KR101280811B1 - Microorganism having zearalenone decomposing activity, method for degrading zearalenone using thereof and feed additives comprising the same - Google Patents

Abstract

The present invention relates to a microorganism having a zeararenone degrading activity, and expresses a gene comprising a nucleotide sequence of SEQ ID NO: 2, variants thereof, or fragments thereof, and a microorganism having a zeararenone degrading activity, using the microorganism Provided are a method for producing a protein having zearenone degrading activity, a method for degrading zearenone, and a feed additive for degrading zearenone.
According to the present invention, an enzyme produced by a microorganism can be used to decompose zearalenone, which is a fungal toxin, or biotransform the fungal toxin using a microorganism, thereby providing an improved control effect of zearalenone.

Description

Microorganism having zearalenone decomposing activity, method for degrading zearalenone using about and feed additives comprising the same}

The present invention relates to a microorganism having a zeararenone degrading activity, a method and a feed additive for zearenone decomposition using the same, and more particularly, a microorganism having a zearenone degrading activity for biologically treating a fungal toxin, zearenone. , Relates to a method for decomposing zearenone and a feed additive using the microorganism.

More than 25% of cereals worldwide are contaminated with mycotoxins and are transmitted to livestock and humans through the food chain, causing serious economic problems.

Zearalenone is a nonsteroidal estrogenous fungal toxin, especially in the fungus of Fusarium, especially F. graminearum (Gibberella zeae), F. culmorum , F. as a real lease three toxin production (F. cerealis), F. Iquique Shetty (F. equiseti), F. keurukeu wells Lawrence (F. crookwellense), F. semi tekteom (F. semitectum) in grain worldwide It is a widely distributed fungal toxin. The chemical structure of zeanenonone and its derivatives is shown in the following formula (1).

[Formula 1]

(a) Zearenone (ZEN), (b) Alpha-Geararenol, (c) Beta-Geararenol, (d) Zearanone, (e) Alpha-Geararanol, and (f) Beta- Zeararanol

Zearalenone is also called F-2 oxy, fusarium toxin, and fermentation extrogenic substrance (FES), and there are about 20 isomers, but trans-a-geralenone occurs mainly in natural grains. Zearalenone ((6- [10-hydroxy-6-oxo-trans-1-undecenyl] -B-rezocyclic acid lactone) is a white crystal having a chemical formula of C ₁₈ H ₂₂ O ₅ , melting point. Is 164-165 ° C. and absorbs at 236, 237, and 316 nm, and forms various isomers by various oxidation and reduction reactions, and is insoluble in water, CS2, carbon tetrachloride, etc., but insoluble in ether, benzene, chloroform, methylene, etc. Soluble in chloride, ethyl acetate, acetonitrile, alcohol, alkali.

Toxicity of zearenone is the same as that of extradiol and natural estrogen, and it is classified as non-acute toxin because it does not die even after 20,000㎍ / kg oral administration to rats in animal experiments. have. Zearenone has been reported to be harmed by the action of sex hormones rather than toxicity, and most of the toxicosis is manifested as hyperhormonosis, cancer and mutations. In particular, it is an important fungal toxin in the livestock industry rather than in humans, which causes infertility in pregnant pigs, ovarian abnormalities, premature infant production, miscarriage, and ovarian changes in pigs with 50ppb feed (Rudolf, 1999).

There are physical, chemical and biological treatment methods for treating fungal toxins, but the physical and chemical treatment methods are not clear in their effects, require separate equipment and space for treatment, and after treatment, It may be said that economic method is inferior because it may need to go through removal process. The chemical treatment method may cause a large number of errors depending on the treatment conditions and the concentration of the treatment, and when the treatment concentration of the chemical agent is low, it is difficult to obtain consistent results for each type of mold toxin.

Biological methods for removing fungal toxins are methods of removing toxins through enzyme decomposition or biotransformation of fungal toxins by microorganisms.

Research on microorganisms with fungal toxin degrading continues to this day, and separation of strains has been carried out under various environmental conditions, such as grains contaminated with fungal toxins, unlike the early rumen microorganisms. Microorganisms with fungal toxin resolution are typically fungi such as Aspergillus niger , yeast and lactic acid bacteria, and have been reported to degrade or to biotransform fungal toxins. There is not much research on the specific role of bacteria (such as enzymes or other metabolites).

Recently, studies have been conducted to inhibit genes, such as hydrolase or epoxidase, from plants or microorganisms whose fungal toxin degradability has been confirmed, from the production stage, but are insufficient. In addition, the development of a product using a microorganism with a confirmed resolution as a feed additive is not much situation so far many products are commercially available as an adsorbent. For yeast, products that adsorb fungal toxins using cell walls have been released, and some studies have reported adsorption effects using yeast cell walls.

Fungal toxins are metabolites produced during the growth of fungi, and killing fungi stops the production of fungal toxins, but the toxins already produced remain in the feed causing toxins. In order not to be affected by mold toxins, only fresh feed that is not contaminated with mold should be fed, but it is practically impossible to reduce the damage by using adsorbents and feed additives. Some mold toxins are generally used through enzymatic treatment. Inactivation methods are also being developed.

The most ideal way to reduce mold toxin contamination is to prevent mold toxin contamination in food, feed, etc., and there are traditional methods to manage them from the seeding stage, but they do not produce satisfactory results.

Therefore, in the present invention, for the effective control of the fungal toxin, in particular, zearenone is to propose an effective fungal toxin removal method through the development of a biological treatment method to replace the existing inefficient physical and chemical treatment methods. Accordingly, the present invention is improved by developing a method of bioconversion or degradation of fungal toxins, in particular zeararenone, by using an enzyme produced by microorganisms, or by using microorganisms. It is intended to provide a way to bring the Lennon control effect.

According to the first aspect of the present invention,

A microorganism is provided that expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2, a variant thereof, or a fragment thereof, and which has zearalenone degrading activity.

According to the second aspect of the present invention,

(a) preparing the microorganism of claim 1 or 2;

(b) culturing the microorganism to produce a protein comprising the amino acid sequence of SEQ ID NO: 1, variants thereof, or fragments thereof having zeararenone degrading activity; And

(c) purifying the protein from the culture

Provided is a method for producing a protein having a zearalenone degrading activity comprising a.

According to the third aspect of the present invention,

The product contaminated with zearalenon

i) a protein comprising the amino acid sequence of SEQ ID NO: 1, a variant thereof, or a fragment thereof having zeararenone degrading activity;

ii) a microorganism which expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2, a variant thereof, or a fragment thereof, and which has zealenone degrading activity; or

iii) mixtures thereof

There is provided a method of dissolving zearenone.

According to the fourth aspect of the present invention,

i) a protein comprising the amino acid sequence of SEQ ID NO: 1, a variant thereof, or a fragment thereof having zearalenone degrading activity;

ii) a microorganism which expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2, a variant thereof, or a fragment thereof, and which has zealenone degrading activity; or

iii) mixtures thereof

There is provided a feed additive for decomposition of zearalenone.

According to the present invention, there is provided a biological treatment method to replace the existing inefficient physical and chemical treatment methods for the effective control of the fungal toxin, in particular zearalenone. The present invention can provide an improved control effect of zearenone by decomposing the fungal toxin zearenone using an enzyme produced by the microorganism or biotransforming the fungal toxin using the microorganism.

Figure 1 shows a schematic of the strain Bacillus subtilis ( Bacillus subtilis ) GB-501 (Accession No .: KCCM10930P, Deposit Date: March 6, 2008) isolated in the present invention,
Figure 2 is the result of evaluating the zearenone degradation activity of each strain according to the present invention culture (control (■); ZEN degradation (▲); Bacillus subtilis GB-501 bacteria (◆),
Figure 3 is the result of evaluating the zeararenone degradation activity of the strain according to the present invention using TLC (1: ZEN standard (10ppm); 2: culture (0 hours, 800ppb); 3: culture (12 hours later) 4: culture (after 24 hours), 5: culture (after 36 hours), 6: culture (after 48 hours),
Figure 4 is the result of measuring the zeararenone degradation activity of the strain according to the invention during liquid fermentation by ELISA,
5 is a result of evaluating zearenone degradation activity of the strain according to the present invention through DDGS solid phase fermentation (control (■); ZEN degradation (▲); microbial growth (◆)),
FIG. 6 shows the results of HPLC measurement of the zearalenone degrading activity of the strain according to the present invention during DDGS solid phase fermentation (A: standard sample; B: 0 hours; C: 12 hours; D: 24 hours; E: 48 time),
7 is a result of evaluating zearenone degrading activity of the fraction obtained from the DEAE-Sepharose column, and
8 shows the result of SDS-PAGE analysis of the separated protein (M: protein standard marker).

Hereinafter, the present invention will be described in detail.

In the present invention, a protein having zeararenone degrading activity, which is a fungal toxin, is provided. The protein comprises the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a gene encoding a protein having the zeararenone degrading activity, the gene comprises the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention is provided with a microorganism having a gerarenone degrading activity, the microorganism expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2. In the present invention, the microorganism is preferably Bacillus subtilis GB-501 (Accession No .: KCCM10930P, Deposit Date: March 6, 2008).

By using the enzyme produced by the novel microbial strain Bacillus subtilis GB-501 provided in the present invention, the fungal toxin zeararenone is decomposed or the microorganism is used to biotransform the fungal toxin. It can provide improved Zelarenone control effect.

In another aspect, the present invention provides a method for producing a protein having a zearalenone degrading activity, the method comprising the steps of (a) culturing the microorganism; (b) expressing a protein comprising the amino acid sequence of SEQ ID NO: 1 having zearalenone degrading activity in the cultured microorganism; And (c) purifying the expressed protein. Herein, the microorganism expresses a gene including the nucleotide sequence of SEQ ID NO: 2, and preferably, Bacillus subtilis GB-501 is used.

In another aspect, the present invention provides a method for dissolving a gerarenone that treats a product contaminated with zearenone, the method comprising: i) a protein comprising the amino acid sequence of SEQ ID NO: 1 having a gerarenone cleavage activity; ii) a microorganism which expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2 and which has zearalenone degrading activity; Or iii) treating with a mixture thereof. Herein, the microorganism is preferably Bacillus subtilis GB-501.

Products that are contaminated with zearalenone during the breakdown of zeararenon are not limited to, i) cereals (e.g. corn, rice, barley, sorghum, wheat, rye, oats, crude, blood, tricale, buckwheat, lupine Seeds and legumes) and their by-products (e.g. cereals, bran, horse flour, barley bran, rice bran, skim rice bran, corn bran, sorghum bran, bran, legume bran, peanut bran, cotton bran, oat bran, almond bran, sunflower bran , Molasses adsorption, etc.), gourds (soybean meal (including soybean processed products), perilla jelly, sesame jelly, rapeseed gourd, cotton thread gourd, peanut gourd, red pepper gourd, flax gourd, palm gourd, sunflower seed gourd, castor Gourd, corn germ meal, wheat germ meal, corn gluten, wheat gluten, alcoholic beverage, beer gourd, soy milk gourd, tofu gourd, oat gourd, k bread gourd, palm oil gourd, starch gourd, etc. and concentrated protein (e.g., soybean, potato) Crops or by-products thereof; ii) fermented feed and fermented foods such as meju, miso, soy sauce and red pepper paste using the crops or by-products thereof; iii) livestock or livestock waste from pig farms or livestock fermentations; Or iv) mixtures thereof.

The treatment method for treating contaminated products may be carried out by methods known to those skilled in the art, but is not limited thereto, by treating the protein, microorganism or a mixture thereof by contacting such as by spraying, mixing or dipping the contaminated product. Can be.

In addition, the present invention is provided with a feed additive for degradation of zeararenone, wherein the feed additive is i) a protein comprising the amino acid sequence of SEQ ID NO: 1 having a zeralenone degradation activity; ii) a microorganism which expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2 and which has zearalenone degrading activity; Or iii) mixtures thereof. Also here, the microorganism is preferably Bacillus subtilis GB-501.

Hereinafter, the present invention will be described in more detail by way of examples.

<Examples>

I. Materials and Methods

1. Toxin Analysis Method (ELISA / HPLC)

(1) Establishment of ELISA assay

In order to analyze zearenone from feedstock, three companies were used among the most commercially available products, and fungal toxin contamination in feedstock was analyzed using each kit (Table 1). The analysis procedure for each kit is summarized in Table 2. In kit C, 5 g of the raw material sample was extracted with 12.5 mL of 70% methanol and used for analysis.

ELISA Kit Types for Immunoassay of Feed Ingredients Manufacturing company name product name Antibody Detection principle A AgarQuant Monoclonal antibody Indirect competitive ELISA B Veratox Monoclonal antibody Indirect competitive ELISA C RIDASCREEN Monoclonal antibodies
* AF: polyclonal antibody Indirect competitive ELISA

Sample Analysis Process by ELISA Kit ELISA kit analysis ELISA Kit Type A B C Sample Preparation ^* 20g / 100mL 70% MeOH 25g / 125mL 70% MeOH 5g / 25mL 70% MeOH percolation Whatman No.1 filter Whatman No.1 filter Whatman No.1 filter Conjugate 200 μl 100 μl 50 μl Antibody - - 50 μl Standard and Sample ^** 100 μl 100 μl 50 μl Mixing and cultivation 10-15 minutes 5 minutes 10 minutes Washing 5 times 5 times 3rd time Substrate 100 μl 100 μl 2 drops (100 μl) culture 5 minutes 5 minutes 5 minutes in the dark Stop solution 100 μl 100 μl 2 drops (100 μl)

* Sample preparation: Sample amount and concentration of MeOH differ depending on each kit and mold toxin type.

** Sample dilution rate also depends on kit and type of fungal toxin.

(2) Establishment of analytical methods using HPLC

10 g of the sample was shaken for 30 minutes using 20 mL of acetonitrile, centrifuged for 5 minutes, and 100 mL of the supernatant was taken.  Elution is performed using an HLB SPE cartridge (200 mg, 6 cc). The eluted sample was dried using N ₂ gas at 50 ° C., redissolved with a mixed solvent of 1 mL of methanol / ammonium acetate (10 mmol / L) (1: 1, v / v), and used as an HPLC sample. It was. Detection of the analytical sample used a fluorescence detector.

(3) Sample of raw material for fungal toxin analysis

In the case of feed material samples, samples were collected by a feed company, and cereals, gourds, and barks were sampled according to the type of raw material according to the country of origin (Table 3). In Table 3 below, "introduction" refers to the case where the import or origin is unclear. Total 26 kinds of feed material samples are widely used in Korea, and the level of fungal toxin contamination in feed materials is examined by examining the immunoassay method and the analysis method using HPLC such as HPLC for each raw material by country of origin. It was.

Feed Ingredients for Fungal Toxin Analysis Classification Raw material name origin sample water Cereals corn United States of America 4 China 4 Wheat China One Introduction One Pomegranate Soybean meal Domestic 3 South America 2 India 2 Palm Park Introduction One River blood Wheat flour Domestic 2 Introduction One Protein Introduction 2 Rice bran Domestic One Introduction 2 system 26

2. Selection of Mold Toxin Degrading Strains for Microbiological Treatment

1) Selection of Mold Toxin-degrading Microorganisms

Samples such as grains contaminated with mold toxins (corn, protein skin, alcoholic beverages, etc.), fermented foods (meju, soybean paste, etc.) and livestock wastes, livestock waste (swine farms and livestock fermentation plants, etc.), industrial wastes, and industrial complex neighborhoods. The microorganisms present in the sample were first selected by spreading them onto nutrient media (nutritional agar, potato dextrose agar, MRS agar) for culturing each microorganism. The colonies of the selected strains were first determined using the respective media (nutrition agar, potato dextrose agar) to determine the type of bacteria (bacteria, fungus, yeast), and all colonies were selected using a medium suitable for characteristics. .

Selected colonies were inoculated in 1 mL of nutritious broth, potato dextrose broth and MRS broth using a 96-well dip plate, and each well was aflatoxin, okratoxin and zearalenone standard (Sigma). Simultaneously added at 40, 40, and 800 ppb and shake cultured at 35 ° C. for 24 hours using a microplate shaker (300 rpm), and then analyzed the fungal toxin resolution of each strain using each mold toxin assay kit (Romer). The strains whose resolution was confirmed were selected secondarily.

Inoculate the second selected strains with confirmed fungal toxin into culture broth of 100 mL, add fungal toxin standard with resolution and shake for 24 hours at 35 ° C, and then use the fungal toxin analysis kit. The resolution was reconfirmed. Colonies whose resolution was reconfirmed were finally selected and characterized for strains.

2) Identification of selection strains

Selection strains were identified by 16S rDNA analysis.

3. Study on characteristics of selection strain

1) Evaluation of Zearenone Degradation Activity of Selected Strains

In order to evaluate the degradation activity according to incubation time of the starter strains, 100 ml of LB broth was inoculated with colonies, and at the same time inoculated with Zearalenone Standard (Sigma) to 900 ppb, shaking culture at 35 ℃ for 24 hours, 5, 12 After 17, 24 hours the change in zearenone concentration was analyzed.

2) Evaluation of Degradation Activity through TLC

TLC analysis was performed using silica 60 plate (Merck), and the sample was developed by mixing chloroform and methanol in a ratio of 97: 3 as a mobile phase. Zearenone was detected after evenly spraying Fast Violet B (Sigma) and heat-treating at 100 ° C. for 5 minutes in a dry oven.

3) Zearenone availability analysis of selected strains

In order to analyze whether the selected strains use zearenone as the sole carbon source, the availability by the strain was evaluated by adding zearenone as the carbon source to the minimal salt media. The composition of the minimal medium is shown in Table 4 below.

Minimal Badge Composition Furtherance Content (g / L) (NH _{4) 2} SO ₄ 0.5 MgSO ₄ 7H ₂ O 0.2 CaCl ₂ 0.05 Na ₂ HPO ₄ 2.44 KH ₂ PO ₄ 1.52 Zeararenon 100 µg / mL

4) Investigation of the Degradation Activity Characteristics of Zearenone Degrading Strains

The division of the liquid culture, the cell (microbial cell), and the culture broth of the selected strains having the gerarenone degrading activity were identified through the classification of the gerarenone decomposition.

4. Establishment of cultivation method of selected microorganisms

1) Establishment of liquid culture conditions for selected strains

A method for establishing optimum liquid fermentation conditions of the selected strains was established. The selected strains were Bacillus strains, which did not have any special constraints on the liquid culture, and established the culture conditions with the optimum degradation activity based on the fungal toxin degradation activity.

In order to confirm the fermentation time of zearenone degradation activity, samples were taken for each hour while incubating in LB broth for 24 hours and analyzed for zearenone degradation activity.

2) Establishment of solid state culture method conditions

Solid phase fermentation conditions for mass production of the selected strains were established at the laboratory level. The fermentation conditions in the solid phase culture conditions were selected as the total fermentation time of 24 hours showing the reduction effect of more than 95% by identifying the fungal toxin reduction effect as the Bacillus strain. For solid phase cultivation, soybean meal is used as a raw material, sterilized by autoclave with initial water content set to 40% level, inoculated with liquid spawn at 1% level, and solid phase fermentation proceeds. Fermentation temperature conditions were determined to be 37 ℃, humidity 70% or more. Through the investigation of the change of the number of bacteria by fermentation time according to the seed inoculation amount, the seed growth was shown to be the same growth pattern from 1-4% except for 0.5% addition, and the seed addition level was set to 1%, and the initial water content was 20-45%. The test was set to 40%.

3) Mold Toxin Degradation Activity Test through DDGS Solid Phase Fermentation

For solid phase fermentation tests, the distillers dried grains with solubles (DGS), which are considered to be the most serious contamination of fungal toxins (about 90% of DDGS in the world) are subjected to solid phase fermentation tests. Fungal toxin degradation effects were assayed.

Add water to the water content of DDGS (water content 12.7%) to about 40%, and kill some of the harmful bacteria in the feed through heat treatment at 100 ° C for 30 minutes to make the liquid culture of the selection strain 1%. After inoculation and incubation at 37 ° C. for 48 hours, drying was performed for 8 hours at 45 ° C. using a dry oven.

4) Validation of degradation activity through HPLC analysis

The fermentation samples by solid phase fermentation (0, 24, 48 hours) were subjected to pretreatment using an immunoaffinity column (EASI-EXTRACT Zearenone, R-biopharm) and analyzed by HPLC. HPLC analysis conditions are shown in Table 5.

HPLC conditions for Zeararenone analysis HPLC settings and conditions column Spherisorb ODS2 (5 μm, 25 cm x 4.6 mm) Mobile phase 50:50 acetonitrile; PH 3.2 with water and acetic acid Flow rate 1.0 mL / min Fluorescent detector settings Excitation: 274 nm, Emission: 418 nm Column burner 25 ℃ Residence time 14 minutes Dose 100 μl

5. Selection and Characterization of Zearenone Degrading Activity Gene

Zearenone (ZEN) degradation activity gene selection method is as follows.

1) Cell culture at 25 ° C. for 1 week at 250 rpm in a nutrient medium containing 100 μg / mL ZEN.

2) After one week, the cultures are transferred to 1 L of fresh nutrient medium containing 25 μg / mL ZEN and incubated at 25 ° C. for one week.

3) Collect cells and transfer to Buffer A (10 mM Tris-HCl, pH 7.5) and sonicate on ice (5 min at level 4).

4) Remove cell debris by centrifugation at 5,000 g.

5) Add ammonium sulfate to the supernatant with 40-60% saturation.

6) Centrifuge at 10,000 ° C. for 1 hour at 4 ° C. to obtain a precipitate.

7) Dialysis overnight in Buffer A.

8) Apply the sample to 5 mL HitrapQcolum (Amersham Bioscience).

9) Elute with a linear gradient of 0-1 M NaCL in 100 mL of buffer A.

10) Active fraction size-fractionation by Superdex75HR10 / 30 (Amersham Bioscience) with buffer A containing 0.1 M NaCl.

11) Apply the catalyst fraction to an anion-exchange column (MonoQHR5 / 5, Amershambioscience) pre-equilibrated with buffer A and elute with a 20 mL NaCl gradient (active fraction elute at 0-0.5M, 0.25M).

12) Repeat the last mono Q step to remove pale contaminated protein.

13) All the above purification steps are carried out at 4 ° C.

II. Results and Discussion

1. Results of Fungal Toxin Analysis in Grain for Feed

Results of Zearenone Content Analysis in Feed Ingredients in Korea Sample number Raw material information ELISA kit (40-1000 ppb) HPLC-FLD
(ppb) A B C One Domestic soybean meal 7.9 18.5 56.9 55.8 2 South American Soybean Meal 23.5 29.0 75.3 61.6 3 Wheat flour 0.1 3.4 37.0 - 4 Rice bran 7.9 14.0 41.2 - 5 Domestic soybean meal 27.2 18.8 69.7 - 6 Wheat flour 3.2 5.3 38.8 - 7 Protein 68.4 66.5 92.3 103.0 8 Rice bran 25.1 16.8 38.3 - 9 Palm Park 4.8 10.5 36.5 - 10 Uncooked corn (whole grain) 5.8 7.4 30.9 - 11 Chinese Corn (3mm) 9.7 14.2 35.5 - 12 Chinese Wheat 25.7 15.8 60.0 tr 13 Indian Soybean Meal 7.3 9.8 42.4 65.0 14 Protein 99.4 82.8 138.2 77.0 15 Uncooked corn (whole grain) 4.6 - 18.8 - 16 Chinese Corn (Whole Grain) 13.5 4.4 45.9 - 17 Rice maize (3mm) 19.3 4.2 31.9 - 18 Chinese Corn (3mm) 37.7 26.2 76.9 - 19 Rice maize (7mm) 10.8 0.3 23.6 - 20 Chinese Corn (5mm) 17.7 2.8 23.3 - 21 Wheat 74.3 36.3 62.5 96.3 22 South American Soybean Meal 22.2 22.8 38.8 69.1 23 Domestic soybean meal 1.5 19.4 33.4 59.0 24 Indian Soybean Meal - 7.7 70.9 - 25 Wheat flour - 0.7 86.9 79.4 26 Rice bran 49.1 64.1 70.3 -

As shown in Table 6, as a result of measuring the fungal toxin (zearenone) in the feed grain it was confirmed that a significant amount of the fungal toxin is detected in both domestic and imported.

2. Selection of Mold Toxin Degrading Strains for Microbiological Treatment

1) Selection of Zearenone Degrading Active Strains

Excellent strains with more than 99% zearalenone degradation activity were selected from livestock fermentation plant, industrial wastewater and industrial area samples, and three strains with more than 90% activity were selected. Among the strains having the highest degradation activity, 16S rDNA analysis was performed, and the selected strains were found to have about 97% of Bacillus subtilis and homology, which can be used as probiotics . The selection strain was named Bacillus subtilis GB-501 and deposited in Korea Microorganism Conservation Center (KCCM) (Accession No .: KCCM10930P, Deposit Date: March 6, 2008).

A schematic diagram of the selected strain Bacillus subtilis GB-501 (hereinafter also referred to simply as 'GB-501') is shown in FIG. 1.

3. Characterization of Selection Strains

1) Evaluation of Zearenone Degradation Activity of Selected Strains

The results of evaluating the degradation activity of the selected strains by incubation time are shown in FIG. 2. There was no by culturing 5 time that the changes in the alas fluorenone concentration, that from the moment the microorganisms bacteria will begin to 10 ⁵ cfu / mL over a sharp change of the Ara fluorenone concentration observed Then, the number of bacteria is to 10 ⁸ cfu / mL At 12 hours of incubation, 95% or more of zearenone was decomposed.

2) Evaluation of Zearenone Degradation Activity Using TLC

Zearenone degradation activity was reconfirmed by TLC analysis, and the results are shown in FIG. 3. As a result, after 12 hours of incubation, no zearenone was detected, and it may be determined that zearenone was decomposed by the strain GB-501.

3) Zearenone availability analysis of selected strains

Selection strain GB-501 and other selection strains (GB-ZEN-14 and GB-ZEN-46) were used as the carbon source in the minimal salt media to analyze zearenone availability as the sole carbon source. Zearenone was added to assess availability by strains (Table 7). It was judged that the mechanism of decomposition of zearalenone could be more easily identified through the verification of zearenone availability, but the availability as a carbon source was not as high as 36%. However, it was judged that the GB-501 strain was not grown in the control group which did not add zearenone as a carbon source, and thus it was found to be useful as a carbon source. This is due to the restriction on the carbon source, and the degree of generation of enzymes for decomposition varies depending on the appropriate number of bacteria. Therefore, it is expected to grow using only the minimum nutrients necessary to grow up to a specific number of bacteria (106 cfu / mL). It is thought to bring about the result, and it is also useful as a carbon source, but it is believed that zearenone is decomposed by a specific enzyme or the like.

Availability of Zeararenone by GB-501 in a Liquid Medium Containing Zearalenone as a Source of Carbon and Energy (37 ° C, 150rpm) Strain Microbial Count (cfu / mL) Usability (%) Early 24 hours GB-ZEN-17 To 10 ² 2.6 x 10 ⁶ 36 GB-ZEN-14 To 10 ² 6.8 x 10 ⁵ 22 GB-ZEN-46 To 10 ² 3.4 x 10 ⁶ 29 Control
(There is no zeararenon) To 10 ² 6.5 x 10 ³

4) Investigation of the Degradation Activity Characteristics of Zearenone Degrading Strains

Part of the liquid culture and selection of cells (microbial cells), cultures and broths of the selected strains having a zealenone degrading activity were identified to determine the parts of the zearenone degrading (Table 8). As a result, it is determined that the most degrading activity is the cell itself, which is decomposed by the enzyme bound to the cell, and the degree of degrading activity of the cell + broth should be culture (cell + broth). This is because the strain itself uses a portion (about 10%) as a carbon source. Therefore, the degrading activity of the selected strain against the zearenone is made by the action of an enzyme bound to the outside of the cell, and some of the strains are used as a nutrient for the strain itself. Therefore, the focus was on enzyme selection at the gene selection stage.

Degradation of Zeararenone (%) by Isolated Strains Strain Culture Cell boundary Intracellular Extracellular (broth) GB-501 99 61 35 25 GB-ZEN-14 97 62 35 21 GB-ZEN-46 99 66 33 19

4. Establishment of cultivation method of selected microorganisms

1) Establishment of liquid culture conditions for selected strains

In order to confirm the fermentation time of zearenone degradation activity, samples were taken for each hour while incubating in LB broth for 24 hours to analyze zearenone degradation activity (FIG. 4). The degradation activity of each culture time tended to increase in proportion to the number of bacteria, and the degradation activity was about 90% at 12 hours of culture, and the number of bacteria was 10 ⁹ cfu / mL or more.

As a result of investigating the growth of fungal toxin-degrading strains in order to set the medium condition for liquid culture, glucose, yeast extract, sodium chloride were considered in consideration of the cost of the medium composition due to the excellent growth in nutrient broth or LB broth. The mixed medium (GB broth) was prepared at an appropriate ratio, and after 24 hours of liquid fermentation, it was selected as a medium for liquid fermentation because it did not show a big difference from other commercial mediums in terms of bacterial count and degradation activity. Used as. Medium conditions for liquid culture are shown in Table 9 below.

Medium for Liquid Fermentation of Selected Strains badge Log CFU / mL Resolution (%) LB Broth 9.28
98-100 Nutrient Broth 9.30 GB Broth 9.15 Tryptic Soi Broth 8.90

2) Establishment of solid phase culture conditions

Solid phase fermentation conditions for mass production of the selected strains were established at the laboratory level. The fermentation conditions in the solid phase culture conditions were selected as the total fermentation time of 24 hours showing the reduction effect of more than 95% by identifying the fungal toxin reduction effect as the Bacillus strain. Soybean meal was used as a raw material for solid phase cultivation and the initial moisture content was adjusted to 40% level, followed by sterilization through autoclave, followed by inoculation with 1% level of liquid spawn and solid phase fermentation. Fermentation temperature conditions were determined to be 37 ℃, humidity more than 70%. However, the fermentation conditions will have to vary slightly depending on the raw materials.

Investigation of the change of the number of bacteria by fermentation time according to spawn inoculation amount showed the same growth pattern up to 1-4% except the addition of 0.5%, and the seed addition level was set to 1%, and the initial water content was up to 20-45%. The test was set to 40%.

3) Mold Toxin Degradation Activity Test through DDGS Solid Phase Fermentation

For the solid phase fermentation test, we tested the fungal toxin degrading effect of feed grains on DDGS (Distillers Dried Grains with Solubles), which is considered to be the most serious toxin contamination. The level of cfu / mL, but increased to 108 cfu / mL level at 24 hours of culture, thereby increasing the fungal toxin degradation activity. Zearenone concentration present in the initial raw material itself was a high level of 250ppb, but from 24 hours of fermentation it was confirmed that the reduction to more than 90% compared to the fungal toxin of the initial raw material (Fig. 5). Thus, fermentation is expected to bring about a reduction in fungal toxins sufficiently contaminated with feed grains.

4) Validation of degradation activity through HPLC analysis

The fermentation samples by solid phase fermentation (0, 24, 48 hours) were subjected to pretreatment using an immunoaffinity column (EASI-EXTRACT Zearenone, R-biopharm) and analyzed by HPLC. The results are shown in Fig.

As a result, zearenone was detected in the 0 hour sample, but rarely detected in the 24 and 48 hour fermentation samples.

5. Selection and Characterization of Zearenone (ZEN) Degrading Activity Proteins

1) Selection of ZEN Degradation Activity Protein

LB broth was added to 100 mL of LB broth to which 1 ppm of ZEN was added, followed by fermentation at 250 rpm at 30 ° C. After 2 days of fermentation, inoculated into 1 L LB broth added to a concentration of 1,000 ppb ZEN and fermented again at 250 ° C for another 3 days.

After fermentation, the cells were recovered by centrifugation, resuspended in 30 mL of buffer [10 mM Tris / HCl (pH 7.5)], and sonicated on ice for 5 minutes. Cell debris was removed by centrifugation, ammonium sulfate was added to 70% by weight of the total weight and placed in a dialysis tube, dialyzed in Tris buffer with PMSF and Peptatin A for 24 hours and centrifuged at 10,000 g for 1 hour. The protein was concentrated. Protein fractions were loaded on a 5 mL HiTrap DEAE column and eluted with NaCl buffer to capture each fraction. From the collected fractions, the fractions with good ZEN degradation activity were separated by size using the Superdex 75 HR 10/30 column through evaluation of the ZEN degradation activity. After separation using Q column, ZEN degradation activity was evaluated and final degradation activity protein was selected.

Selected proteins were transferred to PVDF membranes after electrophoresis and sequenced through N-terminal amino acid sequencing. The identified sequence determined the final protein sequence via NCBI protein blast.

2) Protein Electrophoresis

SDS-PAGE was run according to the method of Laemmli (1970) using a 12.5% polyacrylamide gel. Staining was carried out using brillinant blue R-250, the molecular weight was confirmed through a marker.

3) Results

For ZEN degradation protein selection, preliminary tests were carried out using DEAE-sepharose columns, followed by elution by NaCl concentration to verify ZEN degradation activity, and then columns were selected for separation. ZEN degradation activity is shown in Figure 7 after the analysis by ELISA method.

As a result of the degradation activity test, the ZEN degradation activity in fractions IV and V was about 63%, respectively. The selected fractions IV and V were sequentially selected using only HiTrap Q column, Superdex 75 column, and mono Q column. Finally, the selected fraction IV showed 61% degradation activity and was selected as the final degradation activity protein. The selected cleavage protein was confirmed in size through SDS-PAGE (FIG. 8), and finally transferred to PVDF membrane for analysis of N-terminal amino acid sequence using an amino acid analyzer (N-terminal protein sequencer (Automatic protein sequencer). , Applied Biosystems), Korea Basic Science Institute, Daejeon). Sequence analysis was performed by RVNKLTCVDS, NCBI blast search ( http://blast.ncbi.nlm.nih.gov) and blast search (http://expasy.org) of ExPASy Proteomics Server to confirm protein sequence and function. ), The protein was found using Bacillus sp. B14905 (NCBI Reference Sequence: ZP_01724343.1, ExPASy Sequence Name: A3IBA6) was confirmed to have the same sequence as the unidentified virtual protein, Lysinibacillus sphaericus ([Lysinibacillus sphaericus (strain C3-41)] / NCBI Reference Sequence: YP_001699051.1, ExPASy Sequence Name: B1HRE1] ) and nine sequences were found to be identical (Table 10).

N-terminal Amino Acid Sequence of Isolated Fraction Amino acid sequence Homology search results






RVNKLTCVDS
1. Virtual protein [Bacillus sp.]
Putative uncharacterized protein [BB14905_10835] [Bacillus sp. B14905]

Query 1 RVNKLTCVDS 10
RVNKLTCVDS

Target 90 RVNKLTCVDS 99
1. Virtual protein [Lysinibacillus sphaericus]
Putative uncharacterized protein [Bsph_3429] [Lysinibacillus sphaericus (strain C3-41)]

Query 1 RVNKLTCVDS 10
RV + KLTCVDS

Target 90 RVDKLTCVDS 99

In addition, the final protein sequence and nucleotide sequence were confirmed through the NCBI protein blast as described above, its amino acid sequence is shown in SEQ ID NO: 1, and the expected base sequence thereof is shown in SEQ ID NO: 2.

Korea Microorganism Conservation Center (overseas) KCCM10930P 20080306

Attach an electronic file to a sequence list

Claims (9)

Bacillus subtilis GB-501 (accession number: KCCM10930P, deposited date: March 6, 2008) having zearenone degrading activity, which expresses a gene comprising the nucleotide sequence of SEQ ID NO: 2.
delete (A) preparing a Bacillus subtilis GB-501 of claim 1;
(b) culturing the Bacillus subtilis GB-501 to produce a protein comprising the amino acid sequence of SEQ ID NO: 1 having zearalenone degrading activity; And
(c) purifying the protein from the culture
Method of producing a protein having a zeararenone degrading activity comprising a.
delete delete delete delete delete delete

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