KR100762819B1 - A method for effective fruiting body production of paecilomyces japonica using bombus terrestris and preparation of healthy food comprising the same - Google Patents

Abstract

A method of massively producing Paecilomyces japonica is provided to produce quality Paecilomyces japonica using Bombus terrestris which can be in almost continuous production year-round. Paecilomyces japonica strains are subcultured on PDA medium at 4deg.C, inoculated into a culture medium, cultured at 23 to 25deg.C and 75% humidity for 7 to 15 days in a dark room to produce mycelium which is cultured at 18 to 20deg.C, 85% humidity and 100 to 500lux for 15 to 21 days to form fruit bodies. The culture medium is obtained by soaking PDA medium containing 88% by volume of Bombus terrestris, 10% by volume of rice bran and 2% by volume in water to a moisture content of 60 to 65%, sterilizing the PDA medium at 120 to 122deg.C and 14 to 16Psi for 15 to 25min and cooling to 15 to 20deg.C.

Description

A method for effective fruiting body production of paecilomyces japonica using bombus terrestris and preparation of healthy food comprising the same}

1 is a production process diagram of Bumblebee Cordyceps Sinensis

The present invention relates to a mass cultivation method of Cordyceps sinensis using a bumblebee, and more specifically to mass cultivation of Bumblebee Cordyceps by establishing optimum conditions necessary for cultivation of Cordyceps mycelia and fruiting bodies.

Cordyceps sinensis is a type of fungus that has been infected by insects, which is a kind of filamentous fungus, and is known to grow native to China, Nepal, Japan, South America, and Korea.

The path of infection of cordyceps relies mainly on transdermal infection caused by conidia scattering and attaching to the insect epidermis. After spores adhere to the surface, they germinate, penetrate into the insect's epidermis, and grow into mycelia in the inside. After death, hyphae in the body form fruiting bodies on the epidermis of host insects, and the color varies from species to Cordyceps as white, black, red and brown.

Cordyceps sinensis, which has the same physiological and ecological characteristics as above, has been the subject of much interest and research today because of its pharmacological effects on various diseases.

Cordyceps sinensis has not only been used as an herbal medicine since ancient China, but has also been favored as a valuable medicine for protecting the lungs and strengthening the kidneys. Cordyceps sinensis has been documented to be effective in a wide range of diseases by species. In addition, in the case of Japan, records of Cordyceps sinensis were transmitted to the herbaceous book in 1801, and were sold as a special medicine for lung disease and pleurisy at that time. In addition, according to recent studies in China and Japan, Cordyceps sinensis contains components that enhance immune function. When Cordyceps sinensis extract is administered to the human body, it significantly enhances immune function by promoting cellular immunity and humoral immunity. It has been shown to increase the resistance to infection.

There are hundreds of species of Cordyceps sinensis with these pharmacological components in the world, and many species are known to show different efficacy from each other, but it is expected to be expected as a raw material of natural medicine, but it is smaller and distributed than other mushrooms or medicinal plants. Its low density made it difficult to find and find it in the open mountains.

So, it was developed to artificially cultivate Cordyceps sinensis using silkworms, which gave great vitality to the sheep sleep industry. However, the mulberry cultivated land for silkworms is gradually reduced and this production is also produced only once a year. There was a problem.

The present invention is to enable the cultivation of Cordyceps sinensis by establishing the optimal conditions of mycelial culture and fruiting body culture using the bumblebee that can be produced year round to solve the above problems.

The male of the bumble bee (Bombus terrestris) is dense with yellow hairs, and the center of the breast distribution and the hair of the third back are black.

Adults usually appear from May to October.

The present invention purely isolates the superior strains of Cordyceps fungi subcultured in the PDA slope medium and inoculates the fungus inoculated into Bumblebee medium to incubate the mycelia in the dark room, lowering the temperature of the culture medium to produce fruiting bodies Through the use of bumblebees (ground bees) to achieve mass cultivation of Cordyceps sinensis.

The present invention to mass cultivate bumblebee cordyceps
A seed preparation step for subculture of pure Cordyceps fungi in a PDA slope medium for use while storing at 4 ° C;
PDB medium containing bumblebee 88%, rice bran 10%, and sugar 2% by volume ratio was immersed in water to make 60 ~ 65% water content in the culture vessel for cordyceps production. After inoculated with autoclave for 15 ~ 25 minutes at 14 ~ 16Psi ℃ and inoculated with Cordyceps spp. In a state of cooling to 15 ~ 20 ℃, and mycelial culture step for 7 ~ 15 days incubation at 23% ~ 25 ℃ with 75% humidity.
Cultivation of Cordyceps sinensis using amber bee consisting of the step of lowering Cordyceps under cultivation at the culture temperature of 23 ~ 25 ℃ to 18 ~ 20 ℃ and irradiating 100 ~ 500LUX of illuminance for 15 ~ 21 days to produce fruiting body while maintaining humidity 85% Way.
More detailed description as follows.

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end. Experimental Example

Mushroom Mushroom

The strains used in the present invention were subcultured at 1 month intervals on PDA (Potato Dextrose Agar) slope media by purely separating 7 strains of Cordyceps sinensis from the pre-cultivation strain of Gyeongbuk Agricultural Research Institute, Valley of Gyeongbuk Bonghwa and Cheongsong, and Yasan. It was used keeping at 4 degreeC.
Among them, mycelial growth rate and mycelial density were tested and two species (PJ-1: P. Japonica, CM-2: C, militaris) were selected and used as test strains.

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2. Solid Media Preparation and Culture

For solid plate culture and test tube spawning, PDA medium (39g / L) was used for 20 minutes sterilization at 121 ℃ for 20 minutes at petri dish in sterilized chamber.
On the other hand, the seedling for fruiting body culture was inoculated aseptically on a solid incubator by aliquoting the fruiting body, and in culture, the mycelia for fruiting body production were cultured by solid culture in a growth chamber at a temperature of 24 ± 1 ℃, humidity 60 ~ 65%.

3. Liquid medium production and culture

For liquid culture, PDA medium (39 g / L) was used, or 200 ml of modified medium was added to a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes. During liquid culture, the mycelium is collected from a solid plate or a slope medium using a mushroom cutting knife, etc., and added to a Ø18 mm test tube containing 5 mm glass beads, each of which is partially ground for 10 minutes in a voltex mixer for 7 days. The preculture (Vision Scientific Co. Ltd. rotary shaker temperature 25 ℃, rpm 200 capacity) was incubated at 5% spawn inoculum was used as the seed for the liquid culture experiment.

4. Fruiting body culture

Bumblebees are placed in a bottle or bag. (In a 850ml PP bottle, add 88g of bumblebee, 10g of rice bran, 2g of sugar, and add 2.5g of heat-resistant PP bag of 500g of bumblebee, rice bran, and sugar in the above ratio.) Seal and mix well, adjust the pH of medium to 6.5-7.0, sterilize for 15-25 minutes at 120-122 ℃, cool to 15-20 ℃, inoculate 10-20% of fungus subtilis and inoculate humidity 75%, temperature23 Incubate in dark for 7-15 days at ~ 25 ℃, lower the incubation temperature of 23 ~ 25 ℃ to 18-20 ℃, and adjust the humidity to 85% for 15 ~ 21 days of irradiation with light of 100 ~ 500LUX to produce 3 ~ 4cm fruiting body. When it is harvested.

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I. Establish optimal conditions for mycelial culture

 1. Review of mycelial growth (solid media)

In the case of PDA solid medium, the proliferation was induced on the 3rd day at 25 ° C and 60% humidity, and the rapid mycelial growth proceeded from the 5th day of culture.
Table 1

<Table 1> Primary Growth Patterns of Cordyceps Sinensis in Solid Medium

 Culture times (days)  0  2  4  5  6  7  8  9  10  11  Growth (mm)  12  12  36  45  68  75  80  84  87  89

2. Review mycelial growth

In the case of Paecilomyces japonica, success in cultivation of host cultures (insects, beans, rice and other grains, etc.) is considered to be one of the important success factors considering the affinity of the substrate and the degree of spawn status It is important to secure the quality and quantity of the spawn to increase the quality of the spawn, and for this, good spawn culture is an essential process, and it is closely related to the adaptation of the snow fungus fungus mushroom to the bumblebee and the success of the efficient production of fruiting bodies. As a result of reviewing the status and growth of the seed culture in the primary medium, the 7th day growth was the best in terms of quality and quantity (Table 2).

<Table 2> Primary Growth Patterns of Cordyceps Sinensis in Liquid Medium

 Days  0  One  2  3  4  5  6  7  8  9  SED (%)  5  5  20  70  90  100  100  100  100  100  PMV (%)  0  One  3  4  6  9  9  11.0  11.0  11.0

3. Effect of medium type

Test results for nutrients affecting mycelial growth

The best growth was found in PDA medium (Table 3).

<Table 3> Mycelial Growth and Density of Different Types of Cordyceps Mushrooms (mm / 10 Days)

 GPA  PDA  YM  MCM  CHA  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  60.5  ++  68.3  +++  56.5  ++  59.0  ++  57.0  +

GPA : Glucose Peptone Agar PDA: Potato Dextrose Agar YM : Yeast Extract Media MCM : Mushroom Complete Media CHA : Chapecks Agar

Mycelial density; + Low ++ medium +++ high

Petri dish (9㎝)

4. Effect of pH

The fungus mycelium showed the highest growth rate at 85.0 mm / 10 days at pH 6.0. Mycelial growth and density tended to decrease below pH 6.0, which is acidic rather than neutral (Table 4).

TABLE 4 Mycelial Growth and Density According to pH of Cordyceps Mushrooms (㎜ / 10 Days)

 pH 5.0  pH 5.5  pH 6.0  pH 6.5  pH 7.0  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  67.5  ++  80.5  ++  85.0  +++  84.0  +++  73.5  +++

Mycelial density; + Low ++ medium +++ high

Petri dish (9㎝)

PDA: Potato Dextrose Agar media

5. Effect of temperature

As a result of investigating the effect of temperature on mycelial growth of Cordyceps fungi, the mycelial growth and density were the best at 25 ℃. Mycelial growth was very weak at 30 ℃ and below 15 ℃, but it was very weak. Table 5

TABLE 5 Mycelial Growth and Density According to Temperature of Cordyceps Mushrooms (㎜ / 10 Days)

 5 ℃  0 ℃  15 ℃  20 ℃  25 ℃  30 ℃  35 ℃  40 ℃  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  Mycelial Growth  Mycelial Density  20.0  +  27.0  +  45.5  ++  65.3  ++  68.0  +++  60.0  ++  43.3  +  0  -

Mycelial density; + Low ++ medium +++ high

Petri dish (9㎝)

PDA: Potato Dextrose Agar media

6. Influence of dose

In order to increase the adaptability of the spawn, good spawn culture is an essential process to secure the quality and quantity of spawn, and it is closely related to the adaptation of the snow beetle fungus mushroom to the bumblebee and the success of the bee bee fungus fruit body production. As a result of examining the status and growth of seed culture based on the inoculation amount in the primary medium, the best results were obtained when the inoculation amount was 10% (Table 6). However, the proper inoculum for industrialization was able to induce normal growth at more than 5%.

<Table 6> Mycelial Growth according to Inoculation of Cordyceps Sinensis (PD)

 Inoculation amount (%)  0  One  2.5  5.0  7.5  10.0  15.0  SED (%)  5  5  20  70  90  100  100  PMV (%)  0  4  9  11  12  13  12

7. Effect of Addition of Glass Beads on Growth in Liquid Medium

In general, mushrooms belonging to fungi have a mycelial number due to sheare stress due to the addition of glass bead because the growth of pellets, which is characteristic of growth, causes inadequate mycelial growth in terms of securing the quality and quantity of spawn. As a result of the effect of the glass bead size and the number of additions to increase the results, the larger the glass bead size, the larger the addition amount was able to induce pulpy growth (Table 7.)

TABLE 7 Mycelial Growth and Morphological Changes According to the Number of Glass Bead Additions during Liquid Culture

All. Optimization of Pellet Formation Inhibition Condition of Mushroom Spawn

1. Influences of Growth on the Types of Surfactants

In order to increase the number of mycelia by inducing pulpy growth while minimizing aggregation between cells during elongation and isolation of cell walls during mushroom growth, the effects of anionic, cationic, positive and nonionic surfactants were examined. As a result, SED measurement was the highest when using Twin80, a nonionic surfactant whose hydrophilic group does not dissociate into ions when dissolved in water, resulting in the best results without inhibiting mycelial growth (Table 8).

TABLE 8 Effect of Surfactant Types on the Changes in Mycelial Form during Liquid Culture (PD)

※ Btac 22kc: Behenyl trimethyl ammonium chloride, Arlacel 60: Sorbitan stearate,

AE: Alkylated ethoxylate, Tween 60: Polysorbate 60, Tween 80: Polysorbate 80

LAS: Liner alkyl benzene sulfonate, SLES (70%): Sodium laureth sulfate

SCI: Sodium cocoyl isethionate, SLS: Sodium lauryl sulfate

2. Effect of Growth on the Concentration of Twin80

As a result of examining the optimum concentration of Twin80 in the concentration range where mycelia were not inhibited in liquid culture, the pellet formation rate was lower as the concentration increased during the experimental period. PMV was the highest (Table 4). However, when SED and PMV were low at 0.001%, growth inhibition was observed at concentrations higher than 0.0066%.

TABLE 9 Effect of Surfactant Twin80 Concentration on Mycelial Form Change in Liquid Culture (PD)

la. Establishment of Optimal Conditions for the Growth of Bumblebee Cordyceps Fruit Fruits
Example

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1. Cultivation of Bumblebee Cordyceps Sinensis

PDB (Potato Dextrose Broth) liquid medium was prepared and dispensed into 200ml triangular flasks (500ml), followed by autoclaving and autoclaving at 121 ℃ and 15psi for 20 minutes. Five mycelial fragments were administered as a 7 mm cork borer and used as a spawn for the Erlenmeyer flask culture.

Put in a rotary shaker culture incubator for 7 days at 25 ℃, 130rpm was used as a seed.

2. Detonation Badge

The medium to be used for experiments shall be frozen and stored bees, worker bees, and mixed bees at 0 ° C. When using, withdraw the bees that were previously stored frozen, thaw it for 30 minutes, adjust the moisture content to an average of 60%, and use 100g of medium in a PP bottle. After sterilization for 20 minutes at 121 ℃, 15psi, and then cooled to 20 ℃, inoculated with 10-20 ml of the seed was incubated for 15 days at 25 ℃ humidity 75% temperature.

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At this time, the mycelial growth was the most active among the mixed bees and worker bees with different sizes, and the mycelial growth was favorable in the order of bees and worker bees.

3. Production of Bumblebee Cordyceps Sinensis

As a result of lowering the bee with mycelial mycelium to 18 ± 1 ℃ at 25 ° C incubation temperature and maintaining 85% humidity, the PJ-1 strain induced fruiting by 15 ~ 21 days. Can produce.

On the other hand, CM-2 strains were produced in large quantities in 40 days, on average more than twice as long as PJ-1 strains.

<Liquid culture and fruiting body production of zucchini cordyceps fungi>

 Strain name  Optimum temperature for mycelial growth  Optimal condition for fruiting  Mycelial growth pH  Used badge Snow Cordyceps Sinensis ( Paecilomyces japonica )   Temperature: 23 to 25 ° C Humidity: 75% Incubation period: 7 to 15 days   Temperature: 18 to 20 ° C Humidity: 85% Incubation period: 15 to 21 days   6.5-7.0 (6.8) Pupa cordyceps ( Cordyceps militaris )   Temperature: 23 ~ 25 ℃ Humidity: 75% Cultivation period: 14 days   Temperature: 18 to 20 ° C Humidity: 85% Incubation period: 40 days   5.2 to 5.5   SPY

(Pecilomyces japonica)

 No   Fruiting body cultivation and production process  One    Water content control in bumblebee (initial water content 60 ~ 65%)  2    Prepare a medium by mixing at a volume ratio of bumblebee (88%) + rice bran (10%) + sugar (2%) ⇒ adjust the pH to 6.5-7.0  3    100 g of medium is placed in a heat-resistant PP bottle (clear, for 850 ml) and covered with a lid.  4    Put the PP bottle into the autoclave and sterilize (121 ℃ × 20 minutes)  5    Cool as soon as possible to cool the medium temperature in the bag to 15-20 ℃.  6    Inoculate 20ml of liquid spawn per bottle in aseptic box and close the lid.  7    Shake the bottle appropriately and mix so that the spawn is evenly spread on the medium.  8    Mycelial culture in bottles (23-25 ° C, 75% indoor relative humidity, dark): 7-10 days incubation  9    Mushroom growth in bottles (temperature 18-20 ℃, relative humidity of 85%, light irradiation): incubated for 15-21 days  10    Harvest is possible 27 ~ 35 days after spawn vaccination (experience / visual identification)  11    Sold as raw mushrooms in a bottle or packed / sold after being bottled and dried

Cordyceps bag cultivation method ((Paecilomyces japonica)>

 No  Fruiting body cultivation and production process  One    Water content in bumblebees (initial water content 60-65%)  2    Prepare medium by mixing bumble bee (88%) + rice bran (10%) + sugar (2%) ⇒ Adjust the medium to pH 6.5-7.0  3    500 g of medium is sealed in a heat-resistant PP bag (transparent, for 2.5 kg) with an air filter.  4    Put the bag into the autoclave and sterilize (121 ℃ × 20 minutes)  5    Cool as soon as possible to cool the medium temperature in the bag to 15-20 ℃.  6    Inoculate 100ml of liquid spawn per bag in aseptic box and close the lid. -Make the final medium 75% moisture.  7    Mix and mix the bag properly so that the spawn is evenly spread on the medium.  8    Mycelial culture in bags (23-25 ° C, 75% relative humidity, dark): 7-10 days incubation  9    Open the bag, crush the cultured mass, and spread it over a box of mineral wood.  10    Spray groundwater with a sprayer on the media and cover the box with vinyl.  11    Mushroom growth (Temperature 18 ~ 20 ℃, Relative Humidity 85%, Illumination 200 ~ 500lux Incubate for 15 ~ 21 days  12    The groundwater is sprayed at regular intervals to prevent the drywood on the bottom of the box from drying out.  13    After growing about 15 days 3-4cm in length, harvested and dried.    ※ Harvest after 30 ~ 35 days after spawn inoculation.

<Manufacturing healthy foods using zucchini bee cordyceps Example >

1. Manufacturing Process and Prototype of Health Food (Capsule)

Bumblebee Cordyceps Sinensis Contents (Freeze Dried) 200g + Water 25 Times (5L)

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2hr hydrothermal extraction

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After nonwoven filtration

8,000 rpm 20min centrifugation

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Concentrated under reduced pressure at 60 ℃

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Cordyceps Sinensis Concentrate

     (35 ° brix, 190ml)

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Drying and capsule preparation after addition of subsidiary materials

<Amber zucchini cordyceps capsule composition table>

2. In addition to the capsule manufacturing, the bumblebee cordyceps organism itself as it is / freeze-dried and commercialized, introduced into other insects for moisture and commercialization of application products, development of food materials and additives for food and beverage, X, powder and encapsulation It is also used as a pharmaceutical material through the development of food and extraction of bioactive substances.

As described above, the present invention enables the cultivation of mycelia and fruiting bodies by cultivating mycelial larvae under optimum conditions using the horn bee that enables year-round production. Rather, it will create new market demand for the beekeeping industry.

In addition, there is an advantage that can be utilized as a pharmaceutical material through the extraction of physiologically active substances as well as health foods using cultivated beeworm cordyceps.

Claims (4)

A seed preparation step for subculture of pure Cordyceps fungi in a PDA slope medium for use while storing at 4 ° C; PDB medium containing bumblebee 88%, rice bran 10%, and sugar 2% by volume ratio was immersed in water to make 60 ~ 65% water content in the culture vessel for cordyceps production. After inoculated with autoclave for 15 ~ 25 minutes at 14 ~ 16Psi ℃ and inoculated with Cordyceps spp. In a state of cooling to 15 ~ 20 ℃, and mycelial culture step for 7 ~ 15 days incubation at 23% ~ 25 ℃ with 75% humidity. Cultivation of Cordyceps sinensis using amber bee consisting of the step of lowering Cordyceps under cultivation at the culture temperature of 23 ~ 25 ℃ to 18 ~ 20 ℃ and irradiating 100 ~ 500LUX of illuminance for 15 ~ 21 days to produce fruiting body while maintaining humidity 85% Way delete delete delete

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