxin B ₁ was reduced by 93.7% by treatment with antagonists at 30.2 ppb when treated with Aspergillus paraciticus. 1.9 ppb.

The contents of free fatty acids, free sugars and organic acids of doenjang treated with antagonists and aflatoxin-producing bacteria were similar to those of the control group. The total content of free amino acids was slightly increased by antagonistic treatment, in particular, the amount of glutamic acid was increased to improve the quality slightly.

The flavor component of doenjang was not changed significantly compared to the control when treated with aflatoxin-producing bacteria, but 2-pentyl furan and butanoic acid, which were transformed by antagonistic treatment, disappeared or decreased, and octane Decane (octadecene) compounds were produced. In other words, the isolated antagonists suppressed aflatoxin production in doenjang and improved the quality of soybean paste in terms of glutamic acid and flavor component.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Isolation of Antagonists and Preparation of Spore Suspension and Antagonist Culture Filtrate

Antagonist CP220 (KCTC 8831P) used in the present invention was isolated from soil samples or meju prepared by traditional methods. The above samples were well mixed in 100 ml of tris-HCl buffer (pH 7.5) and then inoculated in nutrient broth (Nutrient Broth) and incubated at 25 ° C. for 30 minutes. A portion of the culture solution was taken, diluted in physiological saline (0.85% NaCl), and applied to an appropriate concentration on nutrient agar (Nutrient Agar), and then cultured at 25 ° C. for 3 days to isolate the bacterial flora. Inoculate Aspergillus Flavers and Aspergillus paraciticus in the center of potato glucose agar medium (Potato Dextrose Agar), and incubate at 25 ° C for 1 day, and continue incubating by inoculating antagonists at each of the four surrounding spots. After 7 days of incubation, strains that formed an inhibition zone were selected as antagonists.

At this time, the degree of antagonism was determined by comparing the mold diameter in the case of replacement culture and mold only culture using the following equation. As a result, the antagonistic degree of the best antagonist CP220 (KCTC 8831P) was 43% and 39% for the two types of fungi.

Inoculate Aspergillus Placebus and Aspergillus paraciticus on potato glucose agar medium for 7 days, incubate for 7 days, add 1 ml of sterile Tween 80 solution (0.1%, v / v) and 5 ml of sterile distilled water. The spores were washed off and filtered through filter paper. The resulting spores were washed three times with sterile distilled water and then suspended again in Tween 80 solution and distilled water to contain 10 ^{7-10 8} spores per ml. Potato glucose medium was used for the production of aflatoxin B _{1. 1} ml of spore suspension was inoculated into 50 ml potato glucose medium and incubated at 25 ° C. for 7 days. For antagonists, SDBP medium (3% soy sauce, 5% glucose, beef extract) was used. 1 ml was inoculated into 50 ml of Treck 0.1% and Proteose peptone 0.1%), followed by incubation at 30 ° C. for 3 days. After culturing the antagonists, the supernatant obtained by centrifugation was sterilized with a 0.22 μm nitrocellulose membrane filter without heat treatment.

Zone of inhibition = (NT-T / NT) × 100

NT: diameter of total flora

TN: mycelial diameter of treatment

Table 1 shows the results of inhibiting the growth of Aspergillus playbus and Aspergillus paraciticus by CP220 (KCTC 8831P) (28 ° C., 7 days culture in potato glucose medium).

(%: Antagonist) Aspergillus Playbus Aspergillus paraciticus CP220 (KCTC 8831P) 43% 39%

The results of inhibition of growth of Aspergillus playbus and Aspergillus paraciticus by CP220 (KCTC 8831P) (28 ° C., 7 days incubation in potato glucose medium) are shown in FIG. 1.

Example 2 Mold Growth Inhibition Effect in Antagonist Culture Filtrate Treatment

Inoculate Aspergillus Placebus and Aspergillus paraciticus to potato glucose agar medium for 1 day, incubate for 1 day, pour the antagonistic culture filtrate on the growing mold surface, and incubate at 25 ° C for 7 days to investigate antagonism. It was. When the untreated heat-treated culture filtrate of antagonist CP220 (KCTC 8831P) was treated with antagonists showed 98% and 93%, respectively.

When the culture filtrate of CP220 (KCTC 8831P) was treated, the results of inhibition of growth of Aspergillus Plovers and Aspergillus paraciticus (28 ° C., 7 days culture in potato glucose medium) are shown in Table 2.

(%: Antagonist) Aspergillus Playbus Aspergillus paraciticus CP220 (KCTC 8831P) 98% 93%

In addition, the results of the inhibition of growth of Aspergillus placebus and Aspergillus paraciticus when treated with the culture filtrate of CP220 (KCTC 8831P) is shown in FIG.

Example 3 Inhibitory Effect on Fungal Culture in Medium Containing Antagonist Culture Filtrates

A medium containing antagonistic culture filtrate was prepared. In the case of liquid medium, 2.5 ml each of the concentrated potato glucose medium in 15 ml captubes was autoclaved and 2.5 ml of the antagonistic culture filtrate was mixed in different cap tubes to prepare 5 ml of media. Spore suspensions of Aspergillus Placebus and Aspergillus paraciticus were inoculated into the medium, and cultured at 30 ° C. for 7 days, and the growth of mold and aflatoxin B ₁ production were compared with the control. In the case of a solid medium, 39 g of powder was dissolved in 250 ml of filtrate corresponding to ¼ of the total volume, and sterilized. After cooling, the remaining 750 ml of filtrate was added to form a medium. The solid medium was inoculated with Aspergillus Placebus and Aspergillus paraciticus, and cultured at 30 ° C. for 7 days. Aspergillus plabus and Aspergillus paraciticus were cultivated in liquid and solid media containing antagonistic culture filtrates. Cyticus was inhibited by 82%, and the liquid medium showed a similar tendency to inhibit the fungal growth of antagonists. Aflatoxin B ₁ production was reduced by 98.1% and 92.5%, respectively.

Inhibition of the growth of Aspergillus placebus and Aspergillus paraciticus on solid medium containing the culture filtrate of CP220 (KCTC 8831P) (28 ° C in potato glucose medium, the result of 7 days culture) is shown in Table 3. It was.

(%: Antagonist) Aspergillus Playbus Aspergillus paraciticus CP220 (KCTC 8831P) 95% 82%

The results of the inhibition of growth of Aspergillus plabus and Aspergillus paraciticus on solid medium containing the culture medium of CP220 (KCTC 8831P) are shown in FIG. 3.

In addition, the results of the inhibition of growth of Aspergillus plabus and Aspergillus paraciticus in the liquid medium containing the culture filtrate of CP220 (KCTC 8831P) are shown in Table 4.

Growth [DCW / 5ml (%)] Aflatoxin production [peak area (%)] Aspergillus Playbus From potato glucose badges 9.2 mg (100%) 107.4 (100%) In CP220 (KCTC 8831P) culture filtrate 0.0 mg (0%) 2.04 (1.9%) Aspergillus paraciticus From potato glucose badges 11.3 mg (100%) 82.1 (100%) In CP220 (KCTC 8831P) culture filtrate 0.0 mg (0%) 6.16 (7.5%)

The results of the inhibition of growth of Aspergillus placebus and Aspergillus paraciticus in the liquid medium containing the culture filtrate of CP220 (KCTC 8831P) are shown in FIG. 4.

Example 4 Inhibition of Aflatoxin B1 Production of Fungi by Co-culture with Antagonists

5 ml of potato glucose medium was inoculated simultaneously with 50 µl each of Aspergillus placebo spore suspension and antagonist spp. The fungal aflatoxin B ₁ production was compared with the control. When co-cultivation of fungi and antagonists in potato glucose medium, aflatoxin B ₁ production decreased to 3.4% and 5.2%, respectively, compared to the control.

Table 5 shows the degree of aflatoxin production (30 ° C., 5 days culture) when CP220 (KCTC 8831P), Aspergillus Placebus, and Aspergillus paraciticus were co-cultured, respectively.

Aspergillus Playbus CP220 + A. f Aspergillus paraciticus CP220 + A. p Aflatoxin production [peak area (%)] 107.4 (100%) 3.65 (3.4%) 82.1 (100%) 4.27 (5.2%)

Example 5 Aflatoxin B1 Degradation Effect by Antagonists

Aspergillus Placebos and Aspergillus paracyticus were cultured in liquid for 4 to 5 days, aflatoxin B ₁ culture medium and antagonist CP220 (KCTC 8831P) culture solution were mixed 1: 1 and incubated at 30 ° C for 5 days. After aflatoxin B ₁ in the culture was analyzed and quantified by TLC method. When the antagonist CP220 (KCTC 8831P) was incubated for 5 days in a fungal culture containing aflatoxin B ₁ , it exhibited a degradation rate of about 95% in Aspergillus placebo culture and 87% in Aspergillus paraciticus culture. The decomposition rate of is shown.

Table 6 shows the effect of aflatoxin degradation by CP220 (KCTC 8831P).

Aspergillus Playbus A. f + CP220 Aspergillus paraciticus A. p + CP220 Peak area 112.1 5.6 88.4 11.5 Dissolution Rate (%) 95% 87%

Example 6 Isolation of Antagonist

Antagonists were produced by culturing antagonist CP220 (KCTC 8831P) in nutrient agar medium at 30 ° C. for 3 days and centrifuging to freeze-dried the supernatant.

1) Solvent Extraction

When the lyophilized culture was extracted with ethyl acetate, acetone, ethanol and methanol and tested for antagonistic activity against aflatoxin-producing bacteria, methanol extracts showed the strongest antagonistic activity against both Aspergillus placebo and Aspergillus paraciticus. Seemed.

2) Separation by Silica Gel Adsorption Chromatography

Silica gel (80 g, 70-230 mesh, Merck), equivalent to about 10 times the amount of the active substance, was made into a slurry with ethyl acetate and charged into a column (1.8 × 48 cm), and then methanol concentration was determined using an ethyl acetate-methanol solvent system. Was tested by increasing the fraction to 50, 60, 70, 80, 90, 100% to test the antagonistic activity. The active part was ethyl acetate-methanol 40:60 (v / v). This active fraction was fractionated with a chloroform-ethyl acetate solvent system to show activity in an elution fraction of chloroform-ethyl acetate 80: 20-70: 30 (v / v).

3) Separation by Sephadex LH-20 Chromatography

Sepadex LH-20 (Sephadex LH-20: 25-100 mesh, Phamacia) was packed into a column (4 × 100 cm) by swelling with methanol-chloroform (4: 1, v / v) solvent system for 12-16 hours. The antagonistic activity was then tested by elution fractionation of the active fraction separated by silica gel adsorption chromatography using the same solvent system. Antagonistic activity was shown at 0.42∼0.60 ratio of elution volumn to total volume (Ve / Vt). Aspergillus Placebuse did not grow for 20 days in the presence of 1 mg of antagonist, and Aspergillus paraciticus also inhibited growth.

Table 7 shows the growth inhibition of CP220 (KCTC 8831P) culture MeOH extract against Aspergillus placebus and Aspergillus paraciticus.

Growth inhibition (clear zone, mm) MeOHextracts * EtOHextracts * EtOACextracts * Acetone * Aspergillus Playbus 21 9 - - Aspergillus paraciticus 19 8 - -

0.5 g eq. extract of CP220 (KCTC 8831P) culture broth 9 mm hole

Example 7 Analysis of Aflatoxin and Flavor Components after Preparation of Doenjang and Aging

Miso is made in our country's traditional way. First, 10 kg of soybeans are soaked to make a shape, and each 10 mL treatment of apertoxin-producing Aspergillus flavus and Aspergillus paraciticus spore suspension, 10 mL treatment of antagonistic culture medium, 10 mL antagonistic culture medium + aflatoxin producing spore suspension 10 mL each was prepared by mixing treatment, initially dried slightly and then fermented in a natural state at about 30 ℃ for 90 days to complete the meju. Doenjang was conventionally prepared by putting 10 kg of meju in 2 L of 23% brine (NaCl) and leaving it at room temperature for 40 days, then removing the brine and aging for 90 days at room temperature.

1) Analysis of Aflatoxin Production

For aflatoxin analysis, 5 g of NaCl and 125 mL of 60% methanol were added to 25 g of soybean paste, and extracted with a brander for 5 minutes, and then adsorbed by passing 2-3 mL per minute through an immunoaffinity column, followed by washing twice with 10 mL of water. And aflatoxin was eluted with 1 mL of methanol. The extract was concentrated and analyzed by HPLC after adding 0.1 mL of trifluoroacetate. HPLC analysis conditions were prodigy 5μ ODS-2 (150 x 4.6 mm) as column, fluorescence photometer (Ex 365 nm, Em 418 nm) as detector, acetonitrile: water (3: 1) as mobile phase, flow rate 0.9 mL / min It was made.

The results for aflatoxin and general components of Doenjang added with antagonists are shown in Table 8. Aflatoxin was not detected in doenjang treated with antagonists alone, but 35.9 ppb was detected in aspergillus pasteus and 30.2 ppb in aspergillus paraciticus. However, aflatoxin B ₁ was treated with aflatoxin B ₁ at 1.1 ppb and 96.9% of aflatoxin was inhibited by antagonistic treatment, and aflatoxin B ₁ was treated with aflatoxin B ₁ at 1.9 ppb. Antagonistic inhibition of aflatoxin production was 93.7%. In addition, aflatoxin levels of doenjang treated with antagonists and aflatoxin-producing bacteria were below 10 ppb, the national standard.

Table 8 shows the analysis of the ingredients of Doenjang prepared with CP220 (KCTC 8831P), Aspergillus Flavor, and Aspergillus paraciticus.

Aspergillus Playbus Aspergillus paraciticus CP220 (KCTC 8831P) Control Aspergillus Playbus Aspergillus paraciticus moisture(%) 52.7 53.4 51.5 53.6 53.7 salt(%) 14.0 13.5 14.1 13.7 14.2 Aflatoxin (ppb) 35.9 30.2 - 1.1 1.9

2) free fatty acid analysis

About 30 g of soybean paste was precisely taken into a round bottom flask, 0.5 N methanolic sodium hydroxide solution was added thereto, and a reflux condenser was installed and heated for 10 minutes. And 14% BF ₃ was added through a reflux condenser and heating continued for 2 minutes. Finally 5 mL of n-peptane was added through a chiller and heated for 1 minute. The heptane layer was taken from the flask, and anhydrous sodium sulfate was added to dehydrate the solution. The test solution was analyzed using GC. DB wax capillary column was used as a column and FID was used as a detector. The inlet temperature was 230 ° C. and the oven temperature was maintained at 165 ° C. for 1 minute, and then heated to 2 ° C. per minute to 200 ° C., and the carrier gas flowed 2 mL of nitrogen per minute. Table 9 shows the free fatty acid contents of doenjang treated with aflatoxin and antagonists. The free fatty acids of doenjang are linoleic acid (51.2 mg%), oleic acid (oleic acid: 21.3 mg%), palmitic acid (10.8 mg%), linolenic acid (8.6 mg%), stearic acid (stearic acid: 5.3 mg%), and arachidonic acid (arachidonic acid: 0.4 mg%). There were no significant changes in the free fatty acids of doenjang by treatment with Aspergillus Placebus or Aspergillus paraciticus. Also, when treated with antagonists, the free fatty acids were not significantly different from the control, but oleic acid was slightly increased.

Table 9 shows the composition of free fatty acids of Doenjang prepared with CP220 (KCTC 8831P), Aspergillus Flavor, and Aspergillus paraciticus.

(mg%) An untreated sheet CP220 (KCTC 8831P) Control Aspergillus Playbus Aspergillus paraciticus Palmitic acid 11.4 10.6 10.7 10.8 Stearic acid 5.3 5.5 5.3 5.9 Oleic acid 20.5 21.2 20.7 21.4 Linoleic acid 50.6 52.3 51.5 50.7 Linolenic acid 9.1 8.9 8.8 9.5 Arachidonic acid 0.4 0.5 0.4 0.4

3) free sugar analysis

10 g of soybean paste was dissolved in 50 mL of water, 200 mL of ethanol was added, followed by extraction for 1 hour in a 70 ° C water bath, followed by filtration. 60 mL of 80% ethanol was added to the filtered residue, which was extracted six times for 1 hour, and then filtered again. The filtrate was concentrated under reduced pressure, ethanol was evaporated, dissolved in distilled water, treated with Sep pak C _18, and 20 μl of the filtrate was injected into HPLC. And analyzed. For analysis, the column uses Prodigy 5μ ODS-2 (150 × 4.6 mm), the detector uses shodex RI-71, the mobile phase uses 80% CH ₃ CN, 20% H ₂ O, and the flow rate is 1.0 mL per minute. It was. Changes in free sugar content of doenjang by aflatoxin-producing bacteria and antagonists are shown in Table 10. Doenjang contains 340.5 mg% of glucose, 85.4 mg% of galactose, and 1.3 mg% of arabinose. There was no significant difference in the composition or content of free sugar in doenjang by aflatoxin producing or antagonistic treatment.

Table 10 shows the free sugar composition of Doenjang prepared with CP220 (KCTC 8831P), Aspergillus Placebus, and Aspergillus paraciticus.

(mg%) An untreated sheet CP220 (KCTC 8831P) Control Aspergillus Playbus Aspergillus paraciticus glucose 340.5 344.3 335.7 347.3 Galactose 85.4 84.7 84.9 85.3 Arabinos 1.3 1.0 1.2 1.4

4) Organic Acid Analysis

5 g of miso was ground and distilled water was added to make 20 mL. After centrifugation and filtration through a 0.45 ㎛ membrane filter, the pigment and protein components were removed with Sep-pak C ₁₈ , and the filtrate was analyzed by injection into HPLC, and the conditions were 0.5-μm on a μ-Bondapak C ₁₈ (3.9 × 30 cm) column. The detector was UV 214 nm at 1.0 mL / min flow rate using% KH ₂ PO ₄ solvent. The organic acid contents of doenjang by aflatoxin-producing bacteria and antagonists are shown in Table 11. The organic acid contents of doenjang were oxalic acid (121.2 mg), followed by malic acid, citric acid, lactic acid, and acetic acid. Organic acids in doenjang did not change significantly due to aflatoxin producing or antagonistic treatment.

Table 11 shows the organic acid composition of Doenjang prepared with CP220 (KCTC 8831P), Aspergillus Flavor, and Aspergillus paraciticus.

(mg%) An untreated sheet CP220 (KCTC 8831P) Control Aspergillus Playbus Aspergillus paraciticus Oxalic acid 121.2 120.9 123.2 118.5 Malic acid 89.5 91.1 88.6 88.3 Lactic acid 19.9 19.8 21.7 20.6 Acetic acid 16.5 17.1 18.5 17.5 Citric acid 32.7 36.9 35.3 34.9

5) Free Amino Acid Analysis

75% ethanol was added to the sample miso, extracted for 30 minutes in a water bath, and concentrated. 30 mL of 25% trichloroacetic acid (TCA) solution was added to remove proteins, and 20 mL of ethyl ether was added to remove TCA and concentrated. The concentrate was adsorbed through the column packed with Amberlite IR20 (H ⁺ ) resin and eluted with ammonia again. The eluate was dried and dissolved in citric acid buffer (pH 2.2) and analyzed with an amino acid autoanalyzer. Column is 3.9 × 150 mm PICO. Analysis using a TAG column. The results of measuring the free amino acid content of the sample miso are shown in Table 12. The free amino acids of control doenjang were 315.1 mg% of glutamic acid, 224.3 mg% of aspartic acid, 174.2 mg% of tryptophan, and 122.3 mg% of leucine. 1654.8 mg%. When only aflatoxin producing bacteria were treated, the total amount of free amino acids was 337.1 to 535.1 mg% lower than the control. However, 53.4 mg% increased in the doenjang treated with antagonists, and 20-27 mg% was higher in the doenjang treated with antagonists and aflatoxins. In particular, the amount of glutamic acid, a glutamic amino acid, was higher by 18.8 mg% when antagonists were added, and 13 to 17 mg% was obtained even when aflatoxin and antagonists were mixed.

Table 12 shows the free amino acid composition (mg%) of Doenjang prepared with CP220 (KCTC 8831P), Aspergillus Placebus, and Aspergillus paraciticus.

(mg%) An untreated sheet CP220 (KCTC 8831P) Control Aspergillus Playbus Aspergillus paraciticus Asp 224.3 232.7 195.4 207.3 Glu 315.1 333.9 328.1 332.4 Ser 12.8 15.4 22.5 21.2 Asn 25.5 29.2 19.4 28.1 Gly 44.4 48.9 34.6 38.5 Gln 30.2 29.2 26.1 25.5 His 19.8 20.2 20.8 17.1 Thr 54.5 62.4 54.6 63.8 Ala 118.2 119.1 116.2 115.0 Arg 13.7 13.7 12.9 11.4 Pro 69.0 71.7 69.4 65.8 Tyr 37.2 39.9 45.1 36.4 Val 86.0 82.0 96.3 91.7 Met 41.5 47.5 49.0 45.0 Cys 0.0 0.0 0.0 0.0 Ile 97.1 83.1 98.6 90.9 Leu 122.3 129.3 132.5 121.0 Phe 58.4 60.4 66.7 73.9 Try 174.2 168.6 173.5 162.9 Lys 110.6 121.0 119.9 126.8 Sum 1,654.8 1,708.2 1,681.6 1,674.7

6) Analysis of Volatile Flavor Compounds

Extraction of volatile flavor components of doenjang was extracted for 2 hours at atmospheric pressure with a continuous distillation extractor. As the extraction solvent, 200 mL of a mixed solvent of n-pentan and diethylether (1: 1) was used. After extraction, the extraction solvent was added with anhydrous Na ₂ SO ₄ and left overnight at 4 ℃ to remove moisture, concentrated and analyzed by GC / MSD. The GC column was HP-5MS (crosslinked 5% HP ME Siloxane), the flow rate was 1.0 mL / min of helium, the oven temperature was maintained at 40 ° C. for 2 minutes, and then raised at 4 ° C. per minute for 10 minutes at 240 ° C. It was. Inlet temperature was 250 ° C., detector temperature was 260 ° C., and slitless. Volatile compounds were identified by comparative analysis of mass spectrum and retention time from GC-MS chromatogram of sample miso (control). A total of 47 kinds of volatile flavor components of Doenjang were identified. The main compounds were 1-octen-3-ol, 2-methyl butanol, and 9,12-octadecadiene. Egg (9,12-octadecadienal), hexadecanoic acid, isobutyl isopentanoic acid ester, 2-pentylfuran, 3-octanone, Benzaldehyde, methyl benzene and the like were found to have high contents.

The aroma toxins of the doenjang treated with aflatoxin-producing bacteria were not significantly different from the control, and the aflatoxin-producing bacteria had no significant effect on the aroma of miso. However, the fragrance components of doenjang treated with antagonistic Cp220 showed a little difference from the control. Antagonistic treatment results in 2-methyl ester, dimethyl trisulfide, 2-pentylfuran, butanoic acid, 2-methyl buthyl ester ), 2-methyl-5-isopropyl pyrazine, acetate 2-phenylethyl ester, 9,12-octadecadienal (9,12-octadecadienal ), No aroma component of 2-octyldodecan-1-ol was detected, butanoic acid ethyl ester, 2,4-decadienal (2,4 -decadienal, hexadecanoic acid ethyl ester, 9-octadecenic acid, 9-octadecenal, 9,17-octadecadienal (9, The fragrance component of 17-octadecadienal) was produced by antagonistic treatment. In addition, 2-heptenal and benzaldehyde O-ethyl esters slightly increased, whereas 3-methyl butanoic acid ethyl esters decreased. . In particular, 2-pentylfuran is a causative agent of soybean oil and was not produced by antagonistic treatment. Butanoic acid, also known as unpleasant, was lost or reduced by antagonistic treatment, but 2-methyl butanoic acid methyl ester disappeared and 2-methyl butanoic acid ethyl ester (2-methyl butanoic acid ethyl ester and 3-methyl butanoic acid ethyl ester were decreased. The octadecane compound, an alkan, which will play an important role in the doenjang fragrance component, was increased by antagonistic treatment. -4.61% of 9-octadecenoic acid, 2.12% of 9-octadecenal, and 3.17% of 9,17-octadecadienal.

The present invention provides a method for biologically inhibiting aflatoxin B ₁ produced by some molds by antagonistic microorganisms, in particular, by separating antagonist CP220 (KCTC 8831P) from various samples in nature, producing aflatoxins in the early stage of mold growth. By inhibiting the antagonistic bacteria and the ability to inhibit the growth of fungi and aflatoxin, it was to improve the taste and flavor components of miso through the expression of antagonism in miso and meju.

Claims (5)

Antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P) or a method for inhibiting aflatoxin-producing mold growth or degrading aflatoxin through 0.1-50% culture thereof The method of claim 1, wherein the aflatoxin producing fungi are Aspergillus playbus and Aspergillus paraciticus. Fermented foods containing soybean paste, soy sauce, cheonggukjang, ssamjang, red pepper paste, chunjang, etc., which inhibit the production of aflatoxin fermented with antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P) and improve taste and aroma. Sources such as instant marinated sauce, pork mixed sauce, beef mixed sauce, fish cutlet sauce, pork cutlet sauce, which suppress the production of aflatoxin fermented with antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P) and improve taste and flavor, Meat processed products such as canned pork and canned vegetables, and foods such as pickles such as sesame leaf and anchovy. Livestock feed inhibiting aflatoxin production containing antagonistic Bacillus subtilis CP220 (Accession No. KCTC-8831P)