CN116555386A

CN116555386A - Screening culture medium and breeding method of strontium-rich black fungus strain

Info

Publication number: CN116555386A
Application number: CN202310633402.4A
Authority: CN
Inventors: 解修超; 孙娅; 王昌钊; 陈荣信; 宋玉; 乔艳明
Original assignee: Shaanxi University of Technology
Current assignee: Shaanxi University of Technology
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-08-08

Abstract

The application provides a screening culture medium and a breeding method of a strontium-rich black fungus strain, wherein the culture medium comprises a primary screening culture medium and a secondary screening culture medium, and the primary screening culture medium comprises potato leaching liquid, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and vitamin B ₁ Strontium salt and distilled water. The screening culture medium can effectively and accurately screen the black fungus strains, is suitable for breeding the black fungus strains with strontium-rich capability, is suitable for large-scale popularization and use, is simple and feasible, is effective, provides powerful support for enriching black fungus resources, and can also promote the added value of the black fungus resources.

Description

Screening culture medium and breeding method of strontium-rich black fungus strain

Technical Field

The application relates to the technical field of strain screening, in particular to a screening culture medium and a breeding method of a strontium-rich black fungus strain.

Background

Strontium is commonly present in nature and human tissue, is a trace element essential to the human body, has chemical properties similar to calcium, and is mainly involved in bone development, 99% of strontium in the human body being present in bone, and 0.7% in blood. In medicine, strontium has effects of promoting bone health, preventing cardiovascular diseases, regulating reproductive health, resisting dental caries, relieving inflammation, resisting oxidation, inhibiting fat, resisting cancer and diabetes, promoting angiogenesis, and protecting heart. Humans consume strontium mainly from drinking water and foods such as grains, nuts, leafy vegetables, dairy products, etc., but the strontium content in these foods is extremely low, and it is indeed difficult to fundamentally improve the strontium deficiency condition only by supplementing strontium with natural foods.

The black fungus is a delicacy food with high value for both medicine and food, is the second largest edible fungus in China, and has the effects of moistening lung, washing intestines and stomach, reducing cholesterol and the like. The traditional black fungus has single consumption form, simple and crude processing mode, and along with the continuous improvement of the requirements of people on food, the existing processing mode and processing products can not meet the requirements of people on the black fungus. Therefore, the method has positive significance for maximally developing the value of the black fungus, researching the strontium-rich characteristic of the black fungus, enriching the species diversity of the black fungus and developing the black fungus resource.

Disclosure of Invention

The application provides a screening culture medium and a breeding method of a strontium-rich black fungus strain, which are used for providing a screening culture medium capable of effectively screening the black fungus strain with strontium-rich capability, and a simple and feasible breeding method of the strontium-rich black fungus,

in a first aspect, the present application provides a screening medium for a strontium-enriched black fungus strain, comprising a primary screening medium and a secondary screening medium, wherein the primary screening medium comprises potato extract, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and vitamin B ₁ Strontium salt and distilled water;

the concentration of potato extract in the primary screening culture medium is 200-220 g/L, the concentration of glucose is 18-22 g/L, the concentration of peptone is 4-5 g/L, the concentration of potassium dihydrogen phosphate is 4.5-5.5 g/L, the concentration of magnesium sulfate heptahydrate is 3-3.5 g/L, and the concentration of vitaminElement B ₁ The concentration of the strontium ions is 8-10 mg/L and the concentration of the strontium ions is 220-1100 mg/L;

the re-screening culture medium comprises dry materials, strontium salt and water, wherein the dry materials comprise, by weight, 85-90 parts of sawdust, 8-14 parts of bran, 2-4 parts of soybean meal, 0.5-0.8 part of quicklime and 0.8-1.5 parts of gypsum powder;

the weight ratio of the dry material to the water is 1:1-1:1.2, and the weight ratio of the strontium salt to the dry material is (0.2-1.1) to 1000; the concentration of strontium salt is based on strontium.

The application provides a screening culture medium of a strontium-rich black fungus strain, which has the following advantages:

(1) The raw materials of each component of the primary screening culture medium are cheap and easy to obtain, the proportion is reasonable, the preparation is easy, the dosage of each component in the primary screening culture medium is low, and the screening cost can be saved to a great extent.

(2) The raw materials of each component of the re-screening culture medium are close to the components of the culture medium in actual cultivation, so that the re-screening culture medium not only can further screen the black fungus strains with excellent strontium-rich capability, but also can adapt to the actual cultivation environment, the survival rate of the actual cultivation of the strontium-rich black fungus strains is effectively improved, and the loss in cultivation is reduced.

In addition, the screening culture medium can effectively and accurately screen the black fungus strains, is suitable for breeding the black fungus strains with strontium-rich capability, and is suitable for large-scale popularization and use.

Optionally, the strontium salt is one or more of strontium chloride, strontium acetate, strontium citrate or strontium gluconate.

Optionally, the sawdust comprises coarse cyclobalanopsis glauca sawdust and fine cyclobalanopsis glauca sawdust;

the weight ratio of the coarse cyclobalanopsis glauca sawdust to the fine cyclobalanopsis glauca sawdust is 1:3-4;

the grain diameter of the crude cyclocarya paliurus sawdust is 0.6-0.8 cm; the grain diameter of the fine cyclobalanopsis glauca sawdust is 0.2-0.4 cm.

In a second aspect, the present application provides a screening medium of a strontium-rich black fungus strain, which is applied to screening of a strontium-rich black fungus strain, a strontium-rich flammulina velutipes strain, a strontium-rich morchella strain and other strontium-rich edible fungus strains.

The application of the screening culture medium of the strontium-rich black fungus strain is not limited to the screening of the strontium-rich black fungus strain, and the screening culture medium can be applied to the screening of other edible fungi, so that the application of the screening culture medium has positive significance in developing the application field of the culture medium of the application and popularizing the application of the screening culture medium of the application.

In a third aspect, the present application provides a method for breeding a strontium-rich black fungus strain, including the following steps:

a. inoculating the black fungus strain to be screened into the primary screening culture medium provided in the first aspect, culturing at a constant temperature of 26 ℃ in a dark place, performing primary screening, and selecting a strain with a hypha growth rate of more than 0.60cm/d as a second primary screening strain;

b. inoculating the second primary screening strain into a second primary screening culture medium, and carrying out shaking culture at a constant temperature and in a light-shielding state at 26 ℃ to carry out second primary screening; after the culture is finished, mycelium is obtained, and a strain with biomass more than 4.00g/L is selected as a re-screening strain;

c. packaging the re-screening culture medium provided in the first aspect into a bag material, inoculating the re-screening strain into the bag material, re-screening under the condition of keeping the temperature at 26 ℃ and avoiding light, and selecting the strain with the mycelium full bag time of less than 35 days as the strontium-rich black fungus strain.

The application provides a breeding method of a strontium-rich black fungus strain, which has the following advantages:

(1) The screening indexes are easy to control, the mycelium growth rate, the biomass of the mycelium and the bag filling time of the mycelium are used as the screening indexes, the screening indexes are easy to measure, and the measuring method is simple and easy to implement.

(2) The screening conditions are strict, the mycelium growth rate is more than 0.6cm/d, the mycelium biomass is more than 4.0mg/L, and the bag filling time of the strain is less than 35d, and a solid foundation is laid for screening the strontium-rich black fungus strain with strong strontium-rich capability, vigorous growth and high yield.

(3) The screening process is simple and easy, the method of the application breeds the black fungus strain with strong strontium-rich capability by carrying out primary screening and secondary screening on the original black fungus strain twice, and the breeding method with simple steps and easy operation is provided on the basis of ensuring the screening effect.

The method for breeding the strontium-rich black fungus is simple and feasible, and effective, provides powerful support for enriching the black fungus resources, and can improve the added value of the black fungus resources.

Optionally, the first preliminary screening comprises:

dividing the primary screening culture medium into a plurality of groups according to the content of strontium salt from low to high to obtain a first primary screening culture medium, respectively inoculating the black fungus strains to be screened into the first primary screening culture medium, and culturing at the constant temperature of 26 ℃ in a dark place for 8-10 d;

and selecting a first primary screening culture medium corresponding to the strain with the largest hypha growth rate as a second primary screening culture medium.

Optionally, the second primary screening is operated as:

the second primary screening strain is inoculated in a second primary screening culture medium, and shake culture is carried out for 8-10 days at a constant temperature of 150r/min in a shaking table at a temperature of 26 ℃ and in a dark place.

Optionally, obtaining mycelium includes:

separating the strain cultured by the second primary screen from the culture medium to obtain mycelium, flushing the mycelium for 3-5 times by distilled water, drying the mycelium in a 60 ℃ oven to constant weight, and weighing to obtain the dry weight of the mycelium, wherein the ratio of the dry weight of the mycelium to the volume of the culture medium is biomass.

Optionally, the inoculation amount of the re-screening strain is 8-9% of the weight of the bag material.

Alternatively, the dry weight of the bag is 500g and the package of the bag is 15cm by 30cm by 0.05mm polyethylene or polypropylene bag.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief description will be given below of the drawings that are needed in the embodiments or the prior art descriptions, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph of the hypha growth rate line during a first screening process for different strains according to one embodiment of the present application;

FIG. 2 is a bar graph of biomass during a second screening process for different strains according to one embodiment of the present application;

FIG. 3 is a graph showing the difference between the growth vigor of mycelia of the "ear 91" strain according to an embodiment of the present application.

FIG. 4 is a diagram showing the difference between the hyphae of the "New family" strain according to an embodiment of the present invention.

Detailed Description

For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application are clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without inventive effort, are also within the scope of the present application based on the embodiments herein.

the concentration of potato extract in the primary screening culture medium is 200-220 g/L, the concentration of glucose is 18-22 g/L, the concentration of peptone is 4-5 g/L, the concentration of potassium dihydrogen phosphate is 4.5-5.5 g/L, the concentration of magnesium sulfate heptahydrate is 3-3.5 g/L, and vitamin B ₁ The concentration of the strontium ions is 8-10 mg/L and the concentration of the strontium ions is 220-1100 mg/L;

In the application, the weight of the strontium salt in the re-screening culture medium is calculated according to strontium, and when the preparation is carried out, the strontium salt can be mixed with water and then is compounded with the dry material, so that the uniform mixing of the strontium salt and the dry material is ensured, and the strontium salt is uniformly distributed in the re-screening culture medium. In one implementation, the primary screening culture medium also contains selenium salt with the concentration of 8-10 mg/L of selenium ions, and the selenium salt can be sodium selenite or selenium yeast.

The strontium salt in the application can be inorganic strontium salt, namely strontium chloride and strontium carbonate, wherein the inorganic strontium salt is easy to ionize in water and the strontium ions are easy to be absorbed by bacterial strain cells; organic strontium salts such as strontium acetate, strontium citrate, strontium gluconate, etc. can also be used, and the organic acid radical in the organic strontium salts can be used as carbon source of bacterial strain cells or can be used for regulating pH value of culture medium.

In the application, sawdust is a main component of the re-screening culture medium, the cyclobalanopsis glauca is made of hard miscellaneous wood, the nutritional ingredients contained in the cyclobalanopsis glauca are more comprehensive than those contained in cork, and a certain amount of crude cyclobalanopsis glauca sawdust is contained in the sawdust, so that the permeability of the re-screening culture medium can be increased, the dissolved oxygen in the re-screening culture medium is improved, and the proliferation of strains is facilitated.

In the application, the first primary screening can detect the tolerance of the auricularia auricula strains to strontium ions, so that auricularia auricula strains suitable for growing in a strontium-containing culture medium are screened, and meanwhile, the initial breeding of the auricularia auricula strains with strontium-rich capability is realized.

The second primary screening uses a shaker to culture the strain screened by the first primary screening and measures the biomass thereof, since the biomass is more or less responsive to the yield of the final fruiting body, thereby screening strains having high yields.

In the process of re-screening, the used re-screening culture medium is the same as the culture medium for the actual culture of the strain, so that the re-screening process can reflect the condition of the strain in the actual culture process, the screening aims at selecting and breeding the normally-grown strontium-enriched strain in the actual production culture process, providing reference for the actual production of the strontium-enriched strain, and simultaneously, the strontium-enriched strain suitable for the actual culture environment can be selected, and the survival rate of the strain is improved.

Both inoculation and cultivation in the methods of the present application are performed under sterile conditions.

Optionally, the first preliminary screening comprises:

In the application, the first primary screening divides the primary screening culture medium into a plurality of groups according to the content of strontium salt from low to high, so that the first primary screening culture medium is obtained, the method can play a role in screening strains, and meanwhile, the strontium ion concentration most suitable for the growth of the strains can be selected. In the application, the primary screening culture medium is divided into a plurality of groups according to the content of strontium salt from low to high, and the optimal grouping mode is to group according to a certain strontium concentration gradient, wherein the number of groups is 2-5.

Optionally, the second primary screening is operated as: the second primary screening strain is inoculated in a second primary screening culture medium, and shake culture is carried out for 8-10 days at a constant temperature of 150r/min in a shaking table at a temperature of 26 ℃ and in a dark place.

In this application, in shaking culture process in shaking table, the culture medium is shaken, and the culture medium liquid level contacts with the air under dynamic condition, takes place the mass transfer with the air, is convenient for promote the dissolved oxygen content in the culture medium, consequently shaking culture can provide abundant oxygen for the growth of hypha in shaking table, does benefit to the growth of hypha.

Optionally, obtaining mycelium includes:

In the application, the size of biomass can reflect the weight of the mycelium fruiting body in the later stage, and strains are screened through the size of biomass, so that the strain with vigorous growth and high yield can be screened.

In the application, when the strain is inoculated in the material bag, the inoculation amount is suitable. Too high results in too fast consumption of nutrients in the bag material and too fast growth of hyphae, which results in strain dysplasia and even influences on screening results in the later stage of culture. Moreover, too high inoculum size is unfavorable for later ear-out, and can lead to smaller individual mature fruiting bodies and lower qualified product yield. If the inoculation amount is low, the strain grows to be full of bags for too long, and the screening result is affected.

In this application, adopt the bag material dry weight and the material bag of above-mentioned requirement, accord with the requirement of re-screening in this application, and can more accurate screening out the strontium-rich edible tree fungus strain that accords with the condition, and in use, the ductility in polyethylene material area is better, can prevent to take the cracked emergence that leads to the unrestrained adverse condition of culture medium at the culture process.

Example 1

A method for breeding a strontium-rich black fungus strain is as follows:

s101, preparing a culture medium. Selecting 200g of potato leaching solution, 20g of glucose, 5g of peptone, 5g of monopotassium phosphate, 3g of magnesium sulfate heptahydrate and vitamin B ₁ 10mg and divided into 5 groups, wherein the first group is added with 220mg of strontium salt, the second group is added with 440mg of strontium salt, the third group is added with 660mg of strontium salt, the fourth group is added with 880mg of strontium salt, and the fifth group is added with 1100mg of strontium salt. Distilled water was added to the material of each group of medium to a volume of 1000mL. Sterilizing the culture medium at 121deg.C under high temperature and high pressure for 30min, and pouring into flat plate for use to obtain the first primary screening culture medium.

Wherein the strontium salt is strontium chloride and the weight of the strontium salt is calculated by strontium.

S102, first preliminary screening. After activating the commercial black fungus strains, each strain is divided into 5 groups, and the 5 groups are respectively inoculated into a first primary screening culture medium sterilized in S101, and round fungus blocks with the diameter of 0.5cm are inoculated into each plate during inoculation. Culturing at 26 deg.C in dark for 8 days, measuring colony diameter of Auricularia auricula mycelium in each plate, and calculating mycelium growth rate. A strain having a hypha growth rate of more than 0.60cm/d was selected as the second primary strain. And simultaneously selecting a first primary screening culture medium corresponding to the strain with the largest hypha growth rate as a second primary screening culture medium. The hypha growth rate was calculated according to the following formula:

s103, second primary screening. Inoculating the second primary screening strain obtained in the step S102 into a second primary screening culture medium (taking 100mL of the second primary screening culture medium, adding the second primary screening culture medium into a triangular flask with the capacity of 250 mL), carrying out shaking culture on 2 round fungus blocks with the diameter of 0.5cm at the constant temperature of 26 ℃ and 150r/min for 10 days, separating mycelia from the second primary screening culture medium by adopting a vacuum filtration mode after the culture is finished, cleaning the mycelia for 3-5 times by adopting distilled water, drying the mycelia in a baking oven at 60 ℃ until the weight is constant, weighing to obtain the weight of the mycelia, wherein the ratio of the weight of the mycelia to the volume of the culture medium is the biomass, and selecting the strain with the biomass of more than 4.00g/L as a rescreening strain.

S104, preparing a rescreening culture medium. Selecting 86.5 parts of sawdust (the weight ratio of coarse cyclobalanopsis glauca sawdust to fine cyclobalanopsis glauca sawdust in the sawdust is 1:3), 10 parts of bran, 2 parts of soybean powder, 0.5 part of quicklime and 1 part of gypsum powder, and uniformly mixing to prepare a dry material; dissolving strontium salt in water to obtain strontium salt solution with the same strontium ion concentration as that in the second preliminary screening culture medium, uniformly mixing 1.1 times of the weight of the dry material with the strontium salt solution to obtain a secondary screening culture medium, subpackaging in 15cm x 30cm x 0.05mm polyethylene bags to obtain bags, wherein the weight of each bag is 500g, sterilizing the bags at 120 ℃ under high temperature and high pressure for 2.5h, and cooling to below 30 ℃ for standby.

S105, re-screening. Inoculating the re-screened strain into the bag material, culturing at 26 deg.c in the presence of light, and selecting the strain with full bag time less than 35 days as the strontium-rich black fungus strain.

Example 2

A method for breeding a strontium-rich black fungus strain is as follows:

s101, preparing a culture medium. Selecting 220g of potato extract, 22g of glucose, 4g of peptone, 4.5g of monopotassium phosphate, 3.5g of magnesium sulfate heptahydrate and vitamin B ₁ 8mg and divided into 4 groups, wherein 250mg of strontium salt is added to the first group, 500mg of strontium salt is added to the second group, and the third group750mg strontium salt and 1000mg strontium salt were added to the fourth group. Distilled water was added to the material of each group of medium to a volume of 1000mL. Sterilizing the culture medium at 121deg.C under high temperature and high pressure for 30min, and pouring into flat plate for use to obtain the first primary screening culture medium.

S102, first preliminary screening. After activating the commercial black fungus strains, each strain is divided into 4 groups, and the 4 groups are respectively inoculated into a first primary screening culture medium sterilized in S101, and round fungus blocks with the diameter of 0.5cm are inoculated into each plate during inoculation. Culturing at 26 deg.C in dark for 10 days, measuring colony diameter of Auricularia auricula mycelium in each plate, and calculating mycelium growth rate. A strain having a hypha growth rate of more than 0.50cm/d was selected as the second primary strain. And simultaneously selecting a first primary screening culture medium corresponding to the strain with the largest hypha growth rate as a second primary screening culture medium. The hypha growth rate was calculated according to the following formula:

S104, preparing a rescreening culture medium. Selecting 85 parts of sawdust (the weight ratio of coarse cyclobalanopsis glauca sawdust to fine cyclobalanopsis glauca sawdust in the sawdust is 1:3), 14 parts of bran, 4 parts of soybean powder, 0.8 part of quicklime and 0.8 part of gypsum powder, and uniformly mixing to prepare a dry material; dissolving strontium salt in water to obtain strontium salt solution with the same strontium ion concentration as the second primary screening culture medium, uniformly mixing the strontium salt solution with the same weight as the dry material to obtain a secondary screening culture medium, subpackaging in 15 cm-30 cm-0.05 mm polyethylene bags to obtain bags, wherein the weight of each bag is 500g, sterilizing the bags at 120 ℃ under high temperature and high pressure for 2.5h, and cooling to below 30 ℃ for later use.

S105, re-screening. Inoculating the re-screened strain into the bag material, culturing at a constant temperature of 26 ℃ and in a dark condition, and selecting the strain with the full bag time of less than 35 days as the strontium-rich black fungus strain.

Example 3

A method for breeding a strontium-rich black fungus strain is as follows:

s101, preparing a culture medium. Selecting 210g of potato extract, 18g of glucose, 4.5g of peptone, 5.5g of monopotassium phosphate, 3.2g of magnesium sulfate heptahydrate and vitamin B ₁ 9mg, and divided into 3 groups, wherein the first group was charged with 300mg of strontium salt, the second group was charged with 600mg of strontium salt, and the third group was charged with 900mg of strontium salt. Distilled water was added to the material of each group of medium to a volume of 1000mL. Sterilizing the culture medium at 121deg.C under high temperature and high pressure for 30min, and pouring into flat plate for use to obtain the first primary screening culture medium.

S102, first preliminary screening. After activating the commercial black fungus strains, each strain is divided into 3 groups, and the groups are respectively inoculated into a first primary screening culture medium sterilized in S101, and round fungus blocks with the diameter of 0.5cm are inoculated into each plate during inoculation. Culturing at 26 deg.C in dark for 9 days, measuring colony diameter of Auricularia auricula mycelium in each plate, and calculating mycelium growth rate. A strain having a hypha growth rate of more than 0.50cm/d was selected as the second primary strain. And simultaneously selecting a first primary screening culture medium corresponding to the strain with the largest hypha growth rate as a second primary screening culture medium. The hypha growth rate was calculated according to the following formula:

S104, preparing a rescreening culture medium. Selecting 90 parts of sawdust (the weight ratio of coarse cyclobalanopsis glauca sawdust to fine cyclobalanopsis glauca sawdust in the sawdust is 1:3.5), 8 parts of bran, 3 parts of soybean powder, 0.5 part of quicklime and 1.0 part of gypsum powder, and uniformly mixing to prepare a dry material; dissolving strontium salt in water to obtain strontium salt solution with the same strontium ion concentration as that in the second preliminary screening culture medium, uniformly mixing 1.05 times of the weight of the dry material with the strontium salt solution to obtain a secondary screening culture medium, subpackaging in 15cm x 30cm x 0.05mm polyethylene bags to obtain bags, wherein the weight of each bag is 500g, sterilizing the bags at 120 ℃ under high temperature and high pressure for 2.5h, and cooling to below 30 ℃ for standby.

S105, re-screening. Inoculating the re-screened strain into the bag material, wherein the inoculation amount is 8.5% of the weight of the bag material, culturing at a constant temperature of 26 ℃ in a dark condition, and selecting the strain with the full bag time less than 35 days as the strontium-rich black fungus strain.

Example 4

A method for breeding a strontium-rich black fungus strain is as follows:

s101, preparing a culture medium. Selecting 200g of potato leaching solution, 19g of glucose, 4g of peptone, 5.2g of monopotassium phosphate, 3g of magnesium sulfate heptahydrate and vitamin B ₁ 9.5mg and divided into 2 groups, wherein the first group was charged with 500mg strontium salt and the second group was charged with 1000mg strontium salt. Distilled water was added to the material of each group of medium to a volume of 1000mL. Sterilizing the culture medium at 121deg.C under high temperature and high pressure for 30min, and pouring into flat plate for use to obtain the first primary screening culture medium.

S102, first preliminary screening. After activating the commercial black fungus strains, each strain is divided into 2 groups, and the groups are respectively inoculated into a first primary screening culture medium sterilized in S101, and round fungus blocks with the diameter of 0.5cm are inoculated into each plate during inoculation. Culturing at 26 deg.C in dark for 8 days, measuring colony diameter of Auricularia auricula mycelium in each plate, and calculating mycelium growth rate. A strain having a hypha growth rate of more than 0.50cm/d was selected as the second primary strain. And simultaneously selecting a first primary screening culture medium corresponding to the strain with the largest hypha growth rate as a second primary screening culture medium. The hypha growth rate was calculated according to the following formula:

S104, preparing a rescreening culture medium. Selecting 87 parts of sawdust (the weight ratio of coarse cyclobalanopsis glauca sawdust to fine cyclobalanopsis glauca sawdust in the sawdust is 1:4), 12 parts of bran, 3 parts of soybean powder, 0.5 part of quicklime and 1.2 parts of gypsum powder, and uniformly mixing to prepare a dry material; dissolving strontium salt in water to obtain strontium salt solution with the same strontium ion concentration as that in the second preliminary screening culture medium, uniformly mixing 1.05 times of the weight of the dry material with the strontium salt solution to obtain a secondary screening culture medium, subpackaging in 15cm x 30cm x 0.05mm polyethylene bags to obtain bags, wherein the weight of each bag is 500g, sterilizing the bags at 120 ℃ under high temperature and high pressure for 2.5h, and cooling to below 30 ℃ for standby.

Example 5

A method for breeding a strontium-rich black fungus strain is as follows:

s101, preparing a culture medium. Selecting 200g of potato leaching solution, 20g of glucose, 5g of peptone, 5g of monopotassium phosphate, 3g of magnesium sulfate heptahydrate and vitamin B ₁ 10mg and divided into 5 groups, wherein the first group is added with 220mg of strontium salt, the second group is added with 440mg of strontium salt, the third group is added with 660mg of strontium salt, the fourth group is added with 880mg of strontium salt, and the fifth group is added with 1100mg of strontium salt. Distilled water was added to the material of each group of the medium to a volume of 1000mL, and sodium selenite was added to the medium of each group so that the concentration of selenium ions was 8mg/L. Sterilizing the culture medium at 121deg.C under high temperature and high pressure for 30min, and pouring into flat plate for use to obtain the first primary screening culture medium.

Example 6

A method for breeding a strontium-rich black fungus strain is as follows:

the rest of the procedure was as in example 5, except that selenium yeast was added to each set of the primary screening media so that the concentration of selenium ions was 8mg/L.

Experimental example 1

Actual screening experiments:

1) First primary screening:

selecting black fungus strains: new family, shennong A8, qin Shan 4#, LS256, ear 91 (deposited by the Shanxi edible fungi engineering center).

The above-mentioned black fungus strains to be selected were screened by the method of the first preliminary screening provided in example 1, 3 in parallel were set for each group, wherein the first preliminary screening results are shown in table 1 and fig. 1:

TABLE 1

As can be seen from the results in Table 1 and FIG. 1, the strains described above each had a difference in growth in the first primary screening medium at different strontium addition concentrations, wherein the growth rates of both the "ear 91" strain and the "New family" strain were less than 0.6cm/d for the strontium concentration treatment used in the experiment, indicating that the growth of both the "ear 91" strain and the "New family" strain was inhibited under strontium-containing conditions. Thus the "ear 91" strain and the "neo-family" strain were excluded by the first preliminary screening. For the three strains "Shennong A8", "Qin Shan # 4" and "LS-256", the growth rate was more than 0.6cm/d at low strontium ions (strontium ion concentration 220 mg/L), so that the strains "Shennong A8", "Qin Shan # 4" and "LS-256" after passing through the first primary screen were selected as the second primary screen strain.

For the "Shennong A8", "Qin Dan 4#" and "LS-256" strains, when the growth rate is maximum, the corresponding concentration of strontium ions in the first primary screening culture medium is 220mg/L, so that the first primary screening culture medium containing 220mg/L is selected as the second primary screening culture medium of the "Shennong A8", "Qin Shan 4#" and "LS-256" strains.

2) Second primary screen

Three strains were selected, "Shennong A8", "Qin Shan 4#" and "LS-256" and screened using the second primary screening method provided in example 1, with 3 replicates in each set, and the second primary screening results shown in FIG. 2.

As can be seen from the results of FIG. 2, the biomass of the "Qin Shan # 4" strain was 3.65g/L, which did not meet the screening conditions established by the method of the present application, and thus the "Qin Shan # 4" strain was excluded by the second primary screening. Two strains of Shennong A8 and LS-256 were selected as rescreening strains by the second primary screen.

3) Double screen

The "Shennong A8" and "LS-256" strains were screened using the re-screening method provided in example 1 above, and 30 bags were repeated for each test, with the results shown in Table 2:

TABLE 2

Strain name	Days per day of full bag
		Shennong A8	32±0.87
LS-256	38±1.42

As can be seen from the results of table 2, the shennong A8 is the strontium-rich black fungus strain that passed the screening this time through the re-screening.

Experimental example 2

1) Auricularia auricula yield measurement

The full-bag Shennong A8 strontium-rich black fungus strain screened in the example 1 is moved to an ear bed (in a greenhouse, full-day intermittent spray cultivation), the opening germination acceleration is concentrated in a room, the surface of the fungus bag is disinfected by 0.1% potassium permanganate solution, a small sterile knife is used for cutting a mouth, the length of the cut mouth is 0.5-1 cm, the germination acceleration treatment is carried out at the interval of 1.5-2 cm and 20-28 ℃, the fungus base is divided into beds after being formed, watering is carried out after 2-3 d, water is sprayed for 10min every 1h in the initial stage of fungus bud formation, water spraying time is sprayed for 15min every 4h when the fruiting body is slightly larger, and no water is sprayed at the night below 16 ℃. The water and ventilation quantity are gradually increased in the fruiting body growth stage, the water spraying state is mist water, and the relative humidity of air is kept at 90-95%.

Harvesting after fruiting body maturing, washing with pure water for three times, drying in a drying oven at 60deg.C to constant weight, and measuring dry weight.

Meanwhile, the non-screened Shennong A8 black fungus strain is subjected to germination acceleration and fruiting culture to maturity according to the method in experimental example 2, mature fruiting bodies are collected, dried and the dry weight is measured. The key time period and yield of the cultivation of the fruiting bodies of the auricularia auricula were measured, and the results are shown in Table 3.

TABLE 3 Table 3

As can be seen from the results in Table 3, the single bag of the Shennong A8 strontium-rich black fungus strain screened by the breeding method of the application has obviously higher yield than the non-bred Shennong A8 black fungus strain, and meanwhile, the method also shows that the screened black fungus strain has the advantage of high yield. The Shennong A8 strontium-rich black fungus bred by the method also has the advantages of short fungus base forming period and short fungus sheet maturing period.

2) Determination of total strontium content in wood ear fruiting body

Grinding dried fruiting bodies of the strontium-enriched agaric into powder, taking 0.1g (accurate to 0.0001 g) of the powder, placing the powder into a digestion tank, adding 7mL of concentrated nitric acid for microwave digestion, taking out the digestion tank after the temperature in the tank is reduced to below 80 ℃ after the digestion is completed as shown in a table 4, slowly opening a tank cover for exhausting, removing acid to 0.5-1 mL on an electric heating plate, transferring digestion liquid into a 50mL volumetric flask, washing the inner tank and an inner cover with ultrapure water, then transferring washing liquid into the 50mL volumetric flask, fixing the volume, shaking uniformly, and simultaneously making blank control. And (3) feeding the micro digestion liquid to an engine, and measuring the strontium content by adopting an inductively coupled plasma atomic emission spectrometer (ICP-AES).

TABLE 4 Table 4

Sequence number	Power supply power/W	Temperature/. Degree.C	Hold time/min
				1	1600	120	2
2	1600	160	3
				3	1600	175	25

Experiments prove that the total strontium content in the wood ear fruiting body is 226.29 +/-4.33 g/kg.

Experimental example 3

Ear 91 and the novel strains were cultured according to the first preliminary screening method described in example 5 and example 6 above, and the hypha growth rate was measured and the results are shown in Table 5.

TABLE 5

As can be seen from the results in Table 5, the addition of selenium salt with a concentration of 8mg/L of selenium ions to the first primary screening medium increased the growth rate of ear 91 in the strontium-containing medium, i.e., increased its tolerance to strontium ions, but the results in Table 5 show that ear 91 grown in the medium supplemented with selenium yeast clearly improved over ear 91 supplemented with sodium selenite. The "ear 91" and "New family" strains were cultured according to the second preliminary screening method of example 6 described above, and the biomass of the "ear 91" strain was found to be 5.23g/L; the biomass of the "New family" strain was 6.42g/L.

FIG. 3 shows that the concentration of strontium ions is 220/mg.L ^-1 In the case of culturing "ear 91" strain according to the first preliminary screening method of example 1, example 5 and example 6, the difference in hypha growth vigor is greatly shown in FIG. 4, which shows that the strontium ion concentration is 220/mg.L ^-1 In the meantime, the difference in hyphae growth vigor when "New family" strains were cultured according to the first preliminary screening breeding methods of example 1, example 5 and example 6 was greatly illustrated.

The practical data shows that the selenium ions are added into the primary screening culture medium of the application to help the growth of the auricularia auricula strains in the strontium-containing culture medium, the tolerance of the auricularia auricula strains to the strontium ions can be improved, and the tolerance of the selenium yeast to the strontium ions is improved to be better than that of the auricularia auricula strains.

Finally, it should be noted that the above embodiments are merely for illustrating the technical solution of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand; the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A screening culture medium of a strontium-rich black fungus strain is characterized by comprising a primary screening culture medium and a secondary screening culture medium, wherein the primary screening culture medium comprises potato leaching liquid, glucose, peptone, monopotassium phosphate, magnesium sulfate heptahydrate and vitamin B ₁ Strontium salt and distilled water;

the concentration of potato extract in the primary screening culture medium is 200-220 g/L, the concentration of glucose is 18-22 g/L, the concentration of peptone is 4-5 g/L, the concentration of potassium dihydrogen phosphate is 4.5-5.5 g/L, and the concentration of magnesium sulfate heptahydrate3 to 3.5g/L and vitamin B ₁ The concentration of the strontium ions is 8-10 mg/L and the concentration of the strontium ions is 220-1100 mg/L;

the re-screening culture medium comprises dry materials, strontium salt and water, wherein the dry materials comprise, by weight, 85-90 parts of sawdust, 8-14 parts of bran, 2-4 parts of soybean powder, 0.5-0.8 part of quicklime and 0.8-1.5 parts of gypsum powder;

the weight ratio of the strontium salt to the dry material is (0.2-1.1) to 1000; the weight of the strontium salt is calculated as strontium.

2. The screening medium of a strontium-rich black fungus strain according to claim 1, wherein the strontium salt is one or more of strontium chloride, strontium acetate, strontium citrate or strontium gluconate.

3. The screening medium of the strontium-rich black fungus strain according to claim 1, wherein the sawdust comprises coarse cyclobalanopsis glauca sawdust and fine cyclobalanopsis glauca sawdust;

the grain diameter of the coarse cyclobalanopsis glauca sawdust is 0.6-0.8 cm; the grain diameter of the fine cyclobalanopsis glauca sawdust is 0.2-0.4 cm.

4. A screening culture medium of strontium-rich Auricularia strain is used for screening strontium-rich Auricularia strain, strontium-rich needle mushroom strain, strontium-rich Morchella strain and other strontium-rich edible fungus strain.

5. A breeding method of a strontium-rich black fungus strain is characterized by comprising the following steps:

a. inoculating the black fungus strain to be screened into the primary screening culture medium according to any one of claims 1-3, culturing at a constant temperature of 26 ℃ in a dark place, performing primary screening, and selecting a strain with a hypha growth rate of more than 0.60cm/d as a second primary screening strain;

c. packaging the rescreening culture medium according to any one of claims 1-3 into a bag material, inoculating the rescreening strain into the bag material, rescreening under the conditions of constant temperature of 26 ℃ and light shielding, and selecting the strain with mycelium full bag time less than 35 days as the strontium-rich black fungus strain.

6. The method for breeding a strontium-rich black fungus strain according to claim 5, wherein the first preliminary screening comprises:

7. The method for breeding a strontium-rich black fungus strain according to claim 6, wherein the second preliminary screening is performed by:

and inoculating the second primary screening strain into the second primary screening culture medium, and shake culturing for 8-10 d at a constant temperature of 150r/min in a shaking table at a temperature of 26 ℃ and in a dark place.

8. The method for breeding a strontium-rich black fungus strain according to claim 5, wherein the obtaining mycelium comprises:

9. The method for breeding a strontium-rich black fungus strain according to claim 5, wherein the inoculation amount of the re-screening strain is 8-9% of the weight of the bag material.

10. The method for breeding a strontium-rich black fungus strain according to claim 5, wherein the dry weight of the bag material is 500g, and the package of the bag material is 15cm x 30cm x 0.05mm polyethylene or polypropylene bag.