KR102114190B1 - New microorganism of Aspergillus oryzae having control of aflatoxin production from Aspergillus flavus and the use thereof - Google Patents

Abstract

The present invention relates to a new microorganism of Aspergillus oryzae and its use to inhibit the production of aflatoxin derived from the Aspergillus flabus strain.
The novel microorganism Aspergillus oryzae M2040 strain of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and aflatoxin derived from the strain Aspergillus sp. It is characterized by having production inhibition and resolution.
According to the present invention, it is possible to provide a new microorganism of Aspergillus oriage that inhibits the growth of Aspergillus flavus and simultaneously inhibits the production of Aflatoxin derived from the strain Aspergillus, When manufacturing fermented foods containing soybeans, it is possible to produce berry more safely from aflatoxin and at the same time, it is possible to prevent contamination of aflatoxin from the soil itself during grain production. It is expected that its use value will be increased as it can be safely used as a material for phosphorus livestock feed.

Description

New microorganism of Aspergillus oryzae having control of aflatoxin production from Aspergillus flavus and the use thereof}

The present invention relates to a new microorganism of Aspergillus oryzae that inhibits the production of aflatoxin derived from a strain of the genus Aspergillus and uses thereof, and more specifically, the production of aflatoxin derived from the strain Aspergillus flabus It relates to a new microorganism and its use aspergillus orize that inhibits or decomposes and simultaneously inhibits the growth of the Aspergillus flabus strain.

The fungus Aspergillus sp. Belongs to aerobic incomplete fungi, and is a storage fungus that generally breeds during storage of agricultural products, and there are also useful fungi used to manufacture fermented foods, such as A. oryzae . In addition, about 60 species of Aspergillus sp. Can cause serious illness in humans and animals. Among them, the species that produces aflatoxin that causes liver cancer, known as hepatoxin, is A. flavus , A. parasigicus, and Aspergillus A. nomius and the like are known.

These aflatoxins are secondary metabolites produced from the strains, and in humans, carcinogenicity, teratogenicity, and mutagenicity have been reported, and are known to be the only one group carcinogen among fungal toxins established by the International Cancer Research Organization (IARC). have.

In particular, there have been many cases where aflatoxin produced from grains such as corn, peanut, pepper, and sesame seeds contaminated with Aspergillus flavus contaminated food and feed and greatly damaged humans and animals who consumed it. It has been reported, and in recent years, aflatoxin exceeding the standard value is also detected in native Meju, and there are cases in which sales are prohibited and recovered.

Accordingly, chemical, biological, and physical methods have been proposed to suppress aflatoxin production or to reduce or eliminate aflatoxin. In some studies, bacteria or fungi are used to stop the growth of aflatoxin-producing bacteria and to produce mycotoxin. It has been reported to impede.

Related prior arts include Korean Registered Patent No. 10-0380535, 'Method for inhibiting aflatoxin production by anti-microbial seed 220 and soybean fermented food and animal feed using it', Korean Registered Patent No. 10-150774, 'Lactobacillus para having antibacterial properties' Casein ML-7 strain and its use 'and Japanese Patent P3040234' New Bacillus microorganism and its use 'are known.

However, the prior art is basically not a food material registered strain (GRAS, generally Regarded as Safe) that can be used in food production, so a separate Bacillus genus strain must be treated to suppress aflatoxin-producing strain. There is a concern that the strain may degrade the taste of fermented food, and it is difficult to completely exclude the possibility of contamination of aflatoxin because it cannot prevent 100% of aflatoxin growth itself.

1. Korean Registered Patent No. 10-0380535 'Method of inhibiting aflatoxin production by antagonistic microbial seed 220 and applied soybean fermented food and animal feed' 2. Korean Registered Patent No. 10-150774 'Antibacterial Lactobacillus Paracasein ML-7 Strains and Uses thereof' 3. Japanese Registered Patent P3040234 'New Bacillus microorganisms and their uses

The object of the present invention is to target the Aspergillus orije strain, which was commonly used in the production of fermented foods using soybeans containing soybeans, and inhibit or decompose the production of aflatoxin derived from Aspergillus flabus. At the same time, it is to provide a new microorganism and its use of Aspergillus orize, which inhibits the growth of Aspergillus flabus itself.

To achieve the above object, the novel microorganism Aspergillus oryzae M2040 strain of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and the genus Aspergillus sp. ) It is characterized by having the ability to inhibit and degrade the production of aflatoxin derived from the strain, and more preferably, the Aspergillus sp. Strain is characterized by being the Aspergillus flavus strain.

Soybean fermented food production composition of the present invention is characterized in that it comprises at least one of the new microorganism Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain or its culture as an active ingredient, the culture is the strain After inoculation into the PDB medium, it is characterized by being incubated for 2 to 3 days at 28 to 30 ° C to be activated.

The microbial preparation of the present invention includes at least one of the above-described new microorganism Aspergillus oryzae M2040 strain or a culture thereof as an active ingredient, and the microbial preparation is sprayed directly onto the soil to aspergill It is possible to produce grains in which growth of Aspergillus flavus is inhibited.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above.

 According to the present invention, it is possible to provide a new microorganism of Aspergillus oriage that inhibits the growth of Aspergillus flavus and simultaneously inhibits the production of Aflatoxin derived from the strain Aspergillus, When producing fermented foods containing soybeans, it is possible to produce berry more safely from aflatoxin, and at the same time, it is possible to prevent contamination of aflatoxin from the soil itself during grain production, making it possible to produce grain safely from aflatoxin, thereby representing grains. It is expected that the value of its use will increase as it can be safely used as a material for livestock feed, a commercialization field.

1 is a view showing the relationship between the existing known reference strains in order to determine the classification position of the strain Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain of Example 1.
FIG. 2 is a view showing the results of the Aspergillus oryzae M2040 strain, an isolated strain of Example 1, which inhibits the production of aflatoxins of Aspergillus flabus and rather degrades aflatoxins.
3 is a view showing the results of inhibiting the growth of the Aspergillus oryzae strain M2040 strain of the isolate strain of Example 1 inhibits the growth of Aspergillus flaverse.
4 is a view showing the results of the culture strain of Aspergillus oryzae M2040 strain, the isolate strain of Example 1, inhibits the production of aflatoxin in Aspergillus flavus .
5 is a view showing the results of comparing the ability to inhibit the aflatoxin production of AperGuardus oryzae M2040 strain, an isolated strain of Example 1 and the existing product AflaGuard ^TM .

Hereinafter, the present invention will be described in detail, and detailed descriptions of well-known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted.

In order to achieve the above object, the present invention provides Aspergillus oryzae M2040 strain having inhibitory ability and decomposition ability of aflatoxin of Aspergillus sp. Strain.

In the strain according to an embodiment of the present invention, the Aspergillus sp. Strain is preferably Aspergillus flavus , but is not limited thereto.

The strain of the present invention confirmed that the additional aflatoxin is not generated when mixed with the aflatoxin-producing Aspergillus flabus strain, but rather decreases, and inhibits the growth itself of the Aspergillus flaverse strain, as it is harmless to the human body When manufacturing soybean fermented foods containing soybeans, it can be safely produced, and in particular, it was confirmed that it exhibits a significantly higher aflatoxin production inhibitory power compared to the existing product AflaGuard ^{TM, which} has an inhibitory ability to produce aflatoxins, and it was confirmed that it can be applied more economically.

Thus, the inventor of the present invention deposited the Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain on October 20, 2017 at the National Academy of Agricultural Science, (Accession number: KACC 93295P), the M2040 strain has SEQ ID NO: 1 or It is characterized by including the base sequence represented by SEQ ID NO: 2.

In addition, the present invention provides a composition for producing soybean fermented food, characterized in that it comprises at least one of the above-described new microorganism Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain or its culture as an active ingredient.

In the present specification, the culture may mean a substance including all substances contained in the medium in which the strain is cultured, for example, a substance containing metabolites or secretions that are the result of strain culture, or a fragment thereof. The strain itself may also be included in the culture. In addition, the culture may be cultured according to a method commonly used in the art, preferably using a PDB culture medium, but is not limited thereto.

The culture according to an embodiment of the present invention is used to inoculate the strain in PDB medium, and then incubated for 2 to 3 days at 28 to 30 ° C. to be used in an activated state. There is a fear that the efficacy to be lowered.

Fermented food in the present specification may mean food fermented by a strain, food in progress for fermentation, or food that promotes fermentation, for example, soy fermented foods such as soy sauce, fish soy sauce, miso, cheonggukjang, red pepper paste, fermented dairy products , Kimchi, salted fish, salted fish such as salted fish or flavoring agents such as seasoning, but may not be limited thereto.

The formulation of the composition according to one aspect of the present invention is not particularly limited, for example, tablets, granules, powders, liquids such as drinks, caramels, gels, bars, etc. may be formulated. The composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective properties It may include colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. The food composition of each formulation can be formulated by appropriately selecting the ingredients commonly used in the field other than the active ingredients according to the formulation or purpose of use without difficulty, and synergistic effects may occur when applied simultaneously with other raw materials.

In addition, the present invention is to provide a microbial agent comprising at least one of the new microorganism Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain or a culture thereof as an active ingredient, the microbial agent is in the soil By spraying directly, Aspergillus flavus is able to produce grains with reduced growth.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

<Example 1> omtA and aflR gene analysis for strain isolation and seed identification

1. Experimental method

1) Strain separation

The fungus generated in Meju, which is fermented at the farmhouse in Jeonnam, was directly separated on MEA medium. That is, after a part of the mold-producing meju was plated on the MEA medium, the grown fungus was transferred to the MEA medium and cultured. Subsequently, the mold was purely separated by a monosporre isolation method.

2) Gene analysis

The sequence of omtA and aflR genes of the aflatoxin biosynthetic gene group suitable for species identification of the Aspergillus section Flavi to which the isolated M2040 strain belongs are analyzed. Afterwards, in order to determine the classification location of the M2040 strain, a flexible relationship diagram was prepared together with the previously known reference strains.

2. Experimental results

As shown in FIG. 1, the new strains selected in Example 1 were identified as the final new microorganism Aspergillus orise strain, and as described in the deposit certificate attached thereto, the M2040 strain is' Aspergillus orije (A spergillus oyrzae ) ', and this strain was deposited with the National Academy of Agricultural Science, Agricultural Microbiology Bank (KACC), and was granted the accession number KACC 93295P on November 20, 2017.

<Experimental Example 1> M2040 strain of Example 1 A.flavus Aflatoxin Production Inhibition and Resolution

1. Experimental materials and methods

Aspergillus flavus NRRL3357 (US ARS culture colletion), which is known as a fungus producing Aspergillus oryzae M2040 strain isolated from Example 1 and aflatoxin, was prepared.

Each of the prepared A. oryzae M2040, A. flavus NRRL3357 strains of 5 × 10 ⁶ conidia were inoculated into 2 Erlenmeyer flasks each containing 100 ml of PDB medium (Potato Dextros Broth, Difco 254920), and then 3 at 30 ° C. It was suspended for a day.

Among them, one of the cultured A. oryzae M2040 cultures was prepared in an inactivated state by heat treatment, and the rest were prepared in an activated state without heat treatment.

Then, after preparing three 250 ml Erlenmeyer flasks containing 50 ml of fresh PDB, the first was added 25 ml of A. flavus NRRL3357 and the second was heat-treated A. oryzae M2040 culture and A. flavus NRRL3357 culture, respectively. 25 ml was added, and in the third, 25 ml of A. oryzae M2040 culture and A. flavus NRRL3357 culture were added without heat treatment, and aflatoxin B1 was measured every 1.5 days while cultured at 30 ° C for 12 days.

2. Experimental results

As shown in Fig. 2, the result of the cultivation of A. oryzae M2040 strain (non-heat-treated) and A. flavus NRRL3357 of Example 1 of the present inventor, the amount of aflatoxin (AFB1) was 434 ppb at day 0 of culture. On the other hand, as shown on the 12th day of culture, 324 ppb indicates that aflatoxin was not produced anymore, but rather, the amount of aflatoxin was decreased. As a result, it was confirmed that it has aflatoxin decomposition ability as well as aflatoxin production inhibitory ability.

On the other hand, when A. flavus NRRL 3357 was cultured alone, 434 ppb was shown at day 0 of culture, whereas 2843.3 ppb was shown at day 12 of culture, and A. flavus NRRL 3357 and A. oryzae M2040 (heat treated) were mixed and cultured. Even in the case of the 12th day of culture, it shows 2503.1 ppb, and it was confirmed that aflatoxin more than 6 times the initial amount was produced.

<Experiment 2> M2040 strain culture filtrate of Example 1 A.flavus Growth inhibition and A.flavus of Confirmation of the ability to inhibit aflatoxin production

1. Experimental materials and methods

Aspergillus flavus NRRL3357 (USA, ARS culture collection), which is known as a fungus that produces Aspergillus oryzae M2040 strain and aflatoxin isolated from Example 1, was prepared.

Two Erlenmeyer flasks containing 100 ml of PDB medium (Potato Dextros Broth, Difco 254920) were inoculated with 5 × 10 ⁶ spores of the prepared A. oryzae M2040 strain, respectively. The A. flavus NRRL3357 strain was inoculated with 5 × 10 ⁶ conidia spores into one Erlenmeyer flask containing 25 ml of PDB medium. The inoculated Erlenmeyer flask was suspended for 3 days.

Two cultures of the A. oryzae M2040 strain grown after the culture were inactivated by heat treatment, and the other was not filtered by heat treatment and prepared by filtering using a 0.22 um filter.

Then, add 25 ml of A. flavus NRRL3357 culture to a 250 ml Erlenmeyer flask with 50 ml of PDB medium, and heat-treat each to add 25 ml of inactivated M2040 culture filtrate and 25 ml of untreated M2040 culture filtrate. The amount of aflatoxin B1 and the biomass of A. flavus NRRL3357 were measured during daily incubation.

2. Experimental results

As shown in FIG. 3, the A. oryzae M2040 strain of Example 1 of the present invention was heat-activated and inactivated, and then A. flavus NRRL3357 grew to 0.074 g in a mixed culture prepared by filtering the filter, whereas A. oryzae It was confirmed that A. flavus NRRL3357 rarely grows to 0.02 g in the mixed culture medium filtered with a filter while the M2040 strain is activated without heat treatment, and is reduced by 78.2% compared to the inactivated culture medium. It was found that the culture filtrate of the activated A. oryzae M2040 strain of Example 1 inhibited the growth itself of the A. flavus NRRL3357 strain and showed antibacterial activity against the A. flavus strain.

In addition, as shown in FIG. 4, 1200 ppb of aflatoxin is detected in the mixed culture using the culture filtrate prepared by filtering the filter with a filter by inactivating the A. oryzae M2040 strain of Example 1 of the present invention, whereas the A. oryzae M2040 strain When it was confirmed that aflatoxin was not produced in the mixed culture prepared by filtering with the culture medium while being activated, the culture filtrate of the activated A. oryzae M2040 strain of Example 1 inhibited the production of aflatoxins generated from the A. flavus NRRL3357 strain. Could know.

<Experiment 3> M2040 strain of Example 1 and AflaGuard ^TM Comparison of Aflatoxin Production Inhibition

1. Experimental materials and methods

Aspergillus flavus NRRL3357 (USA, ARS Culture Collection), which is known as a fungus generating aflatoxin and activated Aspergillus oryzae M2040 strain isolated in Example 1, Aspergillus oryzae AflaGuard ^TM (NRRL) 21882, ARS Culture Collection, USA).

The three Erlenmeyer flasks containing 100 ml of PDB medium (Potato Dextros Broth, Difco 254920) were each inoculated with 5 × 10 ⁶ spores of each of the prepared strains, and then suspended and cultured for 3 days.

Then, 100 ml of PDB medium was mixed with Aspergillus oryzae M2040 strain and Aspergillus flavus NRRL3357 at a ratio of 1:10 and 1: 100, and cultured at 30 ° C for 12 days, respectively. As a comparative example, Aspergillus oryzae AflaGuard ^TM The strain and Aspergillus flavus NRRL3357 were mixed at a ratio of 1:10 and 1: 100, and incubated at 30 ° C for 12 days, respectively, and then aflatoxin B1 was measured.

2. Experimental results

As shown in the experimental results Figure 5, in the Example 1 in a 100-fold dilution A. oryzae strain M2040 was produced in the 1144 ppb 46% compared to the 10-fold dilution AflaGuard AflaGuard ^{TM TM} is generated, but the aflatoxin of 1800ppb M2040 strain is a check does not produce aflatoxin bar, A. oryzae strain M2040 was found that a bar, which indicates a much higher efficacy than the existing for aflatoxin generation suppression product to inhibit the previous product AflaGuard ^TM prepare at least 2.2 times or more and produce aflatoxin.

As shown in the above experimental results, by the present invention, aspergillus oryzae novel that inhibits the production of aflatoxin derived from a strain of genus Aspergillus and simultaneously suppresses the growth of Aspergillus flabus itself As it is possible to provide microorganisms, it is possible to produce berry more safely from aflatoxin in the manufacture of fermented food containing soybeans, and at the same time, it is possible to prevent contamination of aflatoxin in the soil itself during grain production, thereby making grain safe from aflatoxin As production is possible, it can be seen that it can be safely used as a material for livestock feed, a representative commercialization field that applies grain.

The present invention has been described with reference to preferred embodiments, and those of ordinary skill in the art to which the present invention pertains may implement embodiments in different forms from the detailed description of the present invention within the essential technical scope of the present invention. Will be able to. Here, the essential technical scope of the present invention is indicated in the claims, and all differences within the equivalent range should be interpreted as being included in the present invention.

Depository name: National Academy of Agricultural Science, Agricultural Microbiology Bank (KACC)

Accession number: KACC93295P

Date of Deposit: 20171020

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> New microorganism of Aspergillus oryzae having control of          aflatoxin production from Aspergillus flavus and the use thereof <130> P2017-0298 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 546 <212> DNA <213> Unknown <220> <223> Aspergillus oryzae M2040 (omtA gene) <400> 1 acctagtccc acatcactcg acacaatcgt cgccgcaacc ggagtatcag aggatttact 60 acgtataaca ttccatatct atatgccatc ccctgaaagg cagtgcatta atcaatatat 120 aggacggatt cttcgaggat gtgcccagcg cttcattttc gaggaggttg cccctgacca 180 atacgcccac acagatgcct caaagatgct gcgagtgacg ggtattcatg ccttggtagg 240 attctcgtga gtctccttct ttaacaataa actaccagat gtcctcacca gggagaccga 300 gttagatgtg acgaagtgat gcggtcgggt gcctactttt ccgacttctt gcagcagacg 360 aaaggcaaac ctccgagttg gaatgtgcct tcacctttct cattggcatt tgatcgtagg 420 tattccactc tatctagata ggaacacagc tgaaatgcga gaaacagcta ccaaagggct 480 atttgactat tacagcactg tggacgaggt tcgcggccgc cgctttgatc taggtatggg 540 tggcac 546 <210> 2 <211> 866 <212> DNA <213> Unknown <220> <223> Aspergillus oryzae M2040 (aflR gene) <400> 2 caacgcctca tgctcatacc caggcccaca ctcatgctca ttctcatccg caaccgcatc 60 cacaatctca tcctcaatcg aatcaaccac cacacgctct gcccaccccg aatggtagca 120 gtagcgtctc cgccatcttt tctcatcaga gtccgccgcc acccgtggag acccagggcc 180 ttggaggaga tctggctggt caggagcaaa gcaccctgtc ttccctaaca gtcgattcgg 240 aattcggggg ctcgttgcag tcaatggaac acggaaacca tgccgatttc ttggccgagt 300 cgacggggag tcttttcgac gcgtttttgg aagtagggac ccccatgatc gacccgttcc 360 tcgagtcggc cccactacca ccgtttcagg cgcgctattg ctgcttttcg ctagcactac 420 aaacactgac ccacctcttc ccccacgccc cgctgggctg tcaactacgg ctgacggacg 480 gtgaggacag ttcgtacaac ctgatgacga ctgatatggt catctcgggg aacaagaggg 540 ctaccgatgc ggtccggaag atcctcgggt gttcgtgcgc gcaggatggc tacttgctga 600 gcatggtcgt ccttatcgtt ctcaaggtgc tggcatggta tgctgcggca gcaggcaccc 660 agtgtacctc aacggcggcg agtggagaaa ccaacagtgg cagctgtagc aacagtcccg 720 ccaccgtgtc cagtggctgt ctgacggaag agcgcgtgct gcacctccct agtatggtgg 780 gcgaggattg tgtggatgag gaagaccagc cgcgagtggc ggcacagctt gttctgagcg 840 aactgcaccg agtccagtcg ctggtg 866

Claims (6)

It includes a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and has the ability to inhibit and degrade the production of aflatoxin derived from the Aspergillus sp. Strain,
New microorganism Aspergillus oryzae M2040 strain (KACC 93295P). According to claim 1,
The Aspergillus sp. Strain is characterized by being an Aspergillus flavus strain,
New microorganism Aspergillus oryzae M2040 strain (KACC 93295P). Claim 1 or claim 2 of the new microorganism Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain or any one or more of its culture, as an active ingredient,
Soybean fermented food manufacturing composition. According to claim 3,
The culture is characterized in that it was prepared in an activated state by inoculating the strain in PDB medium, and then incubating for 2 to 3 days at 28 to 30 ° C.
Soybean fermented food manufacturing composition. Claim 1 or claim 2 of the new microorganism Aspergillus oryzae ( Aspergillus oryzae ) M2040 strain or any one or more of its culture, as an active ingredient,
Microbial preparation. A method for producing grains in which growth of Aspergillus flavus is inhibited by spraying the microbial agent of claim 5 directly onto the soil.

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