KR20190100711A - A novel hybridized mushroomstrain GBN2WP0970 and fruit body of the same with high inhibition rate of neutral fat absorption - Google Patents

Abstract

The present application relates to a novel hybridized strain GBN2WP0970, fruit body thereof and spore thereof. The present application has a high inhibition rate of triglyceride absorption, and thus can be used in the production of new drugs, functional health foods and general foods.

Description

A novel hybridized mushroomstrain GBN2WP0970 and fruit body of the same with high inhibition rate of neutral fat absorption}

The present application relates to hybrid new strain GBN2WP0970, its fruiting bodies and spores.

A. oyster mushroom is a controversial species classified as Pleurotus eryngii var.ferulae or Pleurotus ferulae of Pleurototaceae. Because it is wild in Awi tree in Xinjiang province, China, it is called Awi entree. Awinita mushroom has been studied for artificial cultivation in India, France, and Germany since 1958, and Kalmer's first successful cultivation in 1958, and succeeded in artificial cultivation by mixing sawdust, cotton thread and bran in China in 1983. In 1990, it secured excellent strains by single spore hybridization, and is widely used in Fujian, Xinjiang, and other places (Lee Dong-hee, 2005). Compared with other mushrooms, Ayuentari mushroom is known to have high edible value due to its excellent shape and rich flavor, and antitumor and hypoglycemic effect (Hong et al. 2004). In addition, medicinal effects of controlling diseases of the obstetrics and gynecology by stopping stomach and kidney disorders, coughing and removing inflammation are known (Kim, Dae-sik, 2002), and they also contain large amounts of dietary fiber, amino acids and other vitamins. High value as a medicinal mushroom.

Existing Ayuitari mushroom, white species Pleurotus eryngii and other oyster mushroom species and mushrooms on the market have a low inhibitory effect on triglyceride absorption. There was a need for the fruiting body obtained through the improvement of new strains that can be used as raw materials in the manufacture and cultivation of the strains.

Prior art documents related to the present application include the Republic of Korea Patent No. 10-2017-0053070, but this relates to the new strain of mushrooms and the cultivation method using a hybrid between the species of Awi locust mushroom and Baeknyeong locust mushroom, Republic of Korea Patent No. 10-1544927 No. 1 relates to A. oyster mushroom new strains and new mushrooms through breeding of these species, and there is no suggestion for the development of mushroom strains excellent in triglyceride absorption inhibition by cross. Do.

Under these circumstances, the present inventors have made intensive studies to produce mushroom strains and fruiting bodies having excellent triglyceride absorption inhibitory effects. As a result, new breed mushrooms (GBN2WP0970, Accession No.) were developed by hybrid breeding of Awi oyster mushroom and white oyster mushroom. KCTC18664P) completed the present application by confirming that the triglyceride absorption inhibitory effect is remarkable.

The object of the present application is to provide a hybrid new strain GBN2WP0970 of Accession No. KCTC18664P.

Another object of the present application is to provide a fruiting body formed from hybrid new strain GBN2WP0970.

Another object of the present application is to provide spores, which are derived from fruiting bodies formed from the new strain strain GBN2WP0970.

Another object of the present application is to provide a descendant strain of new varieties GBN2WP0970 and its fruiting body, which are obtained through cross-spores crossing between spores.

Another object of the present application is to provide a production medium of hybrid new strain GBN2WP0970 (KCTC18664P).

Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. Content not described herein is omitted because it can be sufficiently recognized and inferred by those skilled in the art or similar fields of the present application.

Hereinafter, the content of the present application will be described in detail. In addition, description and embodiment of one embodiment disclosed by this application can be applied also to description and embodiment of other aspect about common matter. In addition, all combinations of the various elements disclosed in the present application are within the scope of the present application. In addition, the scope of the present application is not to be limited by the specific description described below.

In order to achieve the object of the present application, the present application provides a hybrid new strain GBN2WP0970 of KCTC18664P. In one embodiment, to obtain the new strains, it is possible to select excellent strains by using cross-crossing and mixed media and controlling moisture.

In one preferred embodiment of the present invention, the hybrid hybrid strain is a mononuclear mycelium isolated from the fruiting body of the Ayuitari mushroom strain (Bishan No. 1) and mononuclear mycelium isolated from the fruiting body of oyster (white melanoma) mono-mono (mono) -mono) Can be obtained by crossing.

Another aspect of the present invention provides a hybrid new breed (GBN2WP0970) mushroom fruiting body formed from the above strain.

In a preferred embodiment of the present invention, the fruiting body may have a yield of 140g / bottle or more when bottled.

In a preferred embodiment of the present invention, the fruiting body extract has an inhibitory effect on triglyceride absorption at a concentration of 1 to 40 mg / rxn, specifically 5 to 40 mg / rxn, 10 to 40 mg / rxn.

The shade of the fruiting body has a diameter of about 30 to 50 mm, a thickness of 25 to 50 mm, the stage of the fruiting body may have a thickness of about 30 to 70 mm, a length of about 70 to 150 mm, preferably The fruiting shade of the fruiting body is about 33-48 mm in diameter, 30-45 mm in thickness, and the stage of the fruiting body is about 35-60 mm thick and about 90-130 mm long.

Still another aspect of the present invention provides a spore obtained from the fruiting body of the above hybrid new strain (GBN2WP0970).

Another aspect of the present invention provides a next generation strain, characterized in that obtained through the cross-spores made of the spores.

Another aspect of the present invention provides a fruiting body formed from the above generation strains.

Another aspect of the present invention provides a medium prepared by mixing in a volume ratio of sawdust: beet pulp: cottonseed foil: = 50: 30: 20 (%) used for cultivating mushroom fruiting body with hybrid new strain GBN2WP0970 (KCTC18664P). do. The volume ratio may be appropriately changed depending on the variety or quantity of mushroom fruiting body, but is not limited thereto.

The present application has an excellent effect of providing a triglyceride inhibitory effect is very high compared to the conventional mushroom, yielding good yield when breeding the bottle (GBN2WP0970) and fruiting body obtained through the cultivation of the strain.

1 is a graph showing the nucleotide sequence analysis of hybrid new strain GBN2WP0970.
Figure 2 is a photograph showing the results of comparing the nucleotide sequence of the ITS portion of hybrid new strain GBN2WP0970, Awi mushroom (Bishan No. 2) and Pleurotus erythroma (white melanoma).
Figure 3 is an electrophoretic picture showing the genetic polymorphism analysis of the new hybrid strain GBN2WP0970 and maternal (M. Marker, 1. hybridization strain, 2. scattering No. 2, 3. elder (white melanoma), 4. large elk ( Pleurotus eryngii), 5. oyster mushroom (commercially available))
Figure 4 is a graph showing the phylogenetic tree by the parental base sequence of hybrid new strain strain GBN2WP0970.
5 is a photograph of fruiting bodies of hybrid new strain GBN2WP0970.
6 is a graph showing pancreatic lipase inhibitory effect according to the concentration of hybrid new strain GBN2WP0970.
Figure 7 is a graph showing the comparison of pancreatic lipase inhibitory effect of hybrid new strain GBN2WP0970, mash parquet, shedding No. 2, white loin.

Hereinafter, the present invention will be described in more detail through examples according to the present application. However, the following examples of the present application are merely examples of the present application. These examples are intended to illustrate the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention as set forth in the appended claims is not limited by these examples. something to do.

Example 1 Acquisition of the new hybrid strain strain GBN2WP0970 of the present invention

In order to obtain the hybrid new strain GBN2WP0970 according to the present invention, it was isolated from the fruiting body of mononuclear mycelium and commercial oyster (white melanoma) isolated from the fruiting body of the A. oyster mushroom strain (Bisan No. 2), which was nurtured by the National Institute of Horticultural & Herbal Science from 2016. Mononuclear mycelium was cross-mono-mono-mono (mono-mono) cross-breed to control the moisture using a conventional culture medium for mushroom cultivation and hybridization was cultured to select a superior strain with high triglyceride inhibitory effect.

The mating specifically inoculates two primary hyphae to the solid medium by 1-3cm apart and checks the mycelia by microscopic observation 7 to 10 days after contact with both mycelia to obtain a hybrid strain. Here, the growth test of stabilized hybrid strains was performed to select strains with high growth rate, normal flora and high mycelial density. Selected hybrid new strain GBN2WP0970 was deposited with KCTC13476BP at BRC in accordance with Budapest Treaty on February 2, 2018. In addition, the nucleotide sequence (SEQ ID NO: 1) of hybrid new strain GBN2WP0970 was analyzed (FIG. 1).

The hybridization of the new strain GBN2WP0970 according to the present invention and the nucleotide sequence of the ITS part of the Ayuta mushroom (Bishan No. 2) and oyster (white melanoma) are as shown in FIG.

Genetic polymorphism analysis of maternal strains of the hybrid hybrid strains of the present invention GBN2WP0970 and awiweed mushroom (Bishan No. 2), oyster (white herb), commercial large oyster (fly mushroom) and Awi mushroom are shown in FIG.

The phylogenetic tree of the hybrid hybrid strain of GBN2WP0970 of the present invention is shown in FIG. 4.

[Example 2] Cultivation and harvesting of new hybrid strain GBN2WP0970 mushroom fruiting body of the present invention

The present application provides a mushroom fruiting body of the new strain (GBN2WP0970) strain obtained in Example 1 (Fig. 5). The mushroom fruiting body can harvest the oyster mushroom through the bottle growing conditions, the growing conditions are as follows.

Sawdust: Beet pulp: Cottonseed foil: = 50: 30: 20 (%) The medium prepared by mixing in a volume ratio of adjusted to a water content of about 68% to prepare a medium, the medium is bottled and sterilized. The seed medium is inoculated to the medium with a standard of about 15 to 20 cc per bottle, and then incubated at about 22 ° C. for 35 to 30 days. The fruiting body may have a yield of 140g / bottle or more when bottled. After completion of incubation as described above, the germs were scraped, and then the feet were grown at 12 to 18 ° C, the humidity was 90 to 95%, and the mushrooms were maintained for 5 to 7 days in a standing state, and the feet were about 1 cm. As soon as the young mushrooms are formed, they are returned to the standing state in the state of water, and the growth temperature is 4 to 7 days at 16 ℃, humidity is 80 to 85%, roughness 100 to 200 lux, and carbon dioxide 1,200 to 1,500 ppm. The later growth was managed and harvested.

The shade of the fruiting body has a diameter of about 30 to 50 mm, a thickness of 25 to 50 mm, the stage of the fruiting body may have a thickness of about 30 to 70 mm, a length of about 70 to 150 mm, preferably The shade of the fruiting body may be about 33-48 mm in diameter, 30-45 mm thick, and the stem of the fruiting body may have a thickness of about 35-60 mm and a length of about 90-130 mm.

The fruiting body of the hybrid mycobacteria (GBN2WP0970) of the above-mentioned oyster mushroom and oyster (white melanoma) can grow fruiting bodies that can continuously improve pancreatic lipase inhibitory effect in the lower generation.

Furthermore, spores could be formed from the fruiting bodies formed by the hybridization mycobacteria (GBN2WP0970) of the oyster mushroom and oyster (white melanoma) according to the present invention, and the lower generation through the spore breeding or Di-mono breeding formed between the spores. The new strain (GBN2WP0970) of the mushroom strain obtained, and the resulting new strain (GBN2WP0970) mushroom fruiting body was able to grow a fruiting body that can improve the pancreatic lipase inhibitory effect.

[Example 3] Mushroom fruiting body water extract of the present invention new strain GBN2WP0970

Coarse powder of dried new strain GBN2WP0970 mushroom hybrid (Pleurotus eryngii var. Ferulea and Pleurotus sp.) After oxidization, the mixture was placed in a bag made of gauze, and 50 mL of water was mixed as an extraction solvent per 1 g of powder, and then extracted at 37 ° C. for 24 hours in a shake incubator. The supernatant of the shaker was centrifuged at 2500 rpm for 10 minutes, and the filtered supernatant was collected and used as a test material.

Experimental Example 1 Pancreatic Lipase According to the Concentration of Water Extract of Mushroom Fruiting Body of New Bacteria GBN2WP0970

Pancreatic lipase is responsible for separating acyl chain by non-specific recognition of substrates with acyl chain. In order to measure pancreatic lipase activity according to the concentration of Awi mushroom water extract of Example 3, phosin pancreatic lipase prepared from Sigma Chemical was used as an enzyme. Pancreatic lipase decomposed the chromo-genic substrate (p-nirophenylbutyrate), and the change in the activity of enzyme was confirmed through the color reaction. The inhibitory effect of enzyme was measured by the change in absorbance of 405nm using a microplate reader, and calculated based on the 405nm absorbance of the extract untreated group and expressed as%.

It was measured how the activity of the enzyme according to the concentration of the mushroom fruiting body water extract of the new strain GBN2WP0970 of Example 3, as a result of the concentration of the mushroom fruiting body extract of the new strain GBN2WP0970 of the example remarkably increased the activity of the pancreatic lipase It confirmed that it reduced. The pancreatic lipase inhibitory effect of the new strain GBN2WP0970 was much better than the parent strain Awinut mushroom (Bishan No. 2), white locust mushroom, and commercially available mushroom mash mushrooms (Fig. 6, 7).

Specifically, according to FIG. 6, when the concentration of the new strain GBN2WP0970 was 1 to 40 mg / rxn, all showed pancreatic lipase inhibitory effect, and a strong inhibitory effect was observed from the concentration of 5 mg / rxn to confirm that there was an excellent neutral fat inhibitory effect. It was.

Production Example 1 Preparation of Powder

Examples of various formulations using water extracts of the mushroom fruiting body of the new strain GBN2WP0970 of Example 3 of the present invention are described below, but the formulation of the present invention is not limited thereto.

New strain GBN2WP0970, mushroom fruiting body water extract powder 20 mg, lactose 100 mg and talc 10 mg

Powder was prepared by mixing and filling the airtight cloth.

Preparation Example 2 Preparation of Tablet

Tablets were prepared by mixing the components of the mushroom fruiting body water extract powder of the new strain GBN2WP0970, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, followed by compression according to a conventional method for preparing a tablet.

Preparation Example 3 Preparation of Capsule

10 mg of mushroom fruiting body water extract powder of new strain GBN2WP0970, 3 mg of crystalline cellulose, 14.8 mg of lactose and 0.2 mg of magnesium stearate were mixed according to the conventional capsule preparation method and filled into gelatin capsules to prepare capsules. Prepared.

Production Example 4 Preparation of Liquid

20 g of mushroom fruit body water extract powder of new strain GBN2WP0970, 10 g of isomerized sugar, 5 g of mannitol and a suitable amount of purified water were dissolved by adding each component according to the conventional method of preparing a liquid solution, and adjusted to 100 mL by adding lemon flavor and purified water. After filling into a brown bottle sterilized to prepare a liquid.

Production Example 5 Preparation of Injection

10 mg of mushroom fruiting body water extract of New strain GBN2WP0970, mannitol 180 mg, sterile distilled water for injection 3000 mg, Na2HPO4, 25 mg of 12H2O were prepared in an ingredient content of 2 mL per ampoule according to a conventional injection method.

Production Example 6 Production of Health Food

New strain GBN2WP0970 mushroom fruiting body water extract powder 1000 mg, vitamin mixture dose, vitamin A acetate 70 ㎍, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 ㎍, vitamin C 10 Mg, biotin 10 μg, nicotinic acid amide 1.7 mg, folic acid 50 μg, calcium pantothenate 0.5 mg, mineral mixture appropriate amount, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, second Calcium phosphate 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, magnesium chloride 24.8 mg is prepared according to a conventional health food manufacturing method, the composition ratio of the vitamin and mineral mixture is a suitable component for a health food is a preferred manufacturing example The mixing ratio may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health food manufacturing method, and then granules are prepared. It may be prepared according to the health food composition.

Korea Research Institute of Bioscience and Biotechnology KCTC18664P 20180125

<110> KYONGSANGBUK-DO <120> A novel hybridized mushroomstrain GBN2WP0970 and fruit body of          the same with high inhibition rate of neutral fat absorption <130> P6107 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 901 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of GBN2WP0970 <400> 1 agaacgagat cactttggag tttgtttgct ggccttaggg gcatgtgcac gcttcactag 60 tctttcaacc acttgtgaac ttttgataga tctgtgaagt cgtctctcaa gtcgttagac 120 ttggtttgct gggatgtaaa cgtctcggtg tgactacgca gtctatttac ttataacacc 180 ccaaatgtat gtctacgaat gtcatttaaa gggccttgtg cctataaacc ataatacaac 240 tttcaacaac ggatctcttg gctctcgcat cgatgaagaa cgcagcgaaa tgcgataagt 300 aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcaccttg cgccccttgg 360 tattccgagg ggcatgcctg tttgagtgtc attaaattct caaactcact ctggtttttc 420 caattgtgat gtttggattg ttgggggctg ctggccttga caggtcggct cctcttaaat 480 gcattagcag gacttctcat tgcctctgcg catgatgtga taattatcac tcatcaatag 540 cacgcatgaa tagagtctgg ctctctaacc gtccgcaagg acaatttgac aatttgacct 600 caaatcaggt aggactaccc gctgaactta agcatatcta aacccggaaa gaaaaatttt 660 cccccctggg ggggcttcca aaaaacgaac caatttctcc acttattgtt ctgccctggg 720 tccaacttta actggaagaa agtaacaata acctggagaa aaagggagga gaaggggaaa 780 aaaaaaaaag ggaaaaaggg aaaaaggaga ggaggaaaaa gaagagagaa ggggaaaaaa 840 agaggaggaa gaaggaggca agatggagga tgaagagagg gaagagagag gaagagaaaa 900 g 901

Claims (9)

Hybridized new strain GBN2WP0970 of Accession No. KCTC18664P.
[Claim 2] The hybrid new breed strain GBN2WP0970 according to claim 1, wherein the hybrid new breed strain hybridizes mononuclear mycelium isolated from the fruiting body of the A. oyster mushroom strain (Bisan No. 2) and mononuclear mycelium of the white oyster mushroom.
Fruiting body formed from the hybrid new strain GBN2WP0970 of claim 1.
The fruiting body formed from the new strain strain GBN2WP0970, wherein the fruiting body has a higher triglyceride inhibitory effect than that of the maternal awiweed mushroom (Bishan No. 2) and the white-winged chickweed.
According to claim 3, wherein the shade of the fruiting body is about 33 to 48 mm in diameter, 30 to 45 mm in thickness, the stage of the fruiting body is about 35 to 60 mm thick, about 90 to 130 mm in length Fruiting body formed from new strain GBN2WP0970.
Spores derived from fruiting bodies formed from the new strain strain GBN2WP0970 of any one of claims 3 to 5.
7. The descendant strain of the new kind GBN2WP0970 according to claim 6, which is obtained through cross-spores crossing between said spores.
Mushroom fruiting body formed from the descendant strain of claim 7.
Sawdust for cultivating mushroom fruiting bodies with hybrid new strain GBN2WP0970 (KCTC18664P): beet pulp: cotton seedlings with a mixing ratio of 50: 30: 20 (%) by volume.

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