CN109337895B

CN109337895B - Production method of high-quality high-yield selenium-rich hericium erinaceus strain

Info

Publication number: CN109337895B
Application number: CN201811422844.XA
Authority: CN
Inventors: 沙淼; 高雪; 谷玉秋
Original assignee: Wu Zitao; Shanghai Zijing Science & Trade Co ltd
Current assignee: Shanghai Zijing Biotechnology Co.,Ltd.; Wu Zitao
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2021-07-27
Anticipated expiration: 2038-11-27
Also published as: CN109337895A

Abstract

The invention provides a production method of a high-quality high-yield selenium-rich hericium erinaceus strain, belonging to the field of edible fungus production. The method comprises the following process flows: step 1, preparing mother seeds; step 2, preparing shake flask strains; step 3, carrying out shake flask strain electric field and magnetic field co-operation treatment; and 4, producing the selenium-rich fermentation strain. The invention utilizes the liquid fermentation technology to produce the hericium erinaceus strain, changes the traditional solid production method of the hericium erinaceus strain, shortens the production period of the strain, improves the quality and the yield of the strain, reduces the cost, and simultaneously adds the selenium element in the liquid fermentation process to obtain the selenium-enriched hericium erinaceus strain with the organic selenium content. The strain has the advantages of quick fungus growth, good growth and high yield after inoculation, and the produced hericium erinaceus sporocarp has high organic selenium content and great market value.

Description

Production method of high-quality high-yield selenium-rich hericium erinaceus strain

Technical Field

The invention belongs to the field of edible mushroom production, relates to a production method of a high-quality high-yield selenium-rich hericium erinaceus strain, and particularly relates to a method for producing a selenium-rich hericium erinaceus strain by using a liquid fermentation technology.

Background

Hericium erinaceus (A)Hericium erinaceus) Hericium erinaceus, Hedgehog fungus, Cauliflower fungus or Hericium erinaceus belonging to the genus Hericium of the order Aphyllophorales, the family Hydnaceae, the class Basidiomycota, the class Hymenomycetes, the family Hymenomycetes, are often hung on the trunk of a tree because the fruit bodies are round and thick, and are covered with acicular fungus sticks, and the shape of the fruit bodies is very similar to the head of a monkey, so the Hericium erinaceus is obtained. The hericium erinaceus is fresh and tender in fungus meat, fragrant and delicious, is called meat in vegetables, and is called four traditional famous dishes of China together with bear paw, sea cucumber and shark fin. The hericium erinaceus is not only a delicious food material, but also a medicinal material. Chinese medicine believes that monkeyThe hericium erinaceus is neutral in nature and sweet in taste, enters the stomach channel, is beneficial to digestion, benefits five internal organs, supports the healthy energy, has the effects of nourishing brain and refreshing, and can be used for treating diseases such as dyspepsia, gastric ulcer, antral gastritis, stomachache, gastrectasia, neurasthenia and the like as modern medicine proves that the hericium erinaceus has good medicinal value.

The hericium erinaceus has high nutritional value, each 100g of the hericium erinaceus contains 23.6g of protein which is 2 times of that of the lentinus edodes, and the fat is 4.9g, so that the hericium erinaceus is a high-protein low-fat food which is worthy of name. In addition, the Hericium erinaceus is rich in carbohydrates, various vitamins and inorganic salts such as calcium, iron, magnesium, zinc, etc. The hericium erinaceus is rich in nutrition, and has the functions of enhancing immunity, resisting tumors, reducing blood sugar and blood fat, resisting oxidation, aging, radiation and mutation, inhibiting bacteria, protecting the liver, improving the hypoxia tolerance of the organism, resisting fatigue and the like. These functions are mainly the result of the action of active ingredients such as proteins, polypeptides, polysaccharides, terpenes, sterols, etc. The hericium erinaceus polysaccharide is one of the most studied active ingredients of hericium erinaceus at present. A large number of researches show that the hericium erinaceus polysaccharide can remove free radicals, has a remarkable antioxidant function, and is an ideal natural antioxidant resource. The Hericium erinaceus polysaccharide can also enhance human body immunity and inhibit cancer cell proliferation. The hericium erinaceus protein and the polypeptide also have an immunoregulation function and an antioxidant function, and compared with common fungi such as flammulina velutipes, lentinus edodes, agaricus bisporus, coprinus comatus and the like, the hericium erinaceus protein content is higher under the same quality, so that the immunoregulation function and the antioxidant function are also stronger.

Hericium erinaceus is a rare variety of edible fungi. Wild hericium erinaceus mostly grows in deep mountain dense forests, saprophytic in dead trees or dead parts of live trees, and is rare in plain and hilly areas. However, the wild hericium erinaceus is rare in quantity, and in order to meet market demands, the cultivation industry of the hericium erinaceus is developed very quickly, and the development situation at home and abroad is well received. The artificial cultivation of hericium erinaceus in China starts in 1959, and starts to be popularized and applied in the 70 th 20 th century, and currently, the hericium erinaceus is produced in each province in northeast and in Henan, Hebei, Tibet, Shanxi, Gansu, Shanxi, inner Mongolia, Sichuan, Hubei, Guangxi, Zhejiang and other provinces. The hericium erinaceus in China mainly adopts a traditional cultivation mode, fruiting is carried out under natural conditions by inoculating solid strains, but the cultivation mode is limited by factors such as natural climate, seasonality and the like, and the hericium erinaceus is low in mechanization degree, low in cultivation density, complex in seed production process, low in efficiency, incapable of realizing annual production and incapable of meeting the increasing consumption requirements. In recent years, the industrial cultivation of common edible fungi such as oyster mushroom, needle mushroom, seafood mushroom and the like is rapidly developed, the technology is gradually mature, and the scale is gradually enlarged, and liquid strains are produced by adopting a liquid fermentation technology and are used for replacing the traditional solid strains to be applied to the industrial cultivation of the edible fungi. The liquid seeds have the advantages of short production period, multiple hypha development points, quick germination, high yield, quick hypha spreading after inoculation, regular fungus age, low cost and the like, can effectively reduce pollution, and the application of the technology to the industrial cultivation of hericium erinaceus is bound to become a future development trend along with the improvement of the living standard of people and the improvement of the understanding of the health care effect of edible fungi.

Selenium is an indispensable trace element for human bodies, is an antioxidant with strong capability, can effectively remove carcinogenic factors, namely free radicals, and has the reputation of 'king of cancer resistance'. Selenium can improve human immunity, promote proliferation of lymphocyte and synthesis of antibody and immunoglobulin. Selenium has obvious inhibiting and protecting effects on colon cancer, skin cancer, liver cancer, breast cancer and other cancers, and has strong anticancer activity on an intermediate metabolite methyl selenol in a body. Selenium and vitamin E, allicin, linoleic acid, germanium, zinc and other nutrients have synergistic antioxidant effect and increased antioxidant activity. Meanwhile, selenium has the effect of relieving and alleviating heavy metal toxicity. Therefore, in recent years, selenium-rich foods and selenium-rich health care products are very hot. The edible fungi is used as a carrier for enriching selenium, and is widely applied in China due to wide cultivation area, simple and convenient operation and remarkable enrichment effect. The traditional hericium erinaceus selenium enrichment method is that selenium is added into a culture medium, and then selenium-enriched hericium erinaceus sporocarp is obtained. With the application of the edible fungus liquid fermentation technology, selenium element is added in the hericium erinaceus fermentation process, so that selenium-rich hericium erinaceus mycelium and fermentation liquid can be obtained, the selenium-rich hericium erinaceus mycelium and the fermentation liquid can be directly used for extracting active ingredients and serving as a nutritional food additive, and the selenium-rich hericium erinaceus can also be produced as a strain.

Disclosure of Invention

The invention aims to provide a method for producing high-quality and high-yield selenium-rich hericium erinaceus strains by using a liquid fermentation technology, so that the problems of long production period, low efficiency, low yield and high cost of hericium erinaceus in the prior art are solved, the content of organic selenium in the hericium erinaceus is increased, and the requirements of industrial cultivation of the hericium erinaceus and selenium-rich products in the market are met.

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

a production method of high-quality and high-yield selenium-rich hericium erinaceus strains is characterized by comprising the following process flows:

step 1, preparing mother seeds

Screening Hericium erinaceus excellent strains with high hypha growth speed and strong growth vigor, and scooping 1 block of Hericium erinaceus with an inoculating scoop under aseptic condition to obtain strain with size of about 0.5cm²Inoculating the strain blocks on a PDA enriched culture medium slant, culturing at 25 deg.C for 5-7d to obtain slant strain, transferring the slant strain onto a PDA enriched culture medium plate, and culturing at 25 deg.C until mycelia grow over the plate to obtain mother strain;

step 2, preparing shake flask strains

Taking 3-4 hericium erinaceus sheets from a flat-plate cultured hericium erinaceus mother strain by using a perforator with the diameter of 5mm, inoculating the hericium erinaceus mother strain into a first-stage shake flask filled with a liquid culture medium, and culturing to obtain a first-stage shake flask strain; then transferring the first-stage shake flask strain to a second-stage shake flask filled with a liquid culture medium according to the inoculation amount of 7-9% (v/v), and culturing to obtain a second-stage shake flask strain; finally, transferring the second-stage shake flask strain to a third-stage shake flask filled with a liquid culture medium according to the inoculation amount of 7-9% (v/v), and culturing to obtain a third-stage shake flask strain;

step 3, shake flask strain electric field and magnetic field combined treatment

Placing the prepared shake flask strain in a high-voltage electrostatic field of 100KV/m for treatment for 1min, and then placing the shake flask strain in a uniform magnetic field of 50mT for treatment for 5 min;

step 4, producing selenium-rich fermentation strain

Formula of fermentation medium: 30-36g/L of corn flour, 5-9g/L of casein, 15-20g/L of soybean straw powder, 6-10g/L of vinegar residue, 7-10g/L of corn cob powder, 5-6g/L of selenium-rich black fungus bran, 2.4-3.0g/L of citric acid, 6.8-7.4g/L of selenium-rich alfalfa powder and MgSO (MgSO)₄·7H₂O0.9-1.1g/L，KH₂PO₄ 1.8-2.2g/L，Na₂SeO₃37-43 mg/L, 1.4-2.0g/L of astragalus mongholicus dregs, 9-10g/L of lichen powder, 3.6-4.8g/L of fly maggot powder, 2.6-3.0g/L of oyster shell powder and VB₆50-70mg/L of glycerol, 1.7-2.3mL/L of glycerol, 0.25-0.35g/L of edible antifoaming agent and the balance of magnetized water, and the initial pH is adjusted to 5.3-5.7; preparing a fermentation culture medium according to the formula;

cleaning and air extinguishing of a fermentation tank: cleaning the fermentation tank, and sterilizing at 121-;

③ charging and sterilizing: adding prepared fermentation medium into the fermentation tank from a feeding port, wherein the content of the fermentation medium is 50-60% of the capacity of the fermentation tank, stirring for 2-3min by opening an air pump, and sterilizing for 40min at the temperature of 121-;

inoculating: after sterilization is finished, when the fermentation culture medium is cooled to be below 30 ℃, inoculating the treated shake flask strain into a fermentation tank from an inoculation port according to aseptic operation, wherein the inoculation amount is 11-13%;

fermenting and culturing: starting the fermentation tank to enter a culture state, setting the culture temperature to be 25-27 ℃, setting the rotating speed of the stirrer to be 140-160r/min, setting the ventilation rate to be 0.8 v/v/min in 1-3 days, and then setting the ventilation rate to be 1.2 v/v/min until the fermentation is finished, wherein the culture time is 6-8 d.

In the step 1, the PDA enriched medium comprises the following components: potato 180-220g/L, trehalose 17-23g/L, agar 18-22g/L, peptone 3-4g/L, lemon juice 3-5mL/L, malt extract 10-14mL/L, VB₁ 20-30mg/L，VB₂ 20-30mg/L，MgSO₄·7H₂O0.4-0.6g/L，KH₂PO₄0.8-1.2g/L, the balance of tap water, and the pH value is adjusted to 4.8-5.2.

In the step 2, the liquid culture medium comprises the following components: soybean starch 4-6g/L, hydrolyzed protein 0.6-1.0g/L, grape20-26g/L sugar, 14-18g/L sweet potato powder, 10-14mL/L malt extract, 8-9mL licorice juice, 1.4-2.0g/L astragalus residue and MnSO₄0.22-0.26g/L，MgSO₄·7H₂O0.4-0.6g/L，KH₂PO₄0.8-1.2g/L, 0.5-0.7mg/L of choline chloride, 1.3-1.5mg/L of IAA and the balance of tap water, and the pH is adjusted to 4.8-5.2.

In the step 2, the liquid loading amount of each level of shake flask strain is 150mL of liquid culture medium per 500mL of shake flask.

In the step 2, the culture conditions of all levels of shake flask strains are as follows: the temperature of the shaking table is 25-27 ℃, the rotation speed of the shaking table is 140-.

In the step 4, the aseptic operation during inoculation is to wipe the inoculation port with 75% ethanol, burn the inoculation port with 95% ethanol, encircle the inoculation port with flames, and quickly insert the rubber tube on the shake flask onto the inoculation port.

The magnetized water in the step 4 is tap water processed by the magnetic water processor, the model of the magnetic water processor is CFG/L-40, the flow rate is 7.0-9.0t/h, and the vertical central magnetic field intensity is 160 mT.

The beneficial effects of the invention are mainly embodied in the following aspects:

1) the hericium erinaceus strain is produced by a liquid fermentation technology, the production period is short, hyphae grow fast, the yield is high, meanwhile, selenium is added into a fermentation culture medium, the hyphae are absorbed and converted into organic selenium in the fermentation process, the selenium-enriched hericium erinaceus strain is obtained, and the selenium-enriched hericium erinaceus can be obtained after the strain is used for production. Compared with the traditional hericium erinaceus selenium enrichment method, the method disclosed by the invention is high in selenium element utilization rate and conversion rate, and high in organic selenium content in the strain, so that the organic selenium content in the produced hericium erinaceus sporocarp is also improved.

2) Aiming at the growth characteristics of the hericium erinaceus, the liquid fermentation culture process is optimized; the hericium erinaceus shake flask strain is subjected to electric field and magnetic field combined treatment, and magnetized water is used in the selenium-rich fermentation process, so that the accumulation of hericium erinaceus mycelium biomass can be promoted, and the selenium enrichment and conversion capacity of the hericium erinaceus mycelium can be improved.

3) When the selenium-rich hericium erinaceus strain is used for cultivating hericium erinaceus, the content of organic selenium in sporocarp is 87.84mg/kg, which is 3 times of that of the traditional selenium-rich hericium erinaceus, and the selenium-rich effect is obvious.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the present invention is further described with reference to the following specific embodiments.

Some of the indicators in the examples were determined as follows:

1) measurement of hyphal biomass: filtering 100mL of strain liquid, collecting mycelia, washing with distilled water for 3-4 times, centrifuging at 5000r/min for 10min, and separating precipitate from supernatant. And (3) putting the precipitate into an oven, drying at 80 ℃ to constant weight, and weighing the dry weight by an electronic balance.

2) And (3) measuring the diameter of the bacterial balls: 1mL of the inoculum solution was diluted 10-fold with water, 20 random pellets were aligned in a fixed line in a petri dish, the total length was measured with a caliper, and the total length was repeated 3 times, and the average value was taken.

3) And (3) determining the density of the bacteria balls: 1mL of the fermentation broth was diluted 10 times with water, and black square paper was placed under the petri dish to count the number.

4) And (3) total selenium content determination: grinding the mycelium dried to constant weight into uniform powder for digestion treatment. Accurately weighing 1g of the powder, adding 10mL of nitric acid, standing for more than 24h, heating at low temperature to completely dissolve the powder, slightly cooling, adding 3mL of perchloric acid, continuing to heat, cooling, and fixing the volume to 50 mL. Taking 10mL of digested sample, adding water to dilute to 40mL, adjusting the pH to 2-3, adding 2mL of EDTA solution with the mass fraction of 5%, then adding 2mL of 3, 3-diaminobenzidine solution with the mass fraction of 0.5%, and shaking up. And (3) taking out after reacting for 30min in a dark place, adjusting the pH value to 7, adding 10mL of toluene, performing oscillation extraction for 2min, standing for layering, placing the toluene layer into a cuvette, measuring absorbance at 430nm, obtaining corresponding concentration according to a selenium standard curve, and calculating the total selenium content in the hypha.

5) And (3) determining the content of organic selenium: accurately weighing 1g of the mycelium powder, adding distilled water to a constant volume of 50mL without digestion treatment, and determining the content of inorganic selenium according to the method for determining the total selenium content. Organic selenium content = total selenium content-inorganic selenium content.

Example 1

The test is carried out according to the production process of the high-quality high-yield selenium-rich hericium erinaceus strain provided by the invention, and the steps are as follows:

step 1, preparing mother seeds

Screening Hericium erinaceus excellent strain with high hypha growth speed and strong growth vigor, and taking 1 block of Hericium erinaceus with size of about 0.5cm under aseptic condition²Inoculating the strain blocks on a slant of a PDA enrichment medium, culturing for 6d at 25 ℃ to obtain slant strains, transferring the slant strains to a flat plate of the PDA enrichment medium, and culturing at 25 ℃ until hyphae grow over the flat plate to obtain mother strains. The components of the PDA enriched medium are as follows: 200g/L of potato, 20g/L of trehalose, 20g/L of agar, 3.5g/L of peptone, 4mL/L of lemon juice, 12mL/L of malt extract and VB₁ 25mg/L，VB₂ 25mg/L，MgSO₄·7H₂O0.5g/L，KH₂PO₄1.0g/L, the balance being water, the pH being adjusted to 5.0.

Step 2, preparing shake flask strains

Taking 3-4 pieces of Hericium Erinaceus mother seed with a perforator of 5mm in diameter, inoculating into liquid culture medium (containing soybean starch 5g/L, hydrolyzed protein 0.8g/L, glucose 23g/L, sweet potato powder 16g/L, malt wort 12mL/L, radix Glycyrrhizae juice 8.5mL/L, radix astragali residue 1.7g/L, MnSO₄0.24g/L，MgSO₄·7H₂O0.5g/L，KH₂PO₄1.0g/L, 0.6mg/L choline chloride, 1.4mg/L IAA and the balance of water, and the pH is adjusted to 5.0) to obtain a first-stage shake flask strain; then transferring the first-stage shake flask strain to a second-stage shake flask filled with a liquid culture medium according to the inoculation amount of 8% (v/v), and culturing to obtain a second-stage shake flask strain; finally, transferring the second-stage shake flask strain to a third-stage shake flask filled with a liquid culture medium according to the inoculation amount of 8% (v/v), and culturing to obtain a third-stage shake flask strain; the liquid loading of each stage of shake flask is 150mL of liquid culture medium per 500mL of shake flask, and the culture conditions of each stage of shake flask strain are as follows: the temperature of the shaking table is 26 ℃, the rotation speed of the shaking table is 150r/min, and the culture time is 5 d. And (4) after the culture is finished, determining hypha biomass, the diameter of the bacterial balls and the density of the bacterial balls of the strains in the shake flask.

Step 3, shake flask strain electric field and magnetic field combined treatment

The prepared shake flask strain is firstly placed in a high-voltage electrostatic field of 100KV/m for treatment for 1min, and then is placed in a uniform magnetic field of 50mT for treatment for 5 min.

Step 4, producing selenium-rich fermentation strain

Formula of fermentation medium: 33g/L of corn flour, 7g/L of casein, 17.5g/L of soybean straw powder, 8g/L of vinegar residue, 8.5g/L of corn cob powder, 5.5g/L of selenium-rich black fungus bran, 2.7g/L of citric acid, 7.1g/L of selenium-rich alfalfa powder and MgSO (MgSO)₄·7H₂O1.0g/L，KH₂PO₄ 2.0g/L，Na₂SeO₃40mg/L, 1.7g/L of astragalus root dregs, 9.5g/L of lichen powder, 4.0g/L of fly maggot powder, 2.8g/L of oyster shell powder and VB₆60mg/L, 2.0mL/L of glycerin, 0.3g/L of edible antifoaming agent and the balance of magnetized water, and the initial pH is adjusted to 5.5; preparing a fermentation culture medium according to the formula; the magnetized water is tap water processed by a magnetic water processor, the model of the magnetic water processor is CFG/L-40, the flow is 7.0-9.0t/h, and the vertical central magnetic field intensity is 160 mT;

inoculating: after sterilization is finished, when the fermentation culture medium is cooled to be below 30 ℃, inoculating the treated shake flask strain into a fermentation tank from an inoculation port according to aseptic operation (the inoculation port is wiped by 75% ethanol and is burnt by 95% ethanol, a flame ring is sleeved on the inoculation port, and a rubber tube on the shake flask is quickly inserted into the inoculation port), wherein the inoculation amount is 12%;

fermenting and culturing: starting the fermentation tank to enter a culture state, setting the culture temperature to be 26 ℃, setting the rotating speed of the stirrer to be 150r/min, setting the ventilation quantity to be 0.8 v/v/min on the 1 st to 3 rd days, and then setting the ventilation quantity to be 1.2 v/v/min until the fermentation is finished, and setting the culture time to be 7 d. And (4) after fermentation is finished, measuring the mycelium biomass, the total selenium content and the organic selenium content of the fermentation strain.

Example 2

To compare the effect of the liquid culture medium of the present invention and the conventional liquid culture medium on the growth of the shake flask strain of hericium erinaceus, the liquid culture medium used in step 2 of example 1 was replaced with the following conventional medium: medium A (glucose 30g/L, yeast extract 10g/L, KH g/L)₂PO₄ 1g/L、MgSO₄·7H₂O0.5g/L), Medium B (25 g/L of soluble starch, 25g/L, KH of Yeast extract)₂PO₄ 1g/L、MgSO₄·7H₂O1 g/L), medium C (maltose 20g/L, wheat bran 15g/L, peptone 20g/L, yeast powder 2g/L, KH g/L)₂PO₄ 1.5g/L、MgSO₄·7H₂O0.75g/L), other conditions were unchanged, and the hyphal biomass, pellet diameter and pellet density of the shake flask strains were measured and are statistically shown in Table 1 below, together with the results of example 1.

As can be seen from the results in Table 1, when the liquid medium of the present invention is used for shake flask culture, the mycelium biomass and pellet density of the shake flask strain of Hericium erinaceus are both maximized, and the pellet diameter is moderate. As a liquid strain, the larger the mycelium biomass is, the smaller the diameter of a mycelium pellet (1-2 mm) is, the more the mycelium pellets are, the more hypha germination points are in inoculation, the higher the hypha growth speed is, and the better the fermentation effect is. Compared with the conventional liquid culture medium, the liquid culture medium optimizes the components and the proportion of the carbon source and the nitrogen source, and is additionally added with the traditional Chinese medicine components and the growth promoter, so that the liquid culture medium has better effect of promoting the growth of the shake flask strains of the hericium erinaceus.

Example 3

1) In order to prove the positive influence of the electric field and magnetic field co-operation treatment on the selenium-rich fermentation strain, the following treatments were performed on the prepared shake flask strain in the present embodiment: 1) separate electric field treatment; 2) separate magnetic field treatment; 3) without treatment, the hyphal biomass, total selenium content and organic selenium content of the fermentation broths were determined and are counted as in table 2 below, together with the results of example 1.

As can be seen from the results in Table 2, the biomass of mycelia, the total selenium content and the organic selenium content of the fermented strain of Hericium erinaceus were all improved when the electric field and magnetic field co-treatment, the electric field alone treatment and the magnetic field alone treatment were carried out, as compared with the non-treatment. Compared with a single treatment mode, the electric field and magnetic field combined treatment effect is obvious, and the treatment mode is proved to be capable of exciting the hypha activity better when used for treating shake flask strains, and the hypha after inoculation is strong in growth vigor and vigorous in metabolism, so that the selenium enrichment capacity of the hypha is improved.

2) In order to prove the influence of using magnetized water to prepare a fermentation medium on the growth and selenium enrichment of hericium erinaceus fermentation strains, tap water is used to prepare the fermentation medium in the embodiment, other conditions are unchanged, the mycelium biomass, the total selenium content and the organic selenium content of the fermentation strains are measured, and the results of the example 1 are counted as the following table 3.

As can be seen from the results in Table 3, the biomass of mycelia, the total selenium content and the organic selenium content of the hericium erinaceus fermentation strain were improved by using the magnetized water, as compared with the case of using tap water to prepare the fermentation medium. Similar to the magnetic field treatment, the magnetized water also has the functions of stimulating the activity of hyphae, promoting the growth of hyphae and improving the selenium enrichment capacity of hyphae.

Example 4

In order to compare the effects of the fermentation medium of the present invention and the conventional fermentation medium on the growth and selenium enrichment of hericium erinaceus fermentation strains, the fermentation medium used in step 4 of example 1 was replaced with the following conventional medium: culture medium A (sucrose 30g, yeast extract 10g, corn flour 10g, KH)₂PO₄ 1g/L、MgSO₄·7H₂O0.5g/L), culture medium B (glucose 30g/L, bean cake powder 7g/L, wheat bran 2g/L, KH)₂PO₄ 1g/L、MgSO₄·7H₂O0.5g/L), Medium C (malt extract 30mL/L, peptone 20g/L, KH₂PO₄ 1.5g/L、MgSO₄·7H₂O0.75g/L), Na was added in the same manner₂SeO₃40mg/L, the hypha biomass, the total selenium content and the organic selenium content of the hericium erinaceus fermentation strain were measured without changing other conditions, and the statistics together with the results of example 1 are shown in Table 4 below.

The results in table 4 show that the hypha biomass, the total selenium content and the organic selenium content all reach the highest values by adopting the selenium-rich fermentation medium, which proves that the selenium-rich fermentation medium is not only beneficial to the accumulation of the hypha biomass, but also beneficial to the enrichment of selenium. The selenium-rich fermentation medium has rich nutrition, reasonable carbon source and nitrogen source proportion, and is added with Na₂SeO₃As an inorganic selenium source, raw materials with high selenium content, such as selenium-enriched black fungus chaff, selenium-enriched alfalfa powder, astragalus dregs, maggot powder, oyster shell powder and the like, are added as an organic selenium source and are compounded with other nutritional ingredients in a culture medium for use, so that the absorption and conversion of hericium erinaceus hyphae on selenium can be promoted, the growth of the hyphae can be promoted, and a higher effect is achieved.

Example 5

The embodiment is an optimization test of some important influence factors in the production process of the selenium-rich fermentation strain.

1) The influence of the culture temperature on the growth and selenium enrichment of hericium erinaceus fermentation strains is as follows: in the experiment, several gradient fermentation culture temperatures of 20 ℃, 23 ℃, 26 ℃ and 29 ℃ are set, the mycelium biomass, the total selenium content and the organic selenium content of the hericium erinaceus fermentation strain are respectively measured, and the statistics of the results are shown in the following table 5.

The results in table 5 show that the hericium erinaceus fermentation strain can grow in the set temperature range, when the temperature is low, the growth and the propagation of mycelia are slow, the biomass is low, when the temperature is too high, the selenium enrichment effect of the mycelia can be influenced, and when the temperature is 26 ℃, the mycelium biomass, the total selenium content and the organic selenium content reach the maximum, so that the temperature of 26 ℃ is selected as the optimal culture temperature.

2) Influence of the initial pH value of the fermentation medium on the selenium-rich fermentation of the hericium erinaceus liquid: in the test, initial pH values of 4.5, 5.5, 6.5 and 7.5 are set in a gradient manner, the hypha biomass, the total selenium content and the organic selenium content of the hericium erinaceus fermentation strain are respectively measured, and the statistics of the results are shown in the following table 6.

The growth of the mycelium is directly influenced by the initial pH value of the fermentation medium, and the proper pH value is favorable for the rapid growth and propagation of the mycelium. As can be seen from the results in Table 6, the hyphal biomass, the total selenium content and the organic selenium content all reached the maximum at an initial pH of 5.5, so that 5.5 was selected as the optimum initial pH.

3) The influence of the inoculation amount on the selenium-rich fermentation of the hericium erinaceus liquid is as follows: in the experiment, the inoculation amounts of 8%, 12%, 16% and 20% are set in a gradient manner, the mycelium biomass, the total selenium content and the organic selenium content of the hericium erinaceus fermentation strain are respectively measured, and the statistics of the results are shown in the following table 7.

The quantity of the inoculation amount directly influences the yield and the culture period of a target product, the growth of mycelium can be accelerated due to the large inoculation amount, so that the culture period is shortened, but the mycelium grows excessively, nutrient substances are consumed too fast, the late fermentation residual force is insufficient, and the accumulation of biomass and the synthesis of metabolites are finally influenced. As can be seen from the results in Table 7, when the inoculation amount was 12%, the mycelium biomass, the total selenium content and the organic selenium content of the fermented strain of Hericium erinaceus reached the maximum values, and thus 12% was selected as the optimum inoculation amount.

4) Na in fermentation Medium₂SeO₃Influence of concentration on selenium-rich fermentation of hericium erinaceus liquid: this experiment prepares Na₂SeO₃The fermentation culture media with the concentrations of 20, 40, 60 and 80mg/L respectively measure the mycelium biomass, the total selenium content and the organic selenium content of the hericium erinaceus fermentation strains, and the statistics of the results are shown in the following table 8.

As can be seen from the results in Table 8, the selenium content in the mycelia was varied with Na in the fermentation medium₂SeO₃The concentration is increased when Na is added₂SeO₃The concentration of the sodium hydroxide tends to be stable when reaching 40mg/L, and the concentration of the sodium hydroxide tends to be stable when Na is added₂SeO₃When the concentration reaches 60mg/L and higher, the hyphal biomass is reduced, indicating that the Na concentration is high₂SeO₃Can inhibit the growth of hericium erinaceus mycelium, but is not beneficial to the enrichment of selenium by the mycelium. When Na is present₂SeO₃When the concentration is 40mg/L, the mycelium biomass of the hericium erinaceus fermentation strain reaches the maximum value, so that the optimal Na is selected as 40mg/L₂SeO₃And (4) concentration.

Example 6

The selenium-rich hericium erinaceus strain is used for carrying out hericium erinaceus cultivation tests, and the cultivation method is as follows:

1) preparing a culture material according to the following mass ratio: 50% of grass meal, 26% of sawdust, 20% of wheat bran, 2% of gypsum powder, 1% of sugar and 1% of calcium superphosphate, wherein the main material is uniformly mixed, and then other auxiliary materials such as gypsum powder, sugar, calcium superphosphate and the like are dissolved in water and slowly sprayed into the culture material, wherein the material-water ratio is 1: 1.2-1.5, so that the water content reaches about 70%. The pH value of the culture material is controlled between 4 and 5.

2) The culture material is filled into a polyethylene plastic bag, the culture material is compacted during filling, sterilization is carried out under normal pressure after the culture material is pricked into the mouth, the sterilization is thoroughly carried out after the temperature of 100 ℃ is kept for 12 to 14 hours, and inoculation is carried out under aseptic condition when the temperature of the culture material is reduced to below 30 ℃. After inoculation, the fungus bag is moved into a culture room for fungus growth, the temperature in the culture room is controlled at 23-27 ℃, the air humidity is about 65%, and the temperature is reduced to about 20 ℃ in a period of strong hypha growth in a light-shielding culture. When the hypha of the fungus bag is basically full, the fungus bag is timely moved into a mushroom shed for bud forcing and fruiting.

3) The temperature of the mushroom shed is controlled to be 15-20 ℃ in the fruiting stage, the air humidity is controlled to be 85-90%, and scattered light irradiation of 200 and 400lx is given in the fruiting body growth stage.

4) When the hericium erinaceus grows for 7-10 days, the fungus spines are about 0.5cm long, and the hericium erinaceus is harvested before a large number of spores are ejected.

The total selenium content and the organic selenium content of the harvested selenium-rich hericium erinaceus and the traditional solid cultivation selenium-rich hericium erinaceus are measured, and the results are shown in the following table 9.

From the results in table 9, it can be seen that when hericium erinaceus cultivation experiments are carried out by using the selenium-rich hericium erinaceus strain disclosed by the invention, the total selenium content in the harvested hericium erinaceus sporophores is 90.38mg/kg, the organic selenium content is 87.84mg/kg, which is more than 3 times of the organic selenium content in the traditional selenium-rich hericium erinaceus, and the method disclosed by the invention is higher in selenium absorption rate and conversion rate and better in selenium-rich effect.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the invention are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A production method of high-quality and high-yield selenium-rich hericium erinaceus strains is characterized by comprising the following process flows:

step 1, preparing mother seeds

step 2, preparing shake flask strains

step 3, shake flask strain electric field and magnetic field combined treatment

step 4, producing selenium-rich fermentation strain

2. The production method of high-quality high-yield selenium-enriched hericium erinaceus strains according to claim 1, characterized in that in the step 1, PDA enriched culture medium comprises the following components: potato 180-220g/L, trehalose 17-23g/L, agar 18-22g/L, peptone 3-4g/L, lemon juice 3-5mL/L, malt extract 10-14mL/L, VB₁ 20-30mg/L，VB₂ 20-30mg/L，MgSO₄·7H₂O0.4-0.6g/L，KH₂PO₄0.8-1.2g/L, the balance of tap water, and the pH value is adjusted to 4.8-5.2.

3. The production method of high-quality high-yield selenium-enriched hericium erinaceus strains according to claim 1, characterized in that in the step 2, the liquid culture medium comprises the following components: 4-6g/L of soybean starch, 0.6-1.0g/L of hydrolyzed protein, 20-26g/L of glucose, 14-18g/L of sweet potato powder, 10-14mL/L of wort, 8-9mL/L of liquorice juice, 1.4-2.0g/L of astragalus mongholicus dregs, MnSO₄0.22-0.26g/L，MgSO₄·7H₂O0.4-0.6g/L，KH₂PO₄0.8-1.2g/L, 0.5-0.7mg/L of choline chloride, 1.3-1.5mg/L of IAA and the balance of tap water, and the pH is adjusted to 4.8-5.2.

4. The method for producing high-quality high-yield selenium-enriched hericium erinaceus strains according to claim 1, wherein in the step 2, the liquid loading amount of each level of shake flask strains is 150mL of liquid culture medium per 500mL of shake flask.

5. The production method of high-quality high-yield selenium-enriched hericium erinaceus strains according to claim 1, characterized in that in the step 2, the culture conditions of all levels of shake flask strains are as follows: the temperature of the shaking table is 25-27 ℃, the rotation speed of the shaking table is 140-.

6. The method for producing high-quality high-yield selenium-enriched hericium erinaceus strains according to claim 1, wherein in the step 4, the aseptic operation during inoculation is that the inoculation openings are wiped with 75% ethanol and burned with 95% ethanol, the inoculation openings are sleeved with flame rings, and rubber tubes on shake flasks are quickly inserted into the inoculation openings.

7. The production method of high-quality high-yield selenium-enriched hericium erinaceus strains according to claim 1, characterized in that the magnetized water in the step 4 is tap water treated by a magnetic water treatment device, the model of the magnetic water treatment device is CFG/L-40, the flow rate is 7.0-9.0t/h, and the vertical central magnetic field strength is 160 mT.