Culture medium containing rice bran and hull and application of culture medium in culture and rejuvenation of armillaria mellea

Abstract

The invention belongs to the technical field of armillaria mellea culture, and particularly relates to a culture medium containing rice bran and hull and application of the culture medium in armillaria mellea culture and rejuvenation. The culture medium comprises the following raw materials in parts by mass: 50 parts of rice bran and hull, 50 parts of wood chips, 200 parts of potatoes, 20 parts of glucose, 15 parts of agar and 1000 parts of water. According to the culture medium, the original activity of the armillaria mellea can be recovered from a degraded state of poor growth vigor, very strong vitality and relatively high growth speed are kept, the dish filling time is effectively shortened to 10-20 days, the cultured armillaria mellea is large in number and thick, and the quality is obviously improved; the method for rejuvenating armillaria mellea by using the culture medium is simple in process and easy to operate. Meanwhile, raw materials of the culture medium are easy to obtain and low in cost, and the culture medium is suitable for large-scale growth and application.

Description

Technical Field

[0001] The invention belongs to the technical field of honey fungus cultivation, and particularly relates to a culture medium containing bran and application of the culture medium in honey fungus cultivation and rejuvenation. Background Art

[0002] Armillaria mellea is a fungus of the genus Armillaria (Fr.) Staude, belonging to the order Agaricales, the family Trichfomataceae, and the subdivision Basidiomycotina. It is mainly saprophytic, and also parasitic or symbiotic. It is saprophytic in the soil matrix of dead wood and decayed branches or parasitic on the roots of trees. It plays a key role in degradation and reduction in the forest ecosystem. At the same time, Armillaria mellea is a symbiotic fungus of the precious Chinese medicinal materials Gastrodia elata and Polyporus umbellatus. Gastrodia elata and Polyporus umbellatus must rely on the infection of Armillaria mellea to grow and reproduce. Armillaria mellea has many of the same pharmacological effects as Gastrodia elata, such as sedation, hypnosis, and anticonvulsant, and has no toxic side effects.

[0003] The phenomenon that the excellent and typical traits of the original variety are partially or completely lost due to genetic variation during the subculture process of Armillaria is called "strain degeneration". The degeneration of Armillaria will cause a reduction in the yield and quality of Gastrodia elata, thereby affecting the economic income of producers. It is an important issue that currently restricts the health of the Gastrodia elata industry. The main reasons for the degeneration of Armillaria are as follows: Ⅰ. Most of the Armillaria strains in production grow and are preserved on a certain semi-synthetic agar medium. On this type of culture medium, the strain can carry out normal nutritional growth, but cannot grow fruiting bodies. Long-term subculture storage will cause the inherent reproductive growth factors of the strain to not be activated for a long time. Ⅱ. The composition and ratio of the culture medium are unreasonable, which is not suitable for the growth and development of Armillaria, resulting in slow and weak mycelium growth. Long-term artificial culture conditions cannot meet its wild requirements, causing it to lose its self-regulation ability and normal physiological functions, resulting in the inability to express some excellent traits. In order to solve the problem of Armillaria degeneration, the prior art often restores the properties of strains whose growth is inhibited or whose performance shows decline during storage, that is, restores the performance of Armillaria by rejuvenating the strains.

[0004] The commonly used honey fungus rejuvenation medium in the prior art is composed of 50g bran, 20g sawdust, 20g glucose, 15g agar, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, and 1000mL water (Jin Longji, 2023); or the following formula: 200g potato, 4g beef extract, 20g glucose, 1g peptone, 2g potassium dihydrogen phosphate, and 4g magnesium sulfate (Sun Jimin, 2008). However, the above culture medium has limited rejuvenation effect on honey fungus. Some honey fungus strains grow slowly on the above rejuvenation medium, and the rejuvenation effect is not good. Therefore, it is of great significance to find a formula with wide applicability, good rejuvenation effect, and high adaptability for the rejuvenation or cultivation of honey fungus. Summary of the invention

[0005] The object of the present invention is to provide a culture medium containing bran and application thereof in the culture and rejuvenation of Armillaria mellea, wherein the culture medium can be used for the rejuvenation culture of Armillaria mellea, effectively restore the vitality of degenerated Armillaria mellea, and improve the quality of Armillaria mellea.

[0006] The present invention provides a culture medium containing bran, comprising the following raw materials in parts by weight:

[0007] 50 parts of whole chaffs, 50 parts of wood chips, 200 parts of potatoes, 20 parts of glucose, 15 parts of agar and 1000 parts of water.

[0008] Preferably, the wood chips include miscellaneous wood chips.

[0009] The present invention also provides a method for preparing the culture medium described in the above technical solution, comprising the following steps:

[0010] Potatoes, bran, sawdust and water are mixed and boiled, and the obtained mixture is filtered to obtain a filtrate;

[0011] The filtrate and water are mixed so that the total weight fraction reaches 1000 parts to obtain a mixed solution;

[0012] The mixed solution, glucose and agar powder are mixed and sterilized to obtain the culture medium.

[0013] Preferably, the boiling time is 30 minutes.

[0014] The present invention also provides the use of the culture medium described in the above technical solution or the culture medium prepared by the preparation method described in the above technical solution in the culture of Armillaria mellea.

[0015] Preferably, the Armillaria mellea culture includes Armillaria mellea rejuvenation culture.

[0016] Preferably, the Armillaria mellea includes Armillaria mellea A9 and / or Armillaria mellea A25.

[0017] The present invention also provides a method for rejuvenation of Armillaria mellea, comprising: inoculating degenerate Armillaria mellea into a rejuvenation culture medium for rejuvenation culture to obtain rejuvenated Armillaria mellea; the rejuvenation culture medium is the culture medium described in the above technical solution or the culture medium prepared by the preparation method described in the above technical solution.

[0018] Preferably, the Armillaria mellea includes Armillaria mellea A9 and / or Armillaria mellea A25.

[0019] Beneficial effects:

[0020] The present invention provides a culture medium containing bran, comprising the following raw materials in parts by mass: 50 parts of bran, 50 parts of sawdust, 200 parts of potato, 20 parts of glucose, 15 parts of agar and 1000 parts of water. The culture medium of the present invention can restore the original vitality of Armillaria mellea from a degenerate state of poor growth, maintain strong vitality and a faster growth rate, effectively shorten the full plate time to 10-20 days, and the Armillaria cords obtained by culture are large in number and thick, and the quality is significantly improved; and the method for rejuvenating Armillaria mellea using the culture medium is simple in process and easy to operate. At the same time, the raw materials of the culture medium are easily available and low in cost, and are suitable for large-scale growth and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 The statistical results of the germination time of the mycelium of the subculture of Armillaria mellea A9 and A25 in Application Example 1;

[0023] Figure 2 The statistical results of the number of mycelial cords of Armillaria mellea A9 and A25 in the subculture in Application Example 1;

[0024] Figure 3 The statistical results of branching rate of Armillaria mellea A9 and A25 subculture in Application Example 1;

[0025] Figure 4 The statistical results of the branch number of Armillaria mellea A9 and A25 subculture in Application Example 1;

[0026] Figure 5 The statistical results of the average growth rate of the mycelium of Armillaria A9 and A25 in the subculture in Application Example 1;

[0027] Figure 6 The statistical results of the mycelial morphology of the subcultured Armillaria A9 and A25 in Application Example 1;

[0028] Figure 7 The statistical results of the full plate time of the subculture of Armillaria mellea A9 and A25 in Application Example 1;

[0029] Figure 8 The statistical results of dry weight of mycelium of Armillaria mellea A9 and A25 in the subculture in Application Example 1;

[0030] Fig. 9 The statistical results of the germination time of the mycelium of Armillaria mellea A9 in different culture media in Application Example 2;

[0031] Fig.10 The statistical results of the number of mycelial cords of Armillaria mellea A9 in subculture in different culture media in Application Example 2;

[0032] Fig.11 The statistical results of the number of branches of Armillaria mellea A9 in subculture in different culture media in Application Example 2;

[0033] Fig.12 The statistical results of branching rate of Armillaria mellea A9 in application example 2 in subculture in different culture media;

[0034] Fig.13 The statistical results of the average growth rate of the mycelium of Armillaria mellea A9 in different culture media in Application Example 2;

[0035] Fig.14 The statistical results of the mycelial morphology of Armillaria mellea A9 in subculture in different culture media in Application Example 2;

[0036] Fig.15 The statistical results of dry weight of mycelium of Armillaria mellea A9 in different culture media in Application Example 2;

[0037] Fig.16 The statistical results of the time of filling the plate of Armillaria mellea A9 in different culture media in Application Example 2;

[0038] Fig.17 Schematic diagrams of the front (upper figure) and back (lower figure) of the culture dish of the first and second generations of Armillaria mellea A9 subcultured in different culture media in Application Example 2;

[0039] Fig.18 Schematic diagrams of the front (upper figure) and back (lower figure) of the culture dish of the third generation of Armillaria mellea A9 subcultured in different culture media in Application Example 2. DETAILED DESCRIPTION

[0040] The present invention provides a culture medium containing bran, comprising the following raw materials in parts by weight:

[0041] 50 parts of whole chaffs, 50 parts of wood chips, 200 parts of potatoes, 20 parts of glucose, 15 parts of agar and 1000 parts of water.

[0042] In terms of mass fraction, the culture medium of the present invention includes 50 parts of whole bran. The whole bran of the present invention is a byproduct of rice husk and rice bran without separation, wherein the rice husk is rich in cellulose, hemicellulose, lignin, pentosan and silicon dioxide, and the content of nutrients such as protein, fat and vitamins is extremely low, and its surface is hard and resistant to decay. Rice bran is composed of the exocarp, mesocarp, cross-linked layer, endosperm layer and seed coat of rice, accounting for about 5.5%-10% of the total mass of rice, and is rich in high-quality fat, protein, polysaccharide, vitamin, mineral and other nutrients and tocopherol, tocotrienol, oryzanol, octacosane alcohol, α-lipoic acid, squalene, ceramide and other natural antioxidants and physiologically active substances. Therefore, the whole bran combined with the two has comprehensive nutrients and high content of functional components (dietary fiber, polypentose, etc.), which is suitable as a culture raw material for microbial culture, especially microbial rejuvenation.

[0043] The culture medium described in the present invention includes 50 parts of sawdust; the sawdust is preferably miscellaneous sawdust, and the types and sources of the miscellaneous sawdust are not particularly limited. Conventional types of sawdust in the art can be used for combination, and miscellaneous sawdust obtained through commercial channels in the art can be used. For example, the miscellaneous sawdust used in an embodiment of the present invention is miscellaneous sawdust composed of chestnut wood chips and maple wood chips; or miscellaneous sawdust composed of cycad wood chips and walnut wood chips; the present invention has no special restrictions on the component ratio of each sawdust in the miscellaneous sawdust, and any combination can achieve the technical effect in this application.

[0044] The culture medium of the present invention comprises 200 parts of potatoes.

[0045] The culture medium of the present invention comprises 20 parts of glucose.

[0046] The culture medium of the present invention comprises 15 parts of agar.

[0047] The culture medium of the present invention comprises 1000 parts of water.

[0048] The present invention also provides a method for preparing the culture medium described in the above technical solution, comprising the following steps:

[0049] Potatoes, bran, sawdust and water are mixed and boiled, and the obtained mixture is filtered to obtain a filtrate;

[0050] The filtrate and water are mixed so that the total weight fraction reaches 1000 parts to obtain a mixed solution;

[0051] The mixed solution, glucose and agar powder are mixed and sterilized to obtain the culture medium.

[0052] The holding time after boiling in the present invention is preferably 30 minutes. The present invention has no special limitation on the filtering method, and a conventional filtering method in the art can be used. The sterilization temperature in the present invention is preferably 121° C., and the time is preferably 20 minutes.

[0053] The present invention also provides the use of the culture medium described in the above technical solution or the culture medium prepared by the preparation method described in the above technical solution in the culture of Armillaria mellea. The Armillaria mellea culture described in the present invention preferably includes Armillaria mellea rejuvenation culture; the Armillaria mellea includes Armillaria mellea A9 and / or Armillaria mellea A25. The culture medium described in the present invention can restore the original vitality of Armillaria mellea from the degenerate state of poor growth, maintain strong vitality and faster growth rate, and effectively shorten the full plate time to 10-20 days, and the Armillaria cords obtained by culture are large in number and thick, and the quality is significantly improved; and the method for rejuvenation of Armillaria mellea using the culture medium is simple in process and easy to operate. At the same time, the raw materials of the culture medium are easy to obtain and low in cost, and are suitable for large-scale growth and application.

[0054] Based on the above advantages, the present invention also provides a method for rejuvenation of Armillaria mellea, comprising: inoculating degenerate Armillaria mellea into a rejuvenation medium for rejuvenation culture to obtain rejuvenated Armillaria mellea; the rejuvenation medium is the medium described in the above technical solution or the medium prepared by the preparation method described in the above technical solution. The present invention preferably uses a puncher to inoculate degenerate Armillaria mellea into the rejuvenation medium. The present invention does not specifically limit the amount of inoculation and the conditions of culture, and the amount of inoculation and the conditions of culture for rejuvenation culture of conventional Armillaria mellea in the art can be used. The Armillaria mellea described in the present invention preferably includes Armillaria mellea A9 and / or Armillaria mellea A25.

[0055] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] The experimental materials used in the following examples and comparative examples and their sources are as follows:

[0057] Bacteria: Armillaria A9 and A25 were purchased from Shaanxi Chunsen Fungi Co., Ltd.

[0058] Reagents: glucose, agar powder, rice bran (the main components are rice bran and rice husk fragments and a small amount of broken rice, purchased from Taobao Grain and Rice Industry); miscellaneous wood chips (the main components are chestnut and maple wood chips, purchased from Pinduoduo Edible Fungus Spawn Material Store); potatoes (purchased from Panlong District, Kunming City, Yunnan Province).

[0059] Example 1

[0060] A composition and preparation method of a culture medium containing bran are as follows:

[0061] 1. The composition of the culture medium is as follows:

[0062] 50g bran, 50g sawdust, 200g potato, 20g glucose, 15g agar, 1000mL (1000g) water.

[0063] 2. The preparation method of the culture medium in step 1 is as follows:

[0064] Weigh 200g of potato, 50g of rice bran, 50g of sawdust, add 1000mL of distilled water (1000g), boil for 30min, filter, take the filtrate and add distilled water to make up to 1000mL. Add 20g of glucose and 15g of agar powder to the filtrate. Sterilize at 121℃ for 20min to obtain a culture medium containing rice bran.

[0065] 3. Packaging of culture medium: Irradiate the clean bench with ultraviolet light for 20 minutes. After the culture medium is sterilized, pour it into 90mm culture dishes, about 15mL-20mL per dish.

[0066] Comparative Example 1

[0067] The composition and preparation method of a PDA culture medium are as follows:

[0068] 1. The composition of the culture medium is as follows:

[0069] 200 g potatoes, 20 g glucose, 15 g agar, 1000 mL (1000 g) water.

[0070] 2. The preparation method of the culture medium in step 1 comprises the following steps:

[0071] Weigh 200g of potato, add 1000g of water, boil for 30min, filter, take the filtrate and add water to make up to 1000mL. Add 20g of glucose and 15g of agar powder to the filtrate. Dispense into conical bottles and sterilize at 121℃ for 20min in high pressure steam sterilization.

[0072] 3. Packaging of culture medium: Expose the clean bench to ultraviolet light for 20 minutes. After the culture medium is sterilized, pour it into 90mm culture dishes, about 15mL-20mL per dish.

[0073] Comparative Example 2

[0074] The composition and preparation method of a culture medium are as follows:

[0075] 1. The composition of the culture medium is as follows:

[0076] Sawdust 100g, soybean meal 100g, glucose 20g, agar 15g and water 1000mL (1000g).

[0077] 2. The preparation method of the culture medium in step 1 comprises the following steps:

[0078] Weigh 100g of sawdust, 100g of soybean meal, add 1000mL of distilled water (1000g), boil for 30min, filter, take the filtrate and add distilled water to make up to 1000mL. Add 20g of glucose and 15g of agar powder to the filtrate. Sterilize at 121℃ for 20min to obtain the culture medium.

[0079] Packaging of culture medium: Irradiate the clean bench with ultraviolet light for 20 minutes. After the culture medium is sterilized, pour it into 90mm culture dishes, about 15mL-20mL per dish.

[0080] Comparative Example 3

[0081] The composition and preparation method of a culture medium are as follows:

[0082] 1. The composition of the culture medium is as follows:

[0083] Bran 100g, sawdust 100g, beef extract 5g, glucose 20g, agar 15g, water 1000mL (1000g).

[0084] 2. The preparation method of the culture medium in step 1 comprises the following steps:

[0085] Weigh 100g of sawdust and 100g of bran, add 1000mL of distilled water (1000g), boil for 30min, filter, take the filtrate, add 5g of beef extract, heat until completely melted, add distilled water to make up to 1000mL. Add 20g of glucose and 15g of agar powder to the filtrate. Sterilize at 121℃ for 20min to obtain the culture medium.

[0086] Packaging of culture medium: Irradiate the clean bench with ultraviolet light for 20 minutes. After the culture medium is sterilized, pour it into 90mm culture dishes, about 15mL-20mL per dish.

[0087] Application Example 1

[0088] 1. Preparation of degraded strains of Armillaria mellea: The steps are as follows:

[0089] 1) Using tweezers, transfer the Armillaria strain stored in the test tube to the culture dish containing the PDA culture medium prepared in Comparative Example 1.

[0090] 2) After culturing in the dark at 25°C for 10 days, use a cork puncher to punch holes in the culture medium with mycelial cords, inoculate the punched bacterial cake into the PDA culture medium again, and transfer Armillaria mellea three times according to the same method as above to obtain the degenerate strain of Armillaria mellea.

[0091] According to the method of steps 1)-2) above, the degenerated strains of Armillaria mellea A9 and Armillaria mellea A25 were obtained respectively.

[0092] 2. The rejuvenation culture of honey fungus is as follows:

[0093] Use a puncher (the diameter of the puncher is 7 mm) to transfer the degraded strain of Armillaria obtained in step 1 to a culture dish containing rejuvenation medium. Place it at 25°C for culture. The newly cultured rhizomes are the first generation of the strain (recorded as G1). After 10 days, punch holes and inoculate them on the same culture medium. Subculture to the third generation in sequence. The second and third generations are recorded as G2 and G3, respectively. Record the germination time of the rhizomes of each generation of Armillaria. After 10 days of culture of each generation, use a ruler to measure the length of the rhizomes, record the number of rhizomes and the number of branches, and calculate the branching rate, where the rhizomes branching rate (%) = (number of rhizomes / number of branches) × 100; at the same time, count the average growth rate, thickness and full dish time of the rhizomes. After 20 days, harvest the mycelium, dry it, and weigh the weight of the dry mycelium.

[0094] The results of subculturing the degenerate strains of Armillaria mellea A9 and Armillaria mellea A25 in the medium containing bran described in Example 1 by the steps described in step 2 are shown in Tables 1-2 and Figure 1-8 As shown, the data in the following Tables 1-2 are the average values ​​of 4 biological replicates, and the robustness in Tables 1-2 represents the mycelial morphology.

[0095] Table 1 Growth indexes of Armillaria mellea A9 strain in subculture

[0096]

[0097] Note: Data are mean ± standard deviation. Different letters in the same column indicate significant differences (P<0.05). The thickness of Armillaria cordi is represented by "1-5", where "1" means 0-1mm, "2" means 1-2mm, "3" means 2-3mm, "4" means 3-4mm, and "5" means 4-5mm. Table 2 is the same and will not be repeated here.

[0098] Table 2 Growth indexes of Armillaria mellea A25 strain in subculture

[0099]

[0100] From Table 1-2 and Figure 1-8 It can be concluded that the two Armillaria strains grow well on the same bran medium, with a short germination time, thick and white mycelium, strong vitality, fast growth rate, high biomass and not easy to age.

[0101] Application Example 2

[0102] The results of subculturing the degenerated strain of Armillaria mellea A9 in the culture medium of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 respectively by the method of step 2 of Application Example 1 Figure 9-18 As shown, in Figure 9-16 In the formula, formula 1, formula 2 and formula 3 represent the culture medium in comparative example 2, comparative example 3 and embodiment 1 respectively.

[0103] Depend on Figure 9-18 It can be concluded that the rejuvenation culture of Armillaria mellea A9 on the medium containing bran in Example 1 germinates quickly, has long, dense, and branched mycelium, fills the dish in a short time, and has a fast growth rate, which is significantly stronger than that on other culture media.

[0104] It can be concluded from the above examples that the culture medium containing bran of the present invention can be used for rejuvenation of degenerated strains of Armillaria mellea with good effect, and the Armillaria mellea obtained by culture has good growth condition, higher vitality and higher quality.

[0105] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium containing bran, characterized in that Including the following raw materials by mass: 50 parts of whole chaffs, 50 parts of wood chips, 200 parts of potatoes, 20 parts of glucose, 15 parts of agar and 1000 parts of water.

2. The culture medium according to claim 1, characterized in that The wood chips include miscellaneous wood chips.

3. The method for preparing the culture medium according to claim 1 or 2, characterized in that: The steps include: Potatoes, bran, sawdust and water are mixed and boiled, and the obtained mixture is filtered to obtain a filtrate; The filtrate and water are mixed so that the total weight fraction reaches 1000 parts to obtain a mixed solution; The mixed solution, glucose and agar powder are mixed and sterilized to obtain the culture medium.

4. The preparation method according to claim 3, characterized in that: The boiling time is 30 minutes.

5. Use of the culture medium according to claim 1 or 2 or the culture medium prepared by the preparation method according to claim 3 or 4 in culturing Armillaria mellea.

6. The use according to claim 5, characterized in that: The Armillaria mellea culture includes Armillaria mellea rejuvenation culture.

7. The use according to claim 5 or 6, characterized in that: The Armillaria melleae includes Armillaria melleae A9 and / or Armillaria melleae A25.

8. A method for rejuvenation of Armillaria mellea, characterized in that: include: The degenerated Armillaria mellea is inoculated into a rejuvenation culture medium for rejuvenation culture to obtain a rejuvenated Armillaria mellea; the rejuvenation culture medium is the culture medium according to claim 1 or 2 or the culture medium prepared by the preparation method according to claim 3 or 4.

9. The method according to claim 8, characterized in that The Armillaria melleae includes Armillaria melleae A9 and / or Armillaria melleae A25.

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