KR101063754B1 - Cultivation method for Mycoleptodonoides aitchisonii - Google Patents

Abstract

The present invention provides a method for efficiently growing the inoculation bacteria from the seed of the true needle mushroom and an artificial cultivation method capable of short-term occurrence of the true needle mushroom, it is possible to cultivate industrially grown needle needle mushrooms only in the farmhouse By increasing the blood pressure, lowering blood sugar, improving brain function, and anticancer effect, it is possible to contribute to improving farm household income by presenting the possibility of using true needle mushroom as a raw material of various foods and medicines.

Needle Mushroom, Artificial Cultivation

Description

Cultivation method for Mycoleptodonoides aitchisonii}

The present invention relates to a method of artificial cultivation of mushrooms, inter alia, a method of artificial cultivation of true needle mushrooms.

Needle Mushrooms ( Mycoleptodonoides aitchisonii ) is a kind of fresh fruit-flavored edible mushroom called acupuncture mushroom. Fruiting body has no mushroom stalk, and it is strong when it is flexible flesh or dried. Edge is tooth-shaped. The hyphae are two mycelial type of thick and thin film. The fruiting layer film on the lower surface is needle mushroom type, needles are sharp and the length becomes yellowish orange when dried to 3 ~ 10 mm.

Needle mushrooms are expected to be very nutritious with pharmacological effects such as lowering blood pressure, lowering blood sugar, improving brain function, and anticancer, but rarely occur in the hardwoods of hardwoods such as oak trees in Gangwon-do and Jeju-do. It has been known that only natural harvesting has been possible so far, and no artificial cultivation method is known.

The present inventors cultivated the bacteria of true needle mushrooms and studied the optimum native conditions, and invented a method that can easily artificially cultivate true needle mushrooms in farmhouses through experiments that can be mass-produced in a short period of time and the present invention Completed.

It is an object of the present invention to provide a method for efficiently growing inoculation bacteria from the seed of a true needle mushroom, and an artificial cultivation method capable of short-term mushroom generation, so that the needle needle mushroom can be grown industrially in a farm.

The method of cultivating the true needle mushroom of the present invention is the fruit body spawn of the true needle mushroom strain, 0.3 to 3 parts by weight of any one or more carbon source of maltose and sucrose, 100 parts by weight of liquid or solid inoculation medium, any of asparagine and sodium nitrate An inoculator preparation step of culturing the liquid inoculation source or the solid inoculation source by adding 0.02 to 0.2 parts by weight of one or more nitrogen sources, and 0.02 to 0.2 parts by weight of one or more minerals of potassium phosphate and magnesium sulfate; 60 to 90 parts by weight of hardwood sawdust, rice bran, wheat flour, beer gourd and crumbs, based on 100 parts by weight of medium solids, 10 to 30 parts by weight of nutrient sources are mixed, and the water content is 55 to 70% by weight A culture medium preparation step of adding water so as to enter and sterilize; Spawn inoculation step of inoculating the culture or solid inoculation source into the culture medium; A culture step of incubating the spawn inoculated bottle medium in a culture chamber at 18 to 28 ° C. and humidity at 50 to 80%; A low temperature treatment step of treating the inoculated bottle medium inoculated through the culture step at 2 to 12 ° C. and a humidity of 50 to 80% for 6 to 12 days; And a mushroom generation step of generating mushrooms inoculated with the spawn inoculation via the low temperature treatment step at 14 to 20 ° C. and humidity at 85 to 99% for 15 to 25 days.

In the cultivation method of the true needle mushroom of the present invention, the hardwood is characterized in that any one of oak, cherry, maple, alder, mulberry, beech, westwood and poplar.

In the method of cultivating the true needle mushroom of the present invention, the liquid inoculation medium, the carbon source, nitrogen source and minerals are dissolved in distilled water, glucose 5-20g, peptone 5-20g, malt extract 10-30g and yeast extract in distilled water It is characterized in that the glucose peptone medium adjusted to 1 L after adding 5 to 20 g.

In the method of cultivating true needle mushroom of the present invention, the solid inoculation medium is mixed with 60 to 90 parts by weight of hardwood sawdust, 10 to 30 parts by weight of rice bran and wheat flour, based on 100 parts by weight of medium solids, and then the carbon source, nitrogen source and minerals. Dissolved distilled water was added to adjust the water content to 55 to 70% by weight based on the total weight of the medium.

In the method of cultivating the true needle mushroom of the present invention, the hardwood of the hardwood sawdust used in the solid inoculation medium is any one of oak, cherry, maple, mulberry, beech, beech, beech and seo It is done.

The present invention can contribute to the increase of farmers' incomes along with the increase of national health by providing an artificial cultivation method capable of mass production of true needle mushrooms containing beneficial ingredients to the human body, which depended only on natural collection.

Hereinafter, with reference to the drawings will be described in detail the cultivation method of the true needle mushroom of the present invention.

1 is a block diagram showing the process of the true needle mushroom cultivation method of the present invention.

As a first step of the true needle mushroom cultivation method of the present invention, a liquid inoculum or a solid inoculum is prepared. The present invention provides a method of preparing a solid inoculum using a sawdust medium that can be easily produced inoculum without a separate facility in a small farm as well as a method of preparing a liquid inoculum that can produce a large amount of inoculum in a short period of time.

In the present invention, in order to prepare the inoculum in a short period of time with high productivity, 0.3 to 3 parts by weight of one or more carbon atoms of maltose and sucrose, 100 parts by weight of liquid or solid inoculation medium, nitrogen source of 0.02 to one or more of asparagine and sodium nitrate It is preferable to add 0.2 weight part and 0.02-0.2 weight part of minerals of at least one of potassium diphosphate and magnesium sulfate. Particularly preferably, 0.8 to 2.4 parts by weight of sucrose is used as the carbon source, 0.03 to 0.15 parts by weight of asparagine as the nitrogen source, and 0.06 to 0.12 parts by weight of magnesium sulfate as the mineral. By adding the carbohydrate source, nitrogen source and minerals to the liquid or solid inoculum medium, the mycelium weight is increased by 1.5 times, preferably 2 times or more, within a predetermined culture period, thereby shortening the inoculator culture period.

Liquid inoculation media include potato dextrose medium (PDB), malt yeast extract media, mushroom complete media, lily media, glucose tryptone media Glucose Peptone media and the like can be used without limitation. Preferably, it is a potato dextrose medium, a malt yeast extract medium, a glucose tryptone medium, a glucose peptone medium, and most preferably a glucose peptone medium. When comparing mycelial weight of liquid inoculation medium 30 days after inoculation of mycelium mushroom, glucose peptone medium showed more than 7 times mycelial growth of mushroom complete medium or lily medium, potato dextrose medium, malt yeast extract medium, glucose tryptone medium More than 2 times of mycelial growth.

The liquid inoculation medium, for example, in the case of glucose peptone medium, dissolves the carbon source, nitrogen source and minerals in distilled water, and in the distilled water, glucose 5-20g, peptone 5-20g, malt extract 10-30g and yeast extract 5 ~ It can be prepared by adjusting to 1 L after adding 20 g.

The pH for mycelial culture in liquid inoculation medium is 3.5-6.5, more preferably 4.5-6.3, most preferably 6.0. In addition, the culture temperature for mycelial culture is 18 ~ 28 ℃, more preferably 22 ~ 27 ℃, most preferably 24 ~ 25 ℃.

Solid inoculum medium is used by adding nutrients to sawdust medium for convenience in farming. As nutrients, rice bran and soybean powder are generally used, but when wheat flour is used, the growth of mycelia until about 10 days Although there is no significant difference from the use of rice bran, the cultivation of inoculum up to about 25 days results in a significant decrease in mycelial growth compared to the rice bran inoculation medium. . The solid inoculation medium was mixed with 60 to 90 parts by weight of hardwood sawdust and 10 to 30 parts by weight of rice bran per 100 parts by weight of medium solids, and then distilled water was added to adjust the water content to 55 to 70% by weight based on the total weight of the medium. To prepare. The carbon source, nitrogen source and minerals for promoting mycelial growth are preferably dissolved beforehand in the distilled water. The hardwood used as the sawdust of the solid inoculation medium may be oak, cherry, maple, mulberry, cypress, beech, beech, seo, etc. More preferably oak, cherry, maple, mulberry To use.

The liquid or solid inoculation medium is autoclaved for 10 to 100 minutes at 100-130 ° C., 1-1.5 atm before inoculation of the true mushroom seed. If liquid inoculation medium is used, the incubation period for obtaining the inoculum is 10 to 25 days, and if the solid inoculation medium is used, the culture period is 20 to 35 days.

The culture medium for generating the true needle mushroom fruiting body of the present invention is mixed with 60 to 90 parts by weight of hardwood sawdust, rice bran, wheat flour, beer foil and dried nutrients 10 to 30 parts by weight of the nutrient source, water content relative to the total weight of the medium The water is added so as to be 55 to 70% by weight, and then enters and sterilizes. As a nutrient source of the culture medium for generating the true needle mushroom fruit body, rice bran or wheat flour is preferable, and more preferably, rice bran is used, and most preferably 5 to 10 parts by weight of beer foil or dried rubbish To use.

As the hardwood of sawdust used as a culture medium for fruiting body generation, oaks, cherry trees, maples, mulberries, beech, beech, poplar, and erhu may be used. It is preferable to vary the nutritional sources of sawdust medium used according to the type of hardwood, and the most suitable for fruit body generation of oak mushrooms is the addition of wheat flour to oak sawdust, or rice bran to the cherry sawdust.

In addition, when a nutrient source such as a carbon source, a nitrogen source, and minerals is added to the distilled water used in the production of the fruiting body-generating medium, the incubation rate is relatively fast and mushrooms are also excellent. Therefore, 100 parts by weight of the medium for fruiting body generation, 0.3 to 3 parts by weight of carbon source of at least one of maltose and sucrose, 0.02 to 0.2 parts by weight of nitrogen source of at least one of asparagine and sodium nitrate, and any one of potassium diphosphate and magnesium sulfate It is preferable to add 0.02-0.2 weight part of the above minerals.

The culture medium for the generation of fruiting bodies is filled with 50 to 100% by volume in a polypropylene bottle, and the inlet is thoroughly washed to prevent contamination of various germs and then autoclaved at 100 to 150 ° C. for 10 to 100 minutes. The sterilized medium was cooled to 10-20 ° C. to inoculate the inoculator in a clean room and cultured for 15-30 days, more preferably 20-28 days, most preferably 23-27 days, but the culture temperature was 18-28 ° C., Preferably 22-27 ℃, humidity is incubated under 50-80% conditions.

After completion of the incubation, it is preferable to perform a bacterium scraping to form a faithful fruiting body of the normal form. The bacterium scraper induces only the mycelium that has risen from the center of the bottle by removing the original formed on the wall of the bottle, the aged hyphae or the mushroom itself growing on the wall of the bottle.

After the bacterium scraping step or after the incubation, low temperature treatment is performed at 2 to 12 ° C., preferably 4 to 8 ° C., and a humidity of 50 to 80%. The low temperature treatment is an essential step for the generation of fruit needle fruiting body, it is carried out for 6 to 12 days at 2 ~ 12 ℃, but not carried out continuously 1 to 3 times in the middle, preferably 2 to 3 times the initial culture temperature Phosphorus is preferably carried out intermittently by adding a process of 0.5-3 days incubation at 18 ~ 28 ℃. One holding time of the low temperature treatment is 0.5 to 4 days, preferably 2 to 3 days. Most preferably, three days of the first low temperature treatment, two days of the first room temperature incubation, three days of the second low temperature treatment, two days of the second room temperature incubation and three days of the third low temperature treatment. After low temperature treatment, the process of incubating 0.5 to 3 days at the initial culture temperature of 18 ~ 28 ℃ 12 ~ 48 hours, preferably 36 to 3 to 6 times at the temperature is necessary. If the low-temperature treatment is not performed, mushrooms are not formed, and if the intermittent low-temperature treatment is not performed twice, the formation of mushrooms is delayed.

Mushroom raw material is formed through the low temperature treatment step, the mushroom can be harvested after the mushroom is formed by incubation for 15 to 25 days at 14 ~ 20 ℃, preferably 16 ~ 18 ℃, humidity 85 ~ 99%. Below the lower limit of the incubation temperature, the time required for fruiting body harvest is delayed, and when the upper limit of the incubation temperature is exceeded, the fruiting body is not formed. In addition, even when the humidity is less than the lower limit, the fruiting body formation rate of normal quality is low.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples, but the present invention is not limited only to the following Examples.

Preparation Example 1 Preparation of Liquid Inoculum

According to the composition of the medium in Table 1, the material was added to distilled water, adjusted to 1 L, and the mixture was heated and stirred at 100 ° C. After autoclaving at 121 ° C. for 15 minutes, the solution was cooled to 15 ° C. to prepare a liquid inoculation medium, and then inoculated with 20cc of true needle mushroom inoculum and incubated at 25 ° C. for 30 days.

division Preparation Example L1 Preparation Example L2 Preparation Example L3 Preparation Example L4 Preparation Example L5 Preparation Example L6 PDB Mixed Medium 24 g KH2PO4 0.05g 1 g MgSO47H2O 0.05g 0.5 g Glucose 10g 10g 5 g Maltose 10g Dexrose 2 g DL-Asparagine 2 g Eptone 10g 5 g 0.2 g Malt extract 15 g Yeast extract 10g 3g 0.2 g 3g Tryptone 10g

Experimental Example 1: Comparison of Cell Weights of Liquid Inoculation Sources

Preparation Example L1 to Preparation Example L6 After incubating for 30 days at 25 ° C. in a liquid inoculation medium, the cells were filtered and dried until the weight was obtained, and the weight of pure cells was measured for each medium and shown in FIG.

The glucose peptone medium of Preparation Example L2 exhibited 7 times more mycelial growth than the mushroom complete medium of Preparation Example L4 or the lily medium of Preparation Example L5. Potato dextrose medium, malt yeast extract medium, and glucose tryptone medium of Preparation Examples L1, L3, and L6 also showed three times more mycelial growth than Preparation Example L4.

Experimental Example 2: Comparison of cell mass of carbohydrate source, nitrogen source, and mineral inoculated source

Preparation of the best mycelial growth L 100 medium to 100 parts by weight of the carbohydrate source, nitrogen source, minerals and vitamins were added to prepare a liquid inoculation medium as shown in Table 2 below and sterilized as in Preparation Example 1, Inoculation was incubated for 30 days at 25 ℃. After filtering the respective cells and dried until the dosage, the pure cell weight was measured for each medium is shown in Table 2 together.

division Preparation example L2 medium
Added ingredient content
(Parts by weight) Cell weight
(g) Preparation Example L2 - - 0.55 Carbohydrate source maltose

0.5 0.66 1.0 0.63 2.0 0.68 Sucrose

0.5 0.60 1.0 0.79 2.0 0.73 Dextrose

0.5 0.62 1.0 0.56 2.0 0.65 Nitrogen source







Asparagine

0.025 0.58 0.05 0.69 0.1 0.69 Sodium nitrate

0.025 0.67 0.05 0.08 0.1 0.49 Glycine

0.025 0.52 0.05 0.51 0.1 0.64 mineral




Potassium Diphosphate

0.025 0.67 0.05 0.65 0.1 0.73 Magnesium Sulfate Heptahydrate

0.025 0.64 0.05 0.71 0.1 0.78 vitamin

Thiamine

0.025 0.48 0.05 0.57 0.1 0.53

In particular, 1 part by weight of sucrose, 0.05 parts by weight of asparagine, and 0.1 parts by weight of magnesium sulfate hydrate were added to 100 parts by weight of the L2 medium, and sterilized as in Preparation Example 1, followed by inoculation with a true needle strain. When cultured at 30 ° C. for 30 days, 1.07 g of the cell mass was obtained, and 1.95-fold cells were obtained during the same culture period as compared with the culture with L2 medium of Preparation Example.

Experimental Example 3: Optimum pH

Preparation Example L2 medium was added with 1N sodium hydroxide or hydrochloric acid to adjust the pH to 3-7, inoculated with the true needle mushroom strain, and cultured at 25 ° C. for 30 days. It was.

The pH for mycelial culture in liquid inoculation medium was 3.5-6.5, and optimum pH 6.0.

Preparation Example 2 Preparation of Solid Inoculum

80% by weight of one of the six species of alder, oak, poplar, cherry, Chinese maple, and mulberry was mixed with 20% by weight of rice bran or wheat flour, and then distilled water was added to adjust the water content to 65% by weight. The solid inoculation medium with added water was added to a test tube, autoclaved at 121 ° C. for 90 minutes, cooled to 15 ° C. to prepare a solid inoculation medium, and inoculated with 20cc of true needle mushroom inoculum inoculum and incubated at 25 ° C. for 25 days. It was. Each solid inoculation medium was put in a test tube and cultured is shown in FIG. 4.

Experimental Example 4 Comparison of Mycelial Growth of Solid Inoculation Sources

On the 10th and 25th day of culture, the mycelial growth of each solid inoculation source was compared. Until 10 days of cultivation, there was no significant difference in the length of mycelial growth according to the types of hardwoods and nutrient sources. In the experimental group, the mycelial growth of wheat flour in poplar sawdust and wheat flour in alder sawdust Most active (FIG. 5). However, it was confirmed that the growth of mycelia was significantly superior to the addition of wheat flour in all active hot water sawdust except alder, and among them, oak, cherry, Chinese maple, and mulberry sawdust were excellent. It could be confirmed (Fig. 6).

Preparation Example 3: Preparation of fruiting medium

Among the six kinds of alder, oak, poplar, cherry, Chinese maple, and mulberry, 80 wt% of sawdust or wheat flour, 15 wt% of rice bran or 5 wt% of beer gourd are mixed, and then distilled water is added to the water content. The weight percentage was adjusted. The fruiting body culture medium to which water was added was filled with 600 g of a 1000 ml polypropylene bottle, autoclaved at 121 ° C. for 90 minutes, and cooled to 15 ° C. to prepare fruiting body culture medium.

Preparation Example 4 Inoculation, Culture, Bacterial Scratch and Development

The polypropylene bottle containing the sterilized culture medium was transferred to a clean room and sterilized with ultraviolet rays, and then inoculated with a 20cc liquid inoculator and cultured under the following conditions.

After incubation for 25 days under incubation conditions at 25 ± 2 ° C and 70% humidity, the cells were scraped and cultured at 5 ° C for 3 days, 16 ° C for 2 days, 5 ° C for 3 days, 16 ° C for 2 days, and 5 ° C for 3 days. Mushroom primitives were formed.

Mushrooms were harvested after 23 days at 16 ° C. and 95% humidity after mushroom base formation. Fruiting body yield according to the type of hardwood sawdust and nutrient sources in the fruiting body-generating medium of Preparation Example 3 is shown in Figure 7, the photo of the true needle mushroom just before the harvest is shown in Figure 8.

1 is a block diagram showing the process of the true needle mushroom cultivation method of the present invention.

Figure 2 is a graph showing the weight of the mycelia after inoculation of the true needle mushroom strain inoculated in liquid inoculation medium of Preparation Examples L1 to L6 for 30 days.

Figure 3 is a graph showing the weight of the mycelia according to the pH of the liquid inoculation medium of Preparation Example L2.

Figure 4 is a photograph of a state in which each solid inoculation medium is put in a test tube and inoculated.

Figure 5 is a graph showing the growth length of the mycelia on the 10th day of culture according to the type of hardwood species and nutrient sources of solid inoculation medium.

Figure 6 is a graph showing the mycelial growth length of the 25th day of culture according to the type of hardwood species and nutrient sources of solid inoculation medium.

Figure 7 is a graph showing the fruiting body weight according to the type of hardwood species and nutrient sources of fruiting medium for generation.

8 is a photograph of a true needle mushroom just before harvesting produced in Preparation Example 4.

Claims (5)

Fruiting body spawn of true needle strain, 0.3 to 3 parts by weight of at least one carbon source of maltose and sucrose, 0.02 to 0.2 parts by weight of at least one of asparagine and sodium nitrate, to 100 parts by weight of liquid or solid inoculation medium, and Inoculating agent preparation step of culturing the liquid inoculation or solid inoculation source by adding 0.02 to 0.2 parts by weight of at least one mineral of potassium phosphate and magnesium sulfate; 60 to 90 parts by weight of hardwood sawdust, rice bran, wheat flour, beer gourd and crumbs, based on 100 parts by weight of medium solids, 10 to 30 parts by weight of nutrient sources are mixed, and the water content is 55 to 70% by weight A culture medium preparation step of adding water so as to enter and sterilize; Spawn inoculation step of inoculating the culture or solid inoculation source into the culture medium; A culture step of incubating the spawn inoculated bottle medium in a culture chamber at 18 to 28 ° C. and humidity at 50 to 80%; A low temperature treatment step of treating the inoculated bottle medium inoculated through the culture step at 2 to 12 ° C. and a humidity of 50 to 80% for 6 to 12 days; And Cultivation method of a true needle mushroom, characterized in that it comprises a; mushroom generation step of generating a mushroom inoculated bottle medium inoculated through the low temperature treatment step 15 ~ 25 days at 14 ~ 20 ℃, humidity 85 ~ 99%. The method of claim 1, wherein the hardwood is any one of oak, cherry, maple, alder, mulberry, beech, cypress and poplar. The liquid inoculation medium of claim 1, wherein the carbon source, nitrogen source, and mineral are dissolved in distilled water, and 5 to 20 g of glucose, 5 to 20 g of peptone, 10 to 30 g of malt extract, and 5 to 20 g of yeast extract are dissolved in distilled water. Method of cultivating true needle mushroom, characterized in that the glucose peptone medium adjusted to 1 L after the addition. The method of claim 1, wherein the solid inoculation medium is mixed with 60 to 90 parts by weight of hardwood sawdust and 10 to 30 parts by weight of rice bran per 100 parts by weight of medium solids, and then distilled water in which the carbon source, nitrogen source and minerals are dissolved is added. , Cultivation method of true needle mushroom, characterized in that the moisture content is adjusted to 55 to 70% by weight based on the total weight of the medium. The method of claim 4, wherein the hardwood is any one of oak, cherry, maple, mulberry, weft pine, beech, and roe.

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