阅读说明：本技术 一种富硒猴头菇的培育方法 (Cultivation method of selenium-rich hericium erinaceus ) 是由 刘寿峰 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种富硒猴头菇的培育方法,设计在猴头菇培育基质中添加抑菌成分,抑制杂菌,特别是芽孢杆菌类的生长,实现对猴头菇培育基质中杂菌的抑制效果,有助于液态菌液的应用和推广；同时为避免抑菌成分影响猴头菇菌种的生长,利用含有抑菌成分的培养基对猴头菇菌种进行梯度浓度的抗性驯化,以获得的具有抗性的猴头菌菌种进行培育,猴头菇菌丝体生长快,出菇早,产量高,培育出来的猴头菇中富硒含量高、质量高。(The invention discloses a cultivation method of selenium-rich hericium erinaceus, which is characterized in that antibacterial components are added into a hericium erinaceus cultivation matrix to inhibit the growth of mixed bacteria, particularly bacillus, so that the effect of inhibiting the mixed bacteria in the hericium erinaceus cultivation matrix is realized, and the application and popularization of liquid-state bacteria liquid are facilitated; meanwhile, in order to avoid the influence of the antibacterial components on the growth of the hericium erinaceus strains, the hericium erinaceus strains are subjected to resistance domestication with gradient concentration by using a culture medium containing the antibacterial components, so that the obtained hericium erinaceus strains with resistance are cultured, the hericium erinaceus mycelia grow fast, grow early and are high in yield, and the cultured hericium erinaceus are high in selenium content and quality.)

1. The cultivation method of the selenium-rich hericium erinaceus is characterized by comprising the following specific steps:

firstly, culturing hericium erinaceus hyphae:

taking hypha tissue from Hericium Erinaceus, transferring to sterilized PDA test tube, placing at 20-23 deg.C, culturing for 8-14 days without illumination, allowing hypha to grow over the test tube, and purifying by rotating tube to obtain pure hypha, and storing;

(II) strain resistance domestication:

A. adding the antibacterial component into the strain culture medium at a ratio of 0.1-1.5%, and preparing a multi-stage strain culture medium in a mode of increasing the concentration of the antibacterial component;

B. sequentially culturing hericium erinaceus hyphae in a strain culture medium with gradually increased antibacterial component concentration, and performing multi-generation tolerance treatment to obtain a resistant hericium erinaceus strain;

C. carrying out expanded culture on the hericium erinaceus strain with resistance;

and (III) preparing selenium-rich bacterial liquid:

firstly, preparing a liquid culture medium rich in inorganic selenium, and inoculating part of hericium erinaceus strains obtained in the step (II) into the liquid culture medium for culture to obtain organic selenium hericium erinaceus bacterial liquid;

(IV) preparing the selenium-rich culture medium:

adding bacteriostatic components into the hericium erinaceus culture medium comprising a carbon source, a nitrogen source and a phosphate fertilizer, so that the content of the bacteriostatic components in the whole culture medium is kept at 1.0-1.5%, uniformly mixing, adding part of the organic selenium hericium erinaceus bacterial liquid obtained in the step (three), mixing and soaking for a period of time, and then airing to obtain a selenium-rich culture medium;

(V) inoculation and cultivation:

sterilizing the selenium-rich culture medium prepared in the step (four), cooling, inoculating the rest hericium erinaceus strains in the step (two), culturing, and continuing culturing hericium erinaceus after mycelia are mature.

2. The cultivation method of the selenium-enriched hericium erinaceus as claimed in claim 1, wherein the bacteriostatic component is a bacteriostatic concentrated solution obtained by extracting plants with bacteriostatic effects.

3. The cultivation method of selenium-enriched hericium erinaceus as claimed in claim 2, wherein the plants with bacteriostatic effect comprise one or more of garlic, persimmon peel, microcos paniculata and honeysuckle, and are extracted at 40-60 ℃.

4. The cultivation method of the selenium-rich hericium erinaceus as claimed in claim 3, wherein the extraction steps of the bacteriostatic concentrated solution are as follows: pulverizing the plants, grinding, sieving with 60-80 mesh sieve, heating to 40-55 deg.C with 75% ethanol solution, extracting at constant temperature for 2-3 times (each time for 1-2 hr), mixing filtrates, discarding residue, volatilizing ethanol at 40-55 deg.C with rotary evaporator, and concentrating to obtain antibacterial concentrated solution.

5. The cultivation method of the selenium-enriched hericium erinaceus as claimed in claim 1, wherein the bacteriostatic component is nisin, and the effective bacteriostatic concentration is 0.05-1% mg/ml.

6. The cultivation method of the selenium-enriched hericium erinaceus as claimed in claim 1, wherein in the step (two) of strain resistance domestication, the resistance concentration of the domesticated hericium erinaceus strain to the bacteriostatic components is as follows: 12 to 1.5 percent, and the adopted strain culture medium is a PDA culture medium.

7. The cultivation method of selenium-enriched hericium erinaceus as claimed in claim 1, wherein the inorganic selenium in the liquid medium in step (III) is one or a combination of sodium selenite and sodium selenate, and the concentration of selenium salt is 0.1-2 g/L.

8. The cultivation method of the selenium-rich hericium erinaceus as claimed in claim 1, wherein the cultivation method comprises the following steps of: the selenium content of the selenium-rich hericium erinaceus is 50-1000mg/kg (dry weight).

Technical Field

The invention relates to the technical field of hericium erinaceus cultivation, in particular to a cultivation method of selenium-rich hericium erinaceus.

Background

Selenium is a necessary trace element in human life activities, is called 'fire of life', has a tiny content in human bodies, but plays a great role in regulating and controlling human physiological functions and maintaining the optimal nutritional state of the human bodies. Selenium has effects in scavenging free radicals, resisting aging, and enhancing immunity. The absorption of the human body to the selenium is mainly obtained from food, the selenium content in the food determines the effect of supplementing the selenium, and in nature, some organisms have the function of enriching the selenium and can improve the selenium content of the food.

Hericium erinaceus is one of selenium-enriched fungi (see the paper of He Donlan in No. 4 of Hubei agricultural science 2005, "influence of growth of mycelia of Hericium erinaceus such as sodium selenite", and the paper of Shangde's quiet in No. 3 of 1999, "comparison of selenium-enrichment capacity of four edible fungi").

Hericium erinaceus is a famous fungus used as both food and medicine in China, is called as the king of mushroom, is combined with bear's paw, bird's nest and shark's fin into four famous vegetables, and is known as "mountain delicacies" from ancient times. The hericium erinaceus belongs to wood rot fungi, wild hericium erinaceus generally grow on dead trees, the artificially cultured hericium erinaceus mainly take solid raw materials such as wood chips, cottonseed hulls and corncobs as culture matrixes, and hericium erinaceus strains are inoculated to the culture matrixes after sterilization treatment.

The production mode of the hericium erinaceus strain mainly comprises 2 types, one type is traditional solid strain, the other type is liquid strain, compared with the solid strain, the liquid strain has many advantages, for example, the liquid strain has no grade, not only can be used as mother strain to be inoculated into an original strain culture medium, but also can be used as cultivated strain to be directly inoculated into a cultivation bag, the production process is simplified, and the production cost is reduced. However, at present, liquid strains are easy to infect mixed bacteria in an inoculating loop section, so that the mixed bacteria in a culture medium grow, the generation of hypha of hericium erinaceus is influenced, and the application and popularization of the liquid strains in the cultivation process of the hericium erinaceus are limited.

Disclosure of Invention

In order to solve the problems in the technical background, the invention provides a cultivation method of selenium-rich hericium erinaceus, which is designed by adding antibacterial components into a hericium erinaceus cultivation matrix to inhibit the growth of mixed bacteria, particularly bacillus, and simultaneously, in order to avoid the antibacterial components from inhibiting the growth of hericium erinaceus mycelium, resistance domestication is carried out on hericium erinaceus strains, and then cultivation of the hericium erinaceus is carried out on the basis of the strains with resistance.

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

the cultivation method of the selenium-rich hericium erinaceus is characterized by comprising the following specific steps:

firstly, culturing hericium erinaceus hyphae:

taking hypha tissue from Hericium Erinaceus, transferring to sterilized PDA test tube, placing at 20-23 deg.C, culturing for 8-14 days without illumination, allowing hypha to grow over the test tube, and purifying by rotating tube to obtain pure hypha, and storing;

(II) strain resistance domestication:

A. adding the antibacterial component into the strain culture medium at a ratio of 0.1-1.5%, and preparing a multi-stage strain culture medium in a mode of increasing the concentration of the antibacterial component;

B. sequentially culturing hericium erinaceus hyphae in a strain culture medium with gradually increased antibacterial component concentration, and performing multi-generation tolerance treatment to obtain a resistant hericium erinaceus strain;

C. carrying out expanded culture on the hericium erinaceus strain with resistance;

and (III) preparing selenium-rich bacterial liquid:

firstly, preparing a liquid culture medium rich in inorganic selenium, and inoculating part of hericium erinaceus strains obtained in the step (II) into the liquid culture medium for culture to obtain organic selenium hericium erinaceus bacterial liquid;

(IV) preparing the selenium-rich culture medium:

adding bacteriostatic components into the hericium erinaceus culture medium comprising a carbon source, a nitrogen source and a phosphate fertilizer, so that the content of the bacteriostatic components in the whole culture medium is kept at 1.0-1.5%, uniformly mixing, adding part of the organic selenium hericium erinaceus bacterial liquid obtained in the step (three), mixing and soaking for a period of time, and then airing to obtain a selenium-rich culture medium;

(V) inoculation and cultivation:

sterilizing the selenium-rich culture medium prepared in the step (four), cooling, inoculating the rest hericium erinaceus strains in the step (two), culturing, and continuing culturing hericium erinaceus after mycelia are mature.

Further, the bacteriostatic component is a bacteriostatic concentrated solution, and the bacteriostatic concentrated solution is a concentrated solution obtained by extracting plants with bacteriostatic effects.

Further, the plant with antibacterial effect comprises one or more of Bulbus Allii, cortex kaki, folium Microcoris Paniculatae, and flos Lonicerae, and is extracted at 40-60 deg.C.

Furthermore, the extraction steps of the bacteriostatic concentrated solution are as follows: pulverizing the plants, grinding, sieving with 60-80 mesh sieve, heating to 40-55 deg.C with 75% ethanol solution, extracting at constant temperature for 2-3 times (each time for 1-2 hr), mixing filtrates, discarding residue, volatilizing ethanol at 40-55 deg.C with rotary evaporator, and concentrating to obtain antibacterial concentrated solution.

Further, the antibacterial component is nisin, and the effective antibacterial concentration is 0.05-1% mg/ml.

Further, in the step (two) of strain resistance domestication, the resistance concentration of the domesticated hericium erinaceus strain to the antibacterial components is as follows: 12 to 1.5 percent, and the adopted strain culture medium is a PDA culture medium.

Further, in the step (III), the inorganic selenium in the liquid culture medium is one or the combination of sodium selenite and sodium selenate, and the concentration of selenium salt is 0.1-2 g/L.

Further, cultivating to obtain mature selenium-rich hericium erinaceus: the selenium content of the selenium-rich hericium erinaceus is 50-1000mg/kg (dry weight).

Compared with the prior art, the invention has the following advantages:

according to the invention, antibacterial components are added into the hericium erinaceus culture medium to inhibit the growth of mixed bacteria, particularly bacillus, meanwhile, in order to avoid the antibacterial components from inhibiting the growth of hericium erinaceus mycelium, the hericium erinaceus strain is subjected to resistance domestication, and then the hericium erinaceus is cultured on the basis of the resistant strain, so that the hericium erinaceus mycelium grows fast, fruiting is early, the yield is high, and the cultured hericium erinaceus has high selenium content and high quality.

In the preparation process of the selenium-rich bacterial liquid, the sterile environment is difficult to achieve, the inoculation loop is influenced by the air environment of an inoculation place, the inoculation loop is easy to infect mixed bacteria, and the more easily infected mixed bacteria are mostly bacillus. In view of the above, the concentrated extract of garlic, persimmon peel, microcos paniculata, honeysuckle flower and the like contains effective components of antibacterial bacillus, for example, flavonoid and triterpenes contained in persimmon peel, microcos paniculata, honeysuckle flower and the like can achieve the effect of inhibiting the growth of bacillus, so that the effect of inhibiting the mixed bacteria in the hericium erinaceus culture medium is achieved, and the application and popularization of the liquid bacterial liquid are facilitated.

Drawings

FIG. 1 is a schematic representation of the zone of inhibition of a bacteriostatic component to Bacillus;

wherein, the diagram (a) is Bacillus cereus WR1, the diagram (b) is Bacillus subtilis WR2, the diagram (c) is Bacillus aryabhattai WR3, and the diagram (d) is Bacillus belgii WR 4;

the figure shows that the antibacterial component diluents added in the serial numbers 1-5 of the traditional Chinese medicine sensitive paper sheet respectively correspond to nisin, microcos paniculata, honeysuckle, garlic and persimmon peel.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Detailed Description

A cultivation method of selenium-rich hericium erinaceus comprises the following specific steps:

firstly, culturing hericium erinaceus hyphae:

taking hypha tissue from Hericium erinaceus, transferring to sterilized PDA test tube, placing at 22 deg.C, culturing for 10 days without illumination, allowing the hypha to grow over the test tube, purifying by rotating tube to obtain pure hypha, and storing;

(II) strain resistance domestication:

A. adding the bacteriostatic component into PDA strain culture medium at a ratio of 0.1-1.5%, and preparing multi-stage strain culture medium in a mode of increasing the concentration of the bacteriostatic component;

wherein, the concentration gradient of the bacteriostatic component in the PDA strain culture medium is as follows: 0.1-0.3%, 0.3-0.5%, 0.5-0.8%, 0.8-1.1%, 1.1-1.5%.

The culture conditions are as follows: the culture temperature of the PDA strain culture medium is 21 +/-1 ℃, and the culture time is 10 days.

Preparation of PDA culture medium: 100g of potato is taken and added with 1000mL of water for cooking, then filtration is carried out to obtain filtrate, 10g of glucose, 10g of agar, 0.2g of magnesium sulfate, 0.5g of dipotassium hydrogen phosphate, 3g of soybean meal, 2g of peptone and 1g of yeast powder are sequentially added into the filtrate, heating is carried out until dissolution is carried out, water is added until the volume is 1000mL, the obtained culture solution is sterilized for 40min at the temperature of 121 ℃, and then cooling is carried out until the temperature is 20 ℃ to obtain the PDA culture medium.

B. Sequentially carrying out multi-generation cultivation on hericium erinaceus hyphae through a PDA strain culture medium with gradually increased antibacterial component concentration, and carrying out multi-generation tolerance treatment to obtain a resistant hericium erinaceus strain;

C. carrying out expanded culture on the hericium erinaceus strain with resistance;

and (III) preparing selenium-rich bacterial liquid:

firstly, preparing a liquid culture medium rich in inorganic selenium, inoculating part of hericium erinaceus strains obtained in the step (II) into the liquid culture medium for culture to obtain organic selenium hericium erinaceus bacterial liquid;

the culture conditions are as follows: the culture temperature of the liquid culture medium is 20 ℃, and the culture time is about 12 d.

Preparation of liquid medium: the raw materials of each 1000mL of inorganic selenium culture medium comprise: 1g of bean foil powder, 20g of white sugar, 0.5g of peptone, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate and 180mg of inorganic selenium.

Wherein the inorganic selenium is sodium selenite, and the concentration of selenium salt is 1 g/L.

(IV) preparing the selenium-rich culture medium:

adding bacteriostatic components into the hericium erinaceus culture medium comprising a carbon source, a nitrogen source and a phosphate fertilizer, so that the content of the bacteriostatic components in the whole culture medium is kept at 1.0-1.5%, uniformly mixing, adding part of the organic selenium hericium erinaceus bacterial liquid obtained in the step (three), mixing and soaking for a period of time, and then airing to obtain a selenium-rich culture medium;

the hericium erinaceus culture medium comprises: 78% of wood chips, 20% of bran, 1% of cane sugar and 1% of gypsum powder, the water content is about 60%, and the pH is natural.

(V) inoculation and cultivation:

sterilizing the selenium-rich culture medium prepared in the step (four), cooling, inoculating the rest hericium erinaceus strains in the step (two), culturing, and continuing culturing hericium erinaceus after mycelia are mature.

Example 1

In the embodiment, the bacteriostatic component in the step (two) is a bacteriostatic concentrated solution obtained by extracting plants with bacteriostatic effects; the plant with antibacterial effect comprises one or more of Bulbus Allii, cortex kaki, folium Microcorii Paniculati, and flos Lonicerae;

the extraction steps of the bacteriostatic concentrated solution are as follows: pulverizing the plants, grinding, sieving with 80 mesh sieve, heating to 50 deg.C with 75% ethanol solution, extracting at constant temperature for 3 times, each for 1 hr, mixing filtrates, removing residue, volatilizing ethanol at 55 deg.C with rotary evaporator, and concentrating to obtain antibacterial concentrated solution.

(1) The experiment for verifying the bacteriostatic effect of the bacteriostatic concentrated solution and nisin on bacillus comprises the following steps:

respectively inoculating 4 kinds of bacillus on 4 solid culture medium plates in an aseptic environment of a clean bench, standing for 15min after inoculation to ensure that water on the plates is completely absorbed by agar, and sticking drug sensitive paper sheets respectively containing garlic, persimmon peel, microcos paniculata, honeysuckle extract concentrated solution and nisin diluent on the solid culture medium plates.

In addition, a blank drug sensitive paper sheet is pasted at the center of the flat plate and used as a control group, the paper sheets are distributed at equal intervals, the distance between the two paper sheets is not less than 24mm, and the distance between the paper sheets and the edge of the flat plate is not less than 15mm, so that the obtained inhibition zones are prevented from being crossed or incomplete.

The 4 kinds of bacillus are respectively as follows: bacillus cereus WR1, bacillus subtilis WR2, bacillus aryabhattai WR3, bacillus belgii WR 4.

The drug sensitive paper sheet comprises: the paper sheet is a special drug sensitive paper sheet with the diameter of 6.00mm and the water absorption capacity of 20 ul.

The drug sensitive paper sheet is added with concentrated extractive solution of Bulbus Allii, cortex kaki, folium Microcoris Paniculatae, and flos Lonicerae and dilute solution of nisin, and the concentration of diluted extract is 1.5-2%.

The results of the verification are shown in FIG. 1. As shown in the bacteriostatic zone in FIG. 1, the extractive solutions of garlic, persimmon peel, microcos paniculata and honeysuckle all have bacteriostatic effects on Bacillus cereus WR1, Bacillus subtilis WR2, Bacillus aryabhattai WR3 and Bacillus belgii WR 4.

Example 2

The cultivation method of the selenium-rich hericium erinaceus in the specific embodiment is used for cultivating the hericium erinaceus, the hericium erinaceus is used as an experimental group, hericium erinaceus (mycelium and fruiting bodies) in different periods are detected, the hericium erinaceus prepared by a conventional cultivation method is used as a control group for detection, and meanwhile, mixed bacteria in a cultivation matrix in the mycelium period are detected.

TABLE 1 detection results of Hericium erinaceus at different periods

According to the invention, antibacterial components are added into the hericium erinaceus culture medium to inhibit the growth of mixed bacteria, particularly bacillus, meanwhile, in order to avoid the antibacterial components from inhibiting the growth of hericium erinaceus mycelium, the hericium erinaceus strain is subjected to resistance domestication, and then the hericium erinaceus is cultured on the basis of the resistant strain, so that the hericium erinaceus mycelium grows fast, fruiting is early, the yield is high, and the cultured hericium erinaceus has high selenium content and high quality.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.