CN112166952A

CN112166952A - Cordyceps militaris culture method and application thereof

Info

Publication number: CN112166952A
Application number: CN202011107542.0A
Authority: CN
Inventors: 陈剑清; 盖其静
Original assignee: Hangzhou Kedisai Biological Technology Co ltd
Current assignee: Hangzhou Kedisai Biological Technology Co ltd
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2021-01-05

Abstract

The invention discloses a cordyceps militaris culture method and application thereof, and the method comprises the following steps: preparing a culture medium: weighing each component of the culture medium, adding water, mixing, fixing volume, packaging, sterilizing under high pressure, and cooling; inoculation: inoculating cordyceps militaris strains into a culture bottle filled with cordyceps militaris culture medium in a super clean bench; culturing: comprises A dark culture and B light culture; harvesting and re-culturing: after illumination culture, taking out the mature cordyceps militaris sporocarp in the culture bottle, and continuously culturing the rest part of cordyceps militaris sporocarp according to an illumination culture method adopted by 20-35 days until the sporocarp is mature.

Description

Cordyceps militaris culture method and application thereof

Technical Field

The invention belongs to the field of edible fungus cultivation, and particularly relates to a cordyceps militaris cultivation method and application thereof.

Technical Field

Cordyceps militaris belongs to the phylum Eumycota (Fungi), Ascomycota (Ascomycota), Hypocreales (Hypocrea), Clavicipitaceae (Clavicipitaceae), and Cordyceps (Cordyceps). The cordyceps sinensis fungus beverage contains active ingredients such as cordycepin, cordycepic acid, adenosine and polysaccharide, has the effects of resisting tumor, inflammation and virus, enhancing the immunity of the organism and the like, is a medical and edible fungus with nourishing effect, has high medicinal value and nutritive value, is accepted by more and more consumers, and the demand is also increased year by year.

Cordycepin (cordycepin) also known as 3' -deoxyadenosine is an adenosine active substance separated from Cuningham in 1951, and has a molecular formula of C₁₀H₁₃N₅0₃Cordycepin is one of main effective components in cordyceps militaris, has the effects of resisting bacteria, viruses and tumors, resisting fatigue, aging and inflammation, improving the immunity of a human body, inhibiting the activity of monoamine oxidase, and has high medicinal value and wide pharmacological action.

Adenosine (Adenosine) is a type of nucleoside, consisting of ribose (ribofuranose) and a portion of adenine linked by a β -N9-glycosidic bond (β -N9-glycosidic bond). Adenosine plays a biochemical important role, including the transfer of energy in the form of Adenosine Triphosphate (ATP) or Adenosine Diphosphate (ADP), or the signaling of cyclic adenosine monophosphate (cAMP), among others. Adenosine is also an inhibitory neurotransmitter (adenosine), and may promote sleep. The content of adenosine in Cordyceps is higher than that of cordycepin, and adenosine is also the quality standard of Cordyceps (Chinese pharmacopoeia).

Due to the harsh growth environment and resource shortage of cordyceps sinensis, the demand of people on cordyceps militaris as an optimal substitute for cordyceps sinensis is gradually expanding, and researches on culture methods capable of improving the yield of cordyceps militaris sporocarp and the cordycepin content are more and more, but most of the researches focus on the yield of cordyceps militaris sporocarp or only focus on the cordycepin content of a single active ingredient, and neglect the adenosine content serving as the quality standard of cordyceps sinensis.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a cordyceps militaris culture method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a cordyceps militaris culture method is characterized by comprising the following steps: the method comprises the following steps:

1) preparing a culture medium: weighing and taking each component of a culture medium, wherein the culture medium comprises the following components: 10-50g/L of mulberry leaf powder, 50-200g/L of corn grit, 50-100g/L of wheat bran, 5-10g/L of peptone, 1.2-1.5g/L of monopotassium phosphate, 0.8-1.2g/L of sodium dihydrogen phosphate and 1g/L of magnesium sulfate, then adding water, uniformly mixing, fixing the volume to the required volume, subpackaging, sterilizing at 121 ℃ and 1.1MPa for 20-30min, and cooling for later use;

2) inoculation: inoculating cordyceps militaris strains into a culture bottle filled with the cordyceps militaris culture medium in a super clean bench;

3) culturing: a dark culture: culturing in the dark at the temperature of 22 ℃ and the humidity of 75-85% for 3-5 days until cordyceps militaris hyphae overgrow the surface of the cordyceps militaris culture medium; b, light culture: and (3) illumination culture adopted in 5-20 days: in 12 hours in the daytime, the fluorescent lamp is used for illumination, the temperature is 20-22 ℃, the humidity is 80-85%, and the illumination intensity is controlled to be 100-150 Lx; keeping out of the sun at 12 hours at night, wherein the temperature is 18-20 ℃, and the humidity is 75-85%; and (3) illumination culture adopted on 20 th to 35 th days: illuminating by natural light and a fluorescent lamp within 12 hours in the day at the temperature of 20-22 ℃ and the humidity of 75-85 percent, and controlling the total illumination intensity at 1000-1200 Lx; closing light at 12 hours at night, wherein the temperature is 18-20 ℃, and the humidity is 75-85%;

4) harvesting and re-culturing: step 3), after illumination culture, taking out the mature cordyceps militaris sporocarp in a culture bottle, and continuously culturing the rest part of cordyceps militaris sporocarp until the sporocarp is mature by the method of the step 3) for 20-35 days;

preferably, the concentration of each component of the culture medium is as follows: 30g/L of mulberry leaf powder, 100g/L of corn grit, 50g/L of wheat bran, 10g/L of peptone, 1.5g/L of monopotassium phosphate, 1g/L of sodium dihydrogen phosphate and 1g/L of magnesium sulfate.

Preferably, the water is distilled water or deionized water.

Preferably, the cordyceps militaris strain in the step 2) is a cordyceps militaris liquid strain, the density of the cordyceps militaris liquid strain is 3-5 pellets/mL, and the volume ratio of the cordyceps militaris culture medium to the cordyceps militaris liquid strain is 50: 1.

Preferably, the fluorescent lamp illumination in step 3) is scattered light.

Preferably, in the step 3), during light culture, ventilation is performed for 2 times every day, and the ventilation is performed once in the morning and at night, and each time lasts for 20-30 minutes;

the application of the cordyceps militaris cultured by the cordyceps militaris culturing method in food, health-care products and food additives.

The beneficial effects are that: the cordyceps militaris sporocarp cultured by the culture method has high yield, high cordycepin and adenosine content, simple operation and easy popularization, and can meet the requirement of people on improving the content of various active components in cordyceps militaris sporocarp.

Drawings

FIG. 1 is an HPLC chromatogram for detecting an active substance in example 3 of the present invention.

In the figure, the peak No. 1 is adenosine, and the peak No. 2 is cordycepin.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The preparation of the cordyceps militaris culture medium comprises the following steps:

weighing and taking each component of a culture medium, wherein the culture medium comprises the following components: 10-50g/L of mulberry leaf powder, 50-200g/L of corn grit, 50-100g/L of wheat bran, 5-10g/L of peptone, 1.2-1.5g/L of monopotassium phosphate, 0.8-1.2g/L of sodium dihydrogen phosphate and 1g/L of magnesium sulfate, weighing the solid components according to the proportion shown in the following table 1, adding water, uniformly mixing, fixing the volume to the required volume, subpackaging, sterilizing at 121 ℃ and 1.1Mpa for 20-30min, and cooling for later use. This example specifically obtained 3 flasks of 50ml each of 5 different media as shown in Table 1.

TABLE 1 concentration of each component contained in different media (unit g/L)

1. The wheat bran is rich in protein, mineral, vitamin, carbohydrate and dietary fiber. It has high content of coarse fiber and protein and high quality. The amino acid composition is relatively balanced, wherein the contents of lysine, tryptophan and threonine are relatively high, the contents of vitamin B group and vitamin E of wheat bran are extremely high, various nutrient components can be provided, the culture medium can be loosened, and the growth of mycelium of cordyceps militaris at the early stage of culture is facilitated;

2. china is a big country in agriculture and silkworm industry, and wheat bran, corn and mulberry leaves are high in yield, easy to obtain and low in cost;

3. the cordyceps militaris fruiting body obtained by the culture method provided by the invention has high cordycepin adenosine content, high fruiting body yield and stable amount, and the method is simple and easy to popularize.

Example 2

The culture of the cordyceps militaris comprises the following steps:

1. inoculating Cordyceps mycelia (Cordyceps militaris (L) Link) in a super clean bench, controlling the density of liquid strains to be 3-5 pellets/mL, culturing the strains in each culture bottle for 1mL in a dark place for 3-5 days under the conditions of 22 ℃ of temperature and 75-85% of humidity, and enabling the Cordyceps mycelia to quickly grow on the surface of a culture medium;

2. culturing in the illumination mode on day 5-20, illuminating with a fluorescent lamp for 12 hours in the day at 20-22 ℃ (preferably 22 ℃), at 80-85% humidity, and controlling the illumination intensity at 100-150 Lx; and (3) keeping away from light for 12 hours at night, wherein the temperature is 18-20 ℃ (preferably 18 ℃), and the humidity is 75-85%. Ventilating for 2 times every day, and ventilating for 20-30 minutes each time in the morning and at night when the temperature is relatively low. The temperature difference is favorable for the differentiation and formation of primordia in the daytime and at night;

3. illuminating (natural light and fluorescent lamp) for 12 hours in the day at 20-35 days at 20-22 ℃ (preferably 22 ℃), humidity is 75-85%, and illumination intensity is controlled to be 1000-1200 Lx; and closing light at night for 12 hours at the temperature of 18-20 ℃ (preferably 18 ℃), and at the humidity of 75-85%. The time is the growth period of the sporocarp, strong light is beneficial to the growth of the sporocarp, the sporocarp grows in a rod shape, is continuously mature, robust and has bright orange color, ventilation is performed for 2 times every day in the growth period, the ventilation is performed once in the morning and at the evening, and each time is performed for 20-30 minutes when the temperature is relatively low;

4. harvesting and re-culturing, picking mature cordyceps militaris sporocarp (only picking part on the culture medium), continuously culturing the rest part in the culture bottle according to the culture condition and method of 20-35 days, and harvesting again.

Example 3

Determination of cordycepin and adenosine

Oven drying collected fruiting body (water content below 5%), grinding into fine powder, dissolving in water, ultrasonic extracting for 30min, centrifuging at 12000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane. The content of cordycepin and adenosine in the extract was measured by HPLC method and the results are shown in table 2.

TABLE 2 culture of fruiting body with medium for each component adenosine and cordycepin content (average value)

According to the experimental results, the cordyceps militaris sporocarp cultured by the culture medium provided by the invention can effectively improve the contents of two active substances, namely cordycepin and adenosine. Wherein, the cordycepin content is generally higher than 0.6 percent and can reach 0.69 percent at most, the adenosine content can also reach more than 0.5 percent, and some is even higher than the cordycepin content, especially when the mulberry leaf powder content in the culture medium is 30g/L and the corn grit content is 100g/L, the effect is best when the wheat bran content is 50 g/L.

Example 4

The cordyceps militaris cultured by the cordyceps militaris culturing method provided by the invention is applied to the aspects of foods, health-care products, medicines, additives and the like.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention.

Claims

1. A cordyceps militaris culture method is characterized by comprising the following steps: the method comprises the following steps:

1) preparing a culture medium: weighing the components of the culture medium, adding water, mixing, fixing volume, packaging, sterilizing at 121 deg.C under 1.1Mpa for 20-30min, and cooling; the components of the culture medium comprise: 10-50g/L of mulberry leaf powder, 50-200g/L of corn grit, 50-100g/L of wheat bran, 5-10g/L of peptone, 1.2-1.5g/L of monopotassium phosphate, 0.8-1.2g/L of sodium dihydrogen phosphate and 1g/L of magnesium sulfate;

2) inoculation: inoculating cordyceps militaris strains into a culture bottle filled with cordyceps militaris culture medium in a super clean bench;

3) culturing: the method comprises A dark culture and B light culture, wherein the A dark culture comprises the following steps: culturing in dark at 22 deg.C and humidity of 75-85% for 3-5 days until Cordyceps militaris mycelia overgrow the surface of Cordyceps militaris culture medium;

b, light culture: and (3) illumination culture adopted in 5-20 days: in 12 hours in the daytime, the fluorescent lamp is used for illumination, the temperature is 20-22 ℃, the humidity is 80-85%, and the illumination intensity is controlled to be 100-150 Lx; keeping out of the sun at 12 hours at night, wherein the temperature is 18-20 ℃, and the humidity is 75-85%; and (3) illumination culture adopted on 20 th to 35 th days: illuminating by natural light and a fluorescent lamp within 12 hours in the day at the temperature of 20-22 ℃ and the humidity of 75-85 percent, and controlling the total illumination intensity at 1000-1200 Lx; closing light at 12 hours at night, wherein the temperature is 18-20 ℃, and the humidity is 75-85%;

4) harvesting and re-culturing: and 3) after illumination culture, taking out the mature cordyceps militaris sporocarp in the culture bottle, and continuously culturing the rest part of cordyceps militaris sporocarp until the sporocarp is mature according to the illumination culture method adopted in the 20 th to 35 th days in the step 3).

2. The method for culturing cordyceps militaris according to claim 1, wherein the concentration of each component of the culture medium is as follows: 30g/L of mulberry leaf powder, 100g/L of corn grit, 50g/L of wheat bran, 10g/L of peptone, 1.5g/L of monopotassium phosphate, 1g/L of sodium dihydrogen phosphate and 1g/L of magnesium sulfate.

3. The method for culturing Cordyceps militaris (L.) Link, as claimed in claim 1, wherein said water is distilled or deionized water.

4. The cordyceps militaris culture method according to claim 1, wherein the cordyceps militaris strain in step 2) is a cordyceps militaris liquid strain, the density of the cordyceps militaris liquid strain is 3-5 pellets/mL, and the volume ratio of the cordyceps militaris culture medium to the cordyceps militaris liquid strain is 50: 1.

5. The method for culturing Cordyceps militaris (L.) Link as claimed in claim 1, wherein the fluorescent lamp in step 3) is a scattered light.

6. The method for culturing cordyceps militaris according to claim 1, wherein ventilation is performed 2 times a day in the light culture in step 3), and the ventilation is performed once in the morning and at night, and each time lasts for 20-30 minutes.

7. An application of Cordyceps militaris cultured by the method of any one of claims 1 to 6 in food, health product and food additive.