CN114891642A

CN114891642A - Preservation method and preservation culture medium for boletus nigricans strains

Info

Publication number: CN114891642A
Application number: CN202210385341.XA
Authority: CN
Inventors: 丁李春; 林旭; 陈慕松; 李志刚; 陈霞娜; 陈录安
Original assignee: NINGDE AGRICULTURAL SCIENCE RESEARCH INSTITUTE
Current assignee: NINGDE AGRICULTURAL SCIENCE RESEARCH INSTITUTE
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-08-12

Abstract

The invention discloses a preservation method and a preservation culture medium for boletus nigricans strains, and belongs to the field of fungus strain preservation. The culture medium for preserving boletus nigricans sclerotium comprises the following raw materials in parts by weight: 45-48 parts of wheat grains, 15-18 parts of miscellaneous wood chips, 15-17 parts of pine wood chips, 18-19 parts of bran, 1-2 parts of gypsum and 1-2 parts of white sugar. The invention also discloses a preservation method of the boletus aereus strain, which comprises the steps of inoculating the boletus aereus strain into the boletus aereus sclerotium preservation culture medium, culturing to generate sclerotium, and preserving at 15-18 ℃. The preservation method has the advantages of simple operation, easily obtained preservation culture medium components and low production cost, and can preserve the sclerotium activity and the excellent performance of the bolete for four years and half, solve the problem that the bolete is not easy to be preserved simply and conveniently for a long time, and provide technical guarantee for the mass subculture and propagation of the bolete.

Description

Preservation method and preservation culture medium for boletus nigricans strains

Technical Field

The invention relates to the field of fungus strain preservation, in particular to a preservation method and a preservation culture medium for boletus nigricans strains.

Background

The quality of the strains is not only directly related to the yield and quality of edible fungus cultivation, but also affects the success or failure of production. The boletus aereus strain is one of the key links of artificial cultivation and bionic cultivation. The boletus aereus belongs to high-temperature basidiomycetes, hyphae are sensitive to low temperature and can be dissolved by itself to die under the low-temperature condition, the optimal temperature for the growth of the hyphae is 28-30 ℃, the temperature is lower than 15 ℃ or higher than 30 ℃, the growth speed of the hyphae can be reduced rapidly, and the boletus aereus is difficult to preserve under the condition of 0-6 ℃ like other strains. Although the research on the low-temperature long-term preservation of the boletus nigricans strain exists in the prior art, the cold-cooling sectional treatment is usually required, or a plurality of protective agent components are additionally added, so that the defects of complex operation, cost increase and the like exist. In the prior production, the strain is preserved at 15-20 ℃ by a common test tube slant subculture preservation method, and the tube is rotated for 1 time every 3-6 months, so that the technology provides a simple and effective short-term preservation method, but the risk of the strain generating pollution and the variation of morphological and physiological characteristics is increased.

In addition, at present, the excellent boletus aereus strains are mostly obtained through natural breeding, namely, the strains are separated through widely collecting sporocarp in different regions, different growth environments and different development periods, systematic tests are carried out on the aspects of hypha growth speed, culture properties, adaptability of cultivation environment, early and late fruiting period, shape, size, color, texture and the like of each strain, and finally the excellent strains are screened out. Therefore, it is very important to design a method for simply and effectively preserving the boletus nigricans strain and maintaining the excellent properties of the boletus nigricans strain.

Disclosure of Invention

The invention aims to provide a preservation method and a preservation culture medium for boletus aereus strains, which are used for solving the problems in the prior art, can preserve the activity and the excellent performance of boletus aereus sclerotium for four years and a half, solve the problem that the boletus aereus is not easy to be preserved simply and conveniently for a long time, and provide technical support for large-scale subculture and propagation of boletus aereus.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a culture medium for preserving boletus nigricans sclerotium, which comprises the following raw materials in parts by weight: 45-48 parts of wheat grains, 15-18 parts of miscellaneous sawdust, 15-17 parts of pine sawdust, 18-19 parts of bran, 1-2 parts of gypsum and 1-2 parts of white sugar.

The invention also provides a preparation method of the culture medium for preserving the boletus nigricans sclerotium, which comprises the following steps:

(1) boiling wheat grains in water until the wheat grains are saturated in water and the skins are not broken, and filtering to dry;

(2) and mixing the rest raw materials, adding lemon water, adding the filtered and dried wheat grains, and uniformly stirring to obtain the boletus aereus sclerotium preservation culture medium.

Further, the concentration of the lemonade in the step (2) is 20mg/L, and the mass ratio of the lemonade to the rest raw materials is 1: 1.4.

The invention also provides a preservation method of the boletus nigricans strains, which comprises the steps of inoculating the boletus nigricans strains into the boletus nigricans sclerotium preservation culture medium of claim 1, culturing to generate sclerotium, and then preserving at 15-18 ℃.

Further, the boletus nigricans strain is a mother strain obtained by culturing boletus nigricans in a mother strain culture medium.

Further, the mother culture medium comprises the following raw materials: by weight, 30 parts of starch, 1 part of peptone, 20 parts of glucose, 1 part of monopotassium phosphate and 16 parts of agar, and the balance of water.

Further, the mother culture medium is prepared according to the following steps: firstly adding starch into water for dissolving, then adding peptone, potassium dihydrogen phosphate and glucose for dissolving, then adding agar for uniformly stirring, finally adjusting the pH value to 5.0 and adding water for fixing the volume to 1L.

Further, the culture conditions comprise that the culture is carried out for 30 days at 28-30 ℃ under the condition that the humidity is controlled to be 60% -70% and the culture is kept in the dark, then the temperature is adjusted to be 25-27 ℃, the humidity is controlled to be 55% -65% and the illumination intensity is 50-100lx, and the culture is continued for 20-30 days.

Further, the storage condition is that the tea is stored in dark under the condition that the humidity is controlled to be 50% -60%.

The invention discloses the following technical effects:

(1) the sclerotium preservation culture medium provided by the invention takes wheat grains as a main nutrient medium, provides a carbon source, fully absorbs water through the operation of water boiling and draining, is beneficial to thorough autoclaving, can fully release nutrient substances in the wheat grains, provides lignocellulose for miscellaneous wood chips, provides a nitrogen source for bran, adjusts the pH value by citric acid, and has a carbon-nitrogen ratio of 20-25: 1, the growth of boletus aereus hyphae is suitable; the pine sawdust contains aromatic substances which can induce the bolete hyphae to form sclerotium, and the bolete sclerotium is a group of tightly interwoven dormancy bodies which are stored with nutrition, can resist adverse environment better than the mycelium, and is easy for the activity maintenance of strains; the miscellaneous wood chips, the pine wood chips and the bran are fillers of pores among the wheat grains, can further absorb moisture on the surface of the wheat grains, adjust the water content of the wheat grains, prevent the wheat grains from being adhered together and provide space for normal growth of hypha.

(2) The preservation method has simple operation, easily obtained preserved culture medium components and low production cost, and the viability and the cultivation performance of the boletus aereus sclerotium preserved by the preservation method have no obvious difference with the original seed sclerotium, can preserve the viability and the excellent performance of the boletus aereus sclerotium for four and a half years, solves the problem that the boletus aereus is not easy to be simply and conveniently preserved for a long time, and provides technical support for the mass subculture and propagation of the boletus aereus.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The test methods used in the examples are all conventional methods unless otherwise specified; the reagents used are commercially available reagents unless otherwise specified.

The bolete nigricans used in the following examples is bolete portentosus (Phlebopus portentosus) collected by the applicant, which has been deposited in the common microorganism center of the China Committee for culture Collection of microorganisms at 28 th.02/2022 with the deposition number of CGMCC NO.40107 and the deposition address of the institute of microbiology, China academy of sciences, third institute of sciences, North China, West Lu first institute of south Cheng, Naja, Beijing. The preservation method of other common bolete in the market is also applicable.

Example 1

1. Mother culture

1) The mother culture medium formula comprises: 30g of starch, 1g of peptone, 20g of glucose, 1g of monopotassium phosphate, 16g of agar and water, wherein the volume is fixed to 1L and the pH value is 5.0.

2) Preparing a mother culture medium: weighing the components according to the formula 1), firstly adding starch into water to dissolve, then adding peptone, potassium dihydrogen phosphate and glucose to dissolve, finally adding agar, heating while stirring, then adding water to a constant volume of 1L, adjusting the pH value to 5.0 by using 1N hydrochloric acid, subpackaging by using 18mm × 180mm test tubes while hot, packaging 10-12 ml of each test tube, and plugging a silica gel plug.

3) And (3) sterilization: and (3) vertically placing the test tube filled with the culture medium into a sterilization pot, and sterilizing at the temperature of 121 ℃ for 20-30 mm.

4) Preparing a test tube slant of a mother culture medium: and (3) opening the sterilization pot after sterilization, placing the test tube according to a certain inclination (about 10 ℃) while the test tube is hot, and cooling to obtain the test tube inclined plane, wherein the distance from the front end of the culture medium inclined plane to the test tube opening is preferably 40-50 mm.

5) Tissue isolation: selecting a boletus aereus fruiting body which is normal in morphological development, free of diseases and insect pests, not unfolded in pileus margin and 60-80 g in weight as a separation material. Sterilizing the surface by using 75% alcohol, cutting a pileus by using a blade under an aseptic condition, cutting a tissue block of 5mm multiplied by 5mm at the joint of a stipe and the pileus into the center of the culture medium of 2), inoculating, placing in a constant temperature box at 28-30 ℃ for dark culture for 15-20 days, checking the growth condition of hypha once every 2-3 days, and selecting the well-grown tissue as a mother strain.

2. Culture of sclerotium

1) The formula of the sclerotium culture medium is as follows: 48% of wheat grains, 16% of miscellaneous wood chips, 16% of pine wood chips, 18% of bran, 1% of gypsum and 1% of white sugar.

2) Preparation of a sclerotium culture medium: weighing the components according to the formula 1), boiling the wheat grains thoroughly with water to ensure that the wheat grains fully absorb water until the skins are not broken and the insides are transparent and white, and then filtering. Uniformly stirring the mixed wood chips, pine wood chips, bran, gypsum and white sugar, wherein the mass ratio of the materials to the water is 1:1.4 adding 20mg/L of lemon water according to the proportion to adjust the water content of the culture material, and finally adding the wheat grains and stirring uniformly.

3) Tube loading: using a 32mm X200 mm test tube, the contents were loaded to 1/2 mm of the length of the tube, the load was tightened loosely, the tube wall was wiped clean, and the silica plug was stoppered.

4) And (3) sterilization: the test tube filled with the culture material is placed in a sterilizing pot vertically, and is sterilized for 2.5 hours at the temperature of 126 ℃.

5) Inoculation: inoculating the mother strain cultured by the 1 culture under aseptic condition when the temperature of the material is reduced to below 30 ℃.

6) Culturing: and (3) culturing for 30 days at 28-30 ℃ in a dark place with the humidity controlled at 60-70%, adjusting the temperature to 25-27 ℃, controlling the humidity to 55-65% and the illumination intensity to 50-100lx, and continuously culturing for 20-30 days to generate a large amount of sclerotia on the surface of the culture material and the wall of the test tube.

3. Sclerotium preservation

The sclerotium test tube cultured in the step 2 is tightly wrapped by kraft paper and is stored for 42 months in the dark under the temperature of 15 ℃ and the humidity of 50-60 percent.

4. Subculture of sclerotium

Inoculating the long-term preserved sclerotium into mother culture medium tubes under aseptic condition, inoculating one sclerotium per tube, and culturing at 28 deg.C.

Example 2

1. The mother culture procedure is the same as that of example 1, except for the steps of 2. sclerotium culture, 3. sclerotium preservation and 4. sclerotium subculture, which are specifically as follows:

2. sclerotium culture

1) The formula of the sclerotium culture medium is as follows: 45% of wheat grains, 18% of miscellaneous wood chips, 15% of pine wood chips, 19% of bran, 2% of gypsum and 1% of white sugar.

2) Preparation of a sclerotium culture medium: weighing the components according to the formula 1), boiling the wheat grains thoroughly with water to ensure that the wheat grains fully absorb water until the skins are not broken and the interiors are transparent and white, draining the wheat grains, uniformly stirring the wheat grains with miscellaneous wood chips, pine wood chips, bran, gypsum and white sugar, and mixing the materials according to the mass ratio of 1:1.4 adding 20mg/L of lemon water according to the proportion to adjust the water content of the culture material, and finally adding the wheat grains and stirring uniformly.

3) Tube loading: using a 32mm X200 mm test tube, the contents were loaded to 2/3 mm of the length of the tube, the load was tightened loosely, the tube wall was wiped clean, and the silica plug was stoppered.

3. Sclerotium preservation

The sclerotium test tube cultured in the step 2 is tightly wrapped by kraft paper and is stored for 45 months in a dark place at the temperature of 16 ℃ and the humidity of 50-60 percent.

4. Subculture of sclerotium

Inoculating the long-term preserved sclerotium into mother culture medium test tubes under aseptic condition, wherein each test tube is connected with one sclerotium, and culturing at 30 deg.C.

Example 3

2. culture of sclerotium

1) The formula of the culture medium is as follows: 47% of wheat grains, 15% of miscellaneous wood chips, 17% of pine wood chips, 18% of bran, 1% of gypsum and 2% of white sugar.

2) Preparation of a sclerotium culture medium: weighing the components according to the formula 1), boiling the wheat thoroughly with water to ensure that the wheat fully absorbs water until the wheat is not broken and the wheat is transparent and white, draining the wheat, uniformly stirring the wheat with the miscellaneous wood chips, pine wood chips, bran, gypsum and white sugar, and mixing the materials according to the mass ratio of 1:1.4 adding 20mg/L of lemon water according to the proportion to adjust the water content of the culture material, and finally adding the wheat grains and stirring uniformly.

3. Sclerotium preservation

The sclerotium test tube cultured in the step 2 is tightly wrapped by kraft paper and stored for 54 months in the dark at the temperature of 18 ℃ and the humidity of 50-60 percent.

4. Subculture of sclerotium

Comparative example 1

The difference from example 1 is that 2. sclerotium culture 2) in the preparation of the medium, pine wood chips are adjusted to oak wood chips.

Comparative example 2

The difference from the example 1 is that 2. in the sclerotium culture, the formula of the culture medium is as follows: 200g of potato, 1g of peptone, 20g of glucose, 1g of monopotassium phosphate, 16g of agar and 1L of water.

Comparative example 3

The difference from example 1 is that 3. in the case of sclerotium preservation, the preservation temperature was 10 ℃.

Effect verification

And 2, taking the group which is directly inoculated into a mother culture medium test tube for propagation culture without preserving sclerotium obtained in the sclerotium culture as a control group, inoculating the strains which are propagated in the control group, the groups of examples 1-3 and the groups of comparative examples 1-3 into a bagged matrix for artificial culture, and respectively arranging 100 groups in parallel. The growth of the control, examples 1-3 and comparative examples 1-3 was counted daily on schedule, as shown in Table 1.

TABLE 1 growth of strains of the groups

As can be seen from table 1, the hypha germination rate, growth rate, hypha growth vigor and the weight of the single mushroom were not significantly different from those of the first generation sclerotia (control group) after the sclerotia preserved in examples 1 to 3 were subjected to activation culture, which indicates that the hypha vitality and the strain culture characteristics of the sclerotia and the original seed sclerotia were the same when the preservation method of the present invention was used. In the comparative examples 1-2, due to the adjustment of the preservation culture medium, the obtained sclerotium activity and hypha growth vigor are inferior to those of the invention; comparative example 3 adopts low-temperature storage, so that the activity of sclerotia is reduced, the sclerotia is not easy to germinate, and the subsequent growth of hyphae is influenced.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A culture medium for preserving boletus nigricans sclerotium is characterized by comprising the following raw materials in parts by weight: 45-48 parts of wheat grains, 15-18 parts of miscellaneous wood chips, 15-17 parts of pine wood chips, 18-19 parts of bran, 1-2 parts of gypsum and 1-2 parts of white sugar.

2. A method for preparing the culture medium for preserving the sclerotium of boletus nigricans according to claim 1, comprising the steps of:

3. The preparation method according to claim 2, wherein the concentration of the lemonade in the step (2) is 20mg/L, and the mass ratio of the lemonade to the rest raw materials is 1: 1.4.

4. A preservation method of boletus aereus strains is characterized by comprising the steps of inoculating boletus aereus strains into a boletus aereus sclerotium preservation culture medium of claim 1, culturing to generate sclerotium, and preserving at 15-18 ℃.

5. The method for preserving a boletus nigricans strain according to claim 4, wherein the boletus nigricans strain is a mother strain obtained by culturing boletus nigricans in a mother strain culture medium.

6. The method of claim 5, wherein the stock culture medium comprises the following raw materials: by weight, 30 parts of starch, 1 part of peptone, 20 parts of glucose, 1 part of monopotassium phosphate and 16 parts of agar, and the balance of water.

7. The method for preserving a species of boletus nigricans as claimed in claim 6, wherein the mother culture medium is prepared by the steps of: firstly adding starch into water for dissolving, then adding peptone, potassium dihydrogen phosphate and glucose for dissolving, then adding agar for uniformly stirring, finally adjusting the pH value to 5.0 and adding water for fixing the volume to 1L.

8. The preservation method of boletus aereus strain according to claim 4, wherein the culture conditions are that the culture is carried out for 30 days at 28-30 ℃ under the humidity controlled at 60% -70% in the dark, then the temperature is adjusted to 25-27 ℃, the humidity is controlled at 55% -65% and the illumination intensity is 50-100lx, and the culture is continued for 20-30 days.

9. The preservation method of boletus aereus species of claim 4, wherein the preservation condition is that the preservation is carried out under the condition of keeping the humidity between 50% and 60% in the dark.