KR101110487B1 - New mycoleptodonoides aitchisonii strains and artificial method for cultivating same - Google Patents

Abstract

PURPOSE: A method for producing a large amount of fruit bodies of Mycoleptodonoides aitchisonii by artificial culture is provided. CONSTITUTION: A method for culturing Mycoleptodonoides aitchisonii strains comprises: a step of culturing mycelium of Mycoleptodonoides aitchisonii in YMG, MYPA, ME, PDA solid medium or GP or GT liquid medium at 20-25 degrees Celsius and pH 5-6; and a step of culturing mycelium of Mycoleptodonoides aitchisonii in a medium containing 0.1% MgSO_4H_2O, 0.05% of asparagines, 1% of sucrose, rice bran, wheat flour, and corn powder.

Description

New Mycoleptodonoides aitchisonii strains and Artificial method for Cultivating same}

The present invention relates to a novel true needle mushroom strain and a method for artificial culture thereof, and more specifically, to a method for separating a new true needle mushroom strain from wild mushrooms and to thereby artificial culture of the isolated true needle mushroom strain The present invention relates to a method for mass-producing needle mushroom fruiting bodies.

Mushroom is an important food material that has value as a health food and contains a large amount of various useful substances such as antibacterial action, antiviral action, cardiovascular action, and antithrombotic action, so it can be said to have a function as an alternative medicine food suitable for modern taste. . The annual output of edible mushrooms in Korea is about 1 trillion won based on consumer sales, and its proportion to the gross domestic product is continuously increasing.

The most common cultivated in Korea is oyster mushroom, which occupies 50.36% (622ha, 61,965 tons, special crops production in 2003) of the total mushroom cultivation area, and mushroom mushrooms ranked 1st with 12.7% (157ha, 19,790 ton). Occupies. The third place is Pleurotus eryngii, accounting for 10.5% (131 ha, 18,358 tons) of the total mushroom growing area.

Recently, the rapid development of biotechnology has elucidated the mechanism of life phenomenon in more detail. Based on this series of technologies, research on the development of new biomaterials through the interaction of biological cells and cells is being actively conducted. Microorganisms continue to apply subtle chemistry such as endless struggles and symbiosis between species for their survival in special circumstances. Microbes to biologically active substances is the most representative gibberellin (Gibberellin, GA3), which strains (Gibberellin tall bottle of rice in the 1920s, Kurosawa fujikuroi ) is the largest producer of growth regulators (Pesticides Annual Report, 1997). Gibberellin promotes cell growth, seed germination, growth, shoot development and fruit development. On the other hand, auxin, which has functions such as vertical growth, cell division of the cambium, and promoting rooting enzyme activity, is also reported to be produced in many prokaryotes.

Due to the development of biotechnology as described above, research on cultivation technology for mushrooms, which has not been possible to artificially cultivate in the past, continues. Korean Patent Publication No. 10-2010-0013740 relates to a method for preparing a large amount of polysaccharide isolated from mycelial liquid culture of Grifola frondosa IP-7 (KCTC18133P) and this strain, and Patent No. 10-0339701 has accession number: KFCC. It relates to chaga strain 701, characterized in that -11190. This document is of technical significance in that it has developed an artificial culture method for leaf mushrooms or chaga mushrooms, which were not available for artificial cultivation, and isolated strains of new varieties.

Wild mushrooms of economic value as a food and medicine are essential to mushrooms (Mycoleptodonoides aitchisonii). The true needle mushroom is a kind of edible mushroom, the fruiting body has no mushroom sack, and it is strong when it is flexible and dries. The fungi are scalloped or spatula, many being regenerated, the surface is flat, white or pale yellow, and the edges are toothed. The flesh is white with a thickness of 2-5mm butyl acetate but it disappears when dried. Mycelia are two mycelial type of thick and thin film. The fruiting layer of the lower surface is needle mushroom type, the needle is sharp and the length becomes 3-10mm and yellowish orange. It grows in hardwood trees from summer to autumn, causing white swelling of wood. The distribution is native to Korea, Japan, and Cassimil.

Mycoleptodonoides aitchisonii is a kind of fresh fruity edible mushroom called agaric mushroom. The fruiting body has no mushroom stalk, it becomes strong when it is flexible, dries, and the germ is regenerated, and the surface is soft. White or pale yellow, and the edges are toothed. Mycelia are thicker and thinner 2 hyphae type, lower fruiting layer is needle mushroom type, needles are sharp and the length becomes yellowish orange when dried to 3 ~ 10 mm.

The true needle mushroom is expected to have excellent nutritional and pharmacological effects, and various studies have been conducted.To date, blood pressure lowering action (Tsuchida, T., et al., Effect of an aqueous extract from Mycoleptodonoides aitchisonii on human) blood pressure, Jpn. Pharmacol. Ther., 29 (11), 899-906 (2001)), hypoglycemic activity (Tsuchida, T., et al., Efficacy and safety of an aqueous extract from Mycoleptodonoides aitchisonii in the long- term administration, Jpn. Pharmacol. Ther., 30 (1), 31-36 (2002)), Okuyama S. et al., Enhancing effect of Mycoleptodonoides aitchisonii on synthesis of nerve growth factor and releasing dopamine in the rat brain, Nutritional Neuroscience, 7, 41-47 (2004)), Yasukawa, K., et al., Inhibitory effect of methanol extracts from edible mushroom on TPAinduced ear oedema and tumour promotion in pharmacological, such as mouse skin, Phytotherapy Research, 10 (4), 367-369 (1996)) Although it is expected to be very nutritious with its efficacy, it is known to occur rarely in the hardwoods of hardwoods such as oak trees such as Gangwon-do and Jeju-do. So far, only natural harvesting was possible, and no artificial culture method is known.

Therefore, the inventors of the present invention, while continuing to develop a method for mass production of fruiting bodies produced from true needle mushroom strains, a specific culture characteristic from the true needle mushrooms that have been used for medicinal purposes by collecting wild mushrooms that were hardly found in Korea And the present invention was completed by separating the sesame mushroom strain having a morphological feature and newly developing the environmental composition and propagation method for cultivating the strain effectively.

Accordingly, it is an object of the present invention to provide a method for separating true needle mushroom strains from wild mushrooms.

Another object of the present invention is to provide a novel true needle mushroom strain isolated by the separation method of the true needle mushroom strain.

Still another object of the present invention is to provide an artificial cultivation method for mass production of true needle mushroom fruiting bodies from true needle mushroom strains.

In order to achieve the above object, the present invention provides a method for separating the true needle mushroom strain from wild mushrooms of the Maple old tree.

In order to achieve the above another object, the present invention provides a true needle mushroom strain JF33-02 (Accession No. KCTC 11970BP), which has the following characteristics separated according to the above method:

In order to achieve the above another object, in the present invention

(1) incubating the mycelium of the true needle mushroom strain in YMG, MYPA, ME, PDA solid medium, or GP, GT liquid medium at a temperature of 20 to 25 ℃ under pH 5-6 conditions; And

(2) Hardwood sawdust consisting of oak, chestnut, willow and cherry trees contains 0.1% MgSO ₄ -H ₂ O, 0.05% asparagine and 1% sucrose, and dried beer, rice bran, wheat flour, wheat bran and corn flour Culturing the mycelia of the true needle mushroom strain of the subcultured true needle mushroom strain in an added sawdust medium

It provides a cultivation method of a true needle mushroom, characterized in that it comprises a.

According to the present invention, the true needle mushroom strain isolated from the true needle mushroom is expected to be able to mass-produce with mycelial growth and fruiting body formation rate and high quality, high yielding multipotent mushroom superior strain. In addition, the true needle mushroom strain is capable of artificial cultivation, mycelial growth and fruiting body formation rate is fast and high quality can be expected to be very useful in the art.

Figure 1 is a photograph showing the mycelia grown in Korean true needle mushroom.
Figure 2 is a graph showing the mycelial growth of the JF33-01 strain and JF33-02 strain as a mycelial growth characteristics of the true needle mushroom.
Figure 3 shows the mycelial growth of true needle mushroom strains cultured in PDA medium.
Figure 4 is a graph showing the mycelial growth of the true needle mushroom strains in oak, larvae, chestnut, willow sawdust.
Figure 5 is a photograph showing the fruiting body grown in the needle mushroom cultured in sawdust medium.
Figure 6 is a micrograph observing the spore shape of the true needle mushroom strain.
Figure 7 is a micrograph observing the mycelia of the needle mushroom strain.
Figure 8 is a photograph showing the substitution culture texture of JF33-01 (2n) and the needle needle JF33-02 (2n) strain of the present invention.
Figure 9 shows the comparison of the mycelial density by solid culture medium.
Figure 10 shows the dry weight of the hyphae after liquid culture for 30 days at different pH.
11 is a graph showing the effect of temperature on mycelial growth of true needle mushroom strains.
12 is a graph showing the influence of the carbon source on the mycelial growth of true needle mushroom strains.
Fig. 13 is a graph showing the effect of nitrogen source on mycelial growth of true needle mushroom strains.
14 is a graph showing the effect of mineral sources on the mycelial growth of true needle mushroom strains.
Figure 15 shows the dry weight reduction rate of the sawdust medium.
16 is a graph showing mycelial culture period when different titration nitrogen sources are added.
Figure 17 is a photograph showing the mycelial culture of true needle mushroom.
Figure 18 shows the production process of the true needle mushroom.

The technical terms and scientific terms used in the present invention can be construed as meaning ordinary meanings understood by those of ordinary skill in the art without departing from the scope of the present invention.

In the present invention, in the middle of September 2010, the wild mushrooms were found and collected in the old trees of the maple trees in the natural forest around the entrance valley of Woljeongsa in Jinbu-myeon, Pyeongchang-gun, Pyeongchang-gun, Gangwon-do, Korea. The strain obtained by purely separating the bacteria over several times under the condition of inhibiting and inducing true needle mushroom mycelial growth was named 'JF33-02'.

Mycelium of strain JF33-02 isolated in the present invention is 5.8cm, and for comparison, JF33-01 collected in Japan was 6.2cm, showing that JF33-02 strain, a wild gene source collected domestically, was somewhat faster. In addition, JF33-02 was able to confirm that the mycelia became yellow during the culture of PDA (Potato Detrose Agar) medium and formed some biofilm. Is an important process in fruiting, JF33-02 strain may be more favorable conditions for fruiting.

Strain JF33-02 isolated from the present invention showed almost similar mycelial growth in fermented oak, chestnut and willow, but in fermented pine pine, JF33-02 appeared about 2.5 cm faster than JF33-01.

In addition, the fruiting body of strain JF33-02 has a fresh surface and fresh white color, whereas the fresh surface of strain JF33-02 isolated from the present invention grows like a ring and has streaks, and the shade is thick and elastic. JF33-02 also has a very strong fresh fruit flavor compared to JF33-01. And in the shape of the lampshade, the JF33-01 is a carnation and the JF33-02 is a fan. In addition, JF33-02 was larger than JF33-01, but JF33-01 was larger than JF33-01, and the average fresh weight and the number of fruiting bodies were higher than JF33-02. Occupied the sheep.

In addition, the spores of JF33-01 was about 7㎛, but in the case of the JF33-02 isolated in the present invention appeared to have a size of 5㎛. In addition, the mycelia of germinated spores were confirmed to be mononuclear mycelium without connecting cramps by microscopic examination of germinated single spores.

Thus, the needle mushroom strain JF33-02 having the above culture characteristics and morphological characteristics isolated according to the present invention was deposited on June 28, 2011 as the accession number KCTC 11970BP No. It was entrusted.

Needle mushroom strain JF33-02 isolated in the present invention has the characteristics as shown in Table 1 below.

Characteristics Found JF33-02 Fruiting body (diameter) a (horizontal) 140.79mm b (vertical) 91.61 mm Width (width) a (horizontal) 104.71 mm b (vertical) 60.37mm Thickness a (middle part) 6.72mm b (end) 0.82 mm Lampshade Fan
(Value is regular) Gad (with or without edges) have Lampshade (tip of the side cut vertically) level Lampshade (surface color) Ivory Lampshade (shape of the surface) Fiber shape (with scale) Lampshade Overall lower part Lampshade Shell Fruiting body fragrance Fresh fruit flavor Saliva length 5.36mm Average raw weight 104.5g / 600g Bottle Medium Number of fruiting bodies 14.0

In the present invention can be obtained in large quantities by artificial culture of a true needle mushroom strain according to a method comprising the following steps:

(1) incubating the mycelium of the true needle mushroom strain in YMG, MYPA, ME, PDA solid medium, or GP, GT liquid medium at a temperature of 20 to 25 ℃ under pH 5-6 conditions; And

(2) Hardwood sawdust consisting of oak, chestnut, willow and cherry trees contains 0.1% MgSO ₄ -H ₂ O, 0.05% asparagine and 1% sucrose, and dried beer, rice bran, wheat flour, wheat bran and corn flour Cultivating the mycelia of the true needle mushroom strain of the subcultured true needle mushroom strain in an added sawdust medium.

In step 1 of the culture method of true needle mushroom strain of the present invention, after incubating for 14 days in 25 in YMG, MYPA, ME or PDA solid medium, low-temperature preservation at 4 and then passaged twice for activation of mycelia. It is done.

After incubating the sawdust medium in step 2 with a primary culture at a temperature of 15 to 25 and a humidity of 50 to 80%, inducing yellowing, and then performing a second culture at a temperature of 10 to 20 and a humidity of 80 to 99%, the bacterium was scraped. It is characterized by generating a mushroom by puncturing the top or side of the bag.

According to the present invention, the true needle mushroom strain isolated from the true needle mushroom is expected to be able to mass-produce with mycelial growth and fruiting body formation rate and high quality, high yielding multipotent mushroom superior strain. In addition, the true needle mushroom strain is capable of artificial cultivation, mycelial growth and fruiting body formation rate is fast and high quality can be expected to be very useful in the art.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited thereto.

Example 1: Fostering a New Variety of Needle Mushroom Strains

(Step 1) Confirmation of domestic habitat and collecting wild mushrooms

In mid-September 2010, wild mushrooms were found and collected in the old trees of the maple trees in the valley near the entrance of Woljeongsa in Jinbu-myeon, Pyeongchang-gun, Pyeongchang-gun, Gangwon-do. Figure 1 is a photograph showing the mycelia grown in Korean true needle mushroom.

(Step 2) Fungal Pure Separation of Collected Wild Mushrooms

The collected mushrooms were named as 'JF33-02' and were used for the experiments. The mycelial growth characteristics were compared with JF33-01 distributed in Gaya Biofarm. Detailed characteristics were observed. Figure 2 is a graph showing the mycelial growth of the JF33-01 strain and JF33-02 strain as a mycelial growth characteristics of the true needle mushroom.

Then, after inoculating the bacteria (Potato Detrose Agar) medium and cultured for one week. The results are shown in FIG. Figure 3 shows the mycelial growth of true needle mushroom strains cultured in PDA medium. As shown here, JF33-01 (left) was 5.8 cm, and JF33-02 (Right) was 6.2 cm, showing that the JF33-02 strain, a wild gene collected domestically, showed a rather rapid growth. In addition, JF33-02 was able to confirm that the mycelium becomes yellow during the cultivation process, and a slight biofilm is formed. JF33-02 strain may be a more favorable condition for fruiting.

(Step 3) Comparison of Mycelial Growth Characteristics in Sawdust Medium

The mycelial growth of various species of oak, larvae, chestnut and willow sawdust was incubated for 3 weeks, and the results are shown in FIG. 4. Figure 4 is a graph showing the mycelial growth of the true needle mushroom strains in oak, larvae, chestnut, willow sawdust. As seen here, fermented oak, chestnut, and willows appeared almost similarly, but in fermented pine, JF33-02 appeared about 2.5cm faster than JF33-01. However, the true needle mushrooms have host characteristics that are caused by dead wood, so it is necessary to confirm the mushroom development even if the mycelial culture in fermented pine sawdust is good.

(Step 4) Fruiting Body Characteristics of Occurring Mushrooms

As shown in Table 2 and FIG. 5, in the case of JF33-01, the fresh surface of the Korean native strain JF33-02 is streaked and grows like a ring, and the shade is thick and elastic as compared to the smooth and white color of the lampshade. . JF33-02 also has a very strong fresh fruit flavor compared to JF33-01. And in the shape of the lampshade, the JF33-01 is a carnation and the JF33-02 is a fan. In addition, JF33-02 was larger than JF33-01, but JF33-01 was larger than JF33-01, and the average fresh weight and the number of fruiting bodies were JF33-02. Occupied a high volume.

Characteristics Found JF33-02 JF33-01 Fruiting body (diameter) a (horizontal) 140.79mm 136.72mm b (vertical) 91.61 mm 69.20mm Width (width) a (horizontal) 104.71 mm 77.75mm b (vertical) 60.37mm 59.54 mm Thickness a (middle part) 6.72mm 4.41mm b (end) 0.82 mm 0.76 mm Lampshade Fan
(Value is regular) Carnation type
(Irregularly severe) Gad (with or without edges) have have Lampshade level level Lampshade (surface color) Ivory Ivory Lampshade (shape of the surface) Fiber shape (with scale) Fiber shape (with scale) Lampshade Overall lower part Lampshade whole Lampshade Shell clam Fruiting body fragrance Fresh fruit flavor Fresh fruity flavor Saliva length 5.36mm 7.06mm Average raw weight 104.5g / 600g Bottle Medium 53 g Number of fruiting bodies 14.0 6.7

(Step 5) Separation and hybridization of single spores

As shown in Figures 6 and 7, spores of JF33-01 was about 7μm, but JF33-02 was about 5μm in size. In addition, the germination of the germinated spores was verified as shown in FIG. 6, and microscopic examination of the germinated single spores confirmed that it was a single-core hyphae without a cramp as shown in FIG. 7.

Subsequently, in order to confirm that the JF33-01 (n) and JF33-02 (2n) strains are different strains, the resultant is shown in FIG. 8. As shown here, it was confirmed that the formation of a clear opposing line. This is considered to be significant between the two strains.

Strain JF33-02 isolated according to the present invention was deposited on June 28, 2011 as Accession No. KCTC 11970BP to the Gene Bank in Korea Research Institute of Biotechnology.

Example 2: Cultivation of True Needle Mushroom Strain

(Step 1)

After incubation in YMG solid medium for 25 days at pH 5 to 6 conditions for 14 days, the cells were cryopreserved at 4 and then subcultured twice for activation of mycelia.

(Step 2)

Hardwood sawdust consisting of oak, chestnut, willow and cherry trees contains 0.1% MgSO ₄ ? H ₂ O, 0.05% asparagine and 1% sucrose, and sawdust added with dried beer, rice bran, wheat flour, wheat bran and corn flour Incubate in the medium at a temperature of 15 to 25 and a humidity of 50 to 80% to induce yellowing, and then incubate at a temperature of 10 to 20 and a humidity of 80 to 99% to perform bacterium scraping. Perforated to generate mushrooms.

Example 3 Selection of Optimal Flat Media

As a result of examining the mycelial density of each culture medium, as shown in FIG. 9, the YM and ME media showed the strongest mycelial activity, and the YMG media with the best mycelial growth also showed relatively strong mycelial growth.

Example 4: Optimal pH Selection

As shown in FIG. 10, the mycelium mushroom hyphae was incubated for 30 days in GP medium, which is the optimal medium for incubation, and the mycelial dry weight was the highest as 0.38 mg at pH 6, followed by pH 5 The optimum acidity of mycelial growth of needle mushrooms was investigated at pH 5.0 to 6,0. On the other hand, mycelial growth was very low at pH 3.0 and pH 7.0. This result was similar to that of shiitake mushroom and hazelnut mushroom showed optimal growth at pH 5.0 to 6.0 (Kim et al. 2009, E. 1997; Kang et al., 1994).

Example 5 Selection of Optimal Culture Temperature

Five-day cultivation of mushroom fungus hyphae in YMG (Yeast Malt Glucose) medium, which is the best medium for plate culture, showed the best growth rate of 7.7cm at 25 ℃, followed by 6.4cm at 20 ℃. Mycelial growth of These results were similar to the result of mycelial growth of 5.3cm after 9 days of incubation at 25 ° C for cultured mushroom mycelial culture, followed by 4.3cm of mycelial growth at 20 ° C. Showed low mycelial growth similar to 4 ℃, Kim et al. (2006) reported. However, as shown in Figure 11, the true needle mushrooms are rather mycelial growth at 15 ℃ culture than 30 ℃ due to the mycelial growth is likely to show a tendency to fall.

Example 6 Selection of Optimal Nutrient Sources

(A) Selection of carbon source

As a result of investigating the effect of various carbon sources on the mycelial growth of true needle mushrooms, as shown in FIG. It showed growth but no statistical significance. This result was similar to that of Sujong's supplemented mycelial growth in the culture of large mushrooms. In addition to other carbon source additions, mycelial growth was higher than that of untreated treatments. This is similar to the results of Kim et al. (2006) reported that the mycelial growth of taste mushrooms was higher than that of the untreated group when 2% carbon source was added, but the sucrose growth was slightly slower (Kim et al. 2006, Oh). 2003) The different carbon sources needed for different mushrooms were found.

(B) Selection of nitrogen source

As a result of investigating the optimum nitrogen source affecting the mycelial growth of true needle mushroom, as shown in FIG. Some nitrogen sources, sodium nitrate 0.05, 0.1% and glycine 0.025, 0.05%, showed lower mycelial growth than the control. This is consistent with the results of Kim et al. (2006) that valine showed lower mycelial growth when added 0.02% of nitrogen source in cultivation of fungi.

(C) mineral selection

As a result of investigating the optimum minerals affecting the mycelial growth of the true needle mushroom, MgSO ₄ 7H ₂ O 0.1% treatment showed the highest mycelial growth of 0.78mg of the total cell weight when incubated in GP medium for 30 days. . All treatments except thiamine B1 addition showed better mycelial growth than the control.

Example 7: Medium weight loss rate

As shown in Fig. 15, PYR ( Prunus) has the best mycelial growth in sawdust medium. yedoensis + Rice bran) in the medium was very low with a 10.4% weight reduction was relatively mycelial growth is fast PSR (Populus spp . The mycelial growth in sawdust medium did not significantly affect the weight loss rate of the medium, with 28.3% having the highest weight loss.

Example 8: Comparison of Sawdust Medium Mycelial Growth with Addition of Optimum Nutrient Solution

Each sawdust medium and inoculum were prepared in advance (FIGS. 17 and 18) to determine whether the mycelial incubation period was shortened by adding the optimally selected optimal nutrient source to the medium. The period has been shortened. Especially, MgSO ₄ 7H ₂ O 0.1% of the optimum minerals showed the shortest incubation period for sawdust bottle medium to 36 days, followed by 0.05% of asparagine, the optimal carbon source, for 37 days. S + A + M treated with carbon source (Sucrose 1%), nitrogen source (Asparagine 0.05%), and minerals (MgSO ₄ 7H ₂ O 0.1%) selected as the optimal nutrient source was 39 days in cultivation period. Although it was shortened, the incubation period was somewhat delayed compared to the single treatment which added the carbon and nitrogen minerals. Among them, the optimum carbon source asparagine 0.05% supplementation was 42 days incubation period was not significantly different from the control. This was related to the result that 0.05% of asparagine which was investigated as the optimal carbon source was not statistically significant.

According to the present invention, the true needle mushroom strain isolated from the true needle mushroom is expected to be able to mass-produce with mycelial growth and fruiting body formation rate and high quality, high yielding multipotent mushroom superior strain. In addition, the true needle mushroom strain is capable of artificial cultivation, mycelial growth and fruiting body formation rate is fast and high quality can be expected to be very useful in the art.

Korea Research Institute of Bioscience and Biotechnology KCTC11970 20110628

Claims (6)

delete delete delete delete Needle mushroom strain JF33-02 (Accession No. KCTC 11970BP) having the following characteristics:
(1) fruit body diameter [width: 140.79mm, length: 91.61mm]
(2) The width of the shade [width: 107.71mm, length: 60.37mm]
(3) Thickness of shade [Middle part: 6.72mm, Tip part: 0.82mm]
(4) The shape of the shade is regular fan type,
(5) there is a gap in the edge of the shade,
(6) the shape of the tip of the cut length of the shade is horizontal,
(7) The surface color of the shade is ivory and the shape of the surface is fibrous,
(8) The fruiting body has a light fruit flavor,
(9) the length of the needle is 5.36mm,
(10) The number of fruiting bodies is 14.0.

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