Method for recovering species of subculture degenerative auricularia auricula strain and application thereof
By culturing black fungus strains using specific culture media and temperature conditions at different stages, the problem of restoring the genetic characteristics of degenerated strains through subgeneration was solved, resulting in a significant improvement in mycelial growth rate, enzyme activity, and yield.
Patent Information
- Application Number
- CN202410635492.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-21
- Publication Date
- 2025-11-07
- Estimated Expiration
- 2044-05-21
AI Technical Summary
There is a lack of effective methods in the current technology to restore the species characteristics of subgenerated degenerated black fungus strains, which leads to reduced mycelial growth rate, reduced enzyme activity, reduced fruiting yield and decreased fruiting body quality.
By using different culture media and temperature conditions at different stages, the nutritional conditions and environment were gradually changed, including barren sawdust medium, PDA medium, sawdust medium and cPDA medium, combined with the gradual increase of temperature, to restore the species characteristics of the black fungus strain.
Rapid restoration of the strain's characteristics was achieved, with significant improvements in mycelial growth rate, enzyme activity, yield per bag, and sensory evaluation score of fruiting bodies, restoring them to pre-subgeneration levels.
Smart Images
Figure CN118355833B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of edible fungus cultivation, and particularly relates to a method for recovering the strain nature of a degraded Auricularia auricula-judae strain and application thereof. BACKGROUND
[0002] Degradation of edible fungus strain refers to the phenomenon that the excellent characteristics and typicality of edible fungus strain are gradually lost in the process of subculture due to the genetic material variation of mycelium under normal cultivation conditions. Degradation of edible fungus strain causes changes in mycelium morphology, physiology and biochemistry, mitochondria, nucleic acid and related genes, and especially causes the decrease of yield and quality of edible fungus.
[0003] The main reasons for degradation of edible fungus strain include impure strain, harmful mutation, virus infection, unsuitable culture environment, excessive subculture times and improper strain preservation, and the excessive subculture times is one of the main reasons. In edible fungus production, the more the subculture times of edible fungus strain, the greater the possibility of causing degradation of edible fungus strain.
[0004] The degraded strain loses the original excellent characteristics. For example, multiple subcultures lead to degradation of Auricularia auricula-judae strain, and after the strain degradation, the mycelium growth rate is reduced, the colony edge is irregular, the aerial mycelium is increased, the enzyme activity of mycelium is reduced, the ear bud is slow, the ear yield is reduced, the quality of fruiting body is reduced, the nutritional components of fruiting body are changed, and the strain is no longer suitable for production. Some mushroom farmers do not understand the harm of unlimited subculture, and continue to use the strain subcultured for many times, resulting in large area yield reduction and serious loss.
[0005] The measures for preventing degradation of edible fungus strain mainly include preventing strain mixing, ensuring pure culture of strain, controlling subculture times of strain, ensuring strain activity, well preserving strain, delaying the degradation speed of strain, creating suitable living environment, promoting the growth of strain, changing the formula and nutritional conditions of culture medium, recovering the characteristics of strain, regularly separating strain, and maintaining the excellent characteristics of strain.
[0006] However, there is no report on the effective recovery method for the degraded edible fungus strain. SUMMARY
[0007] Therefore, the purpose of the present application is to provide a method for recovering the strain nature of a subcultured degraded Auricularia auricula-judae strain, which changes the nutritional conditions by changing the formula of culture medium, and changes the environmental conditions, so as to achieve the purpose of recovering the strain nature of Auricularia auricula-judae.
[0008] The present application provides a method for recovering the strain nature of a subcultured degraded Auricularia auricula-judae strain, which comprises the following steps: inoculating the activated subcultured degraded Auricularia auricula-judae strain into a poor sawdust culture medium, and culturing at 18-20℃ to obtain first mycelium;
[0009] The first mycelium is inoculated into a barren PDA culture medium and first activated culture is carried out at 20-22℃ to obtain a second mycelium;
[0010] The second mycelium is inoculated into a sawdust culture medium and second cultivation culture is carried out at 22-25℃ to obtain a third mycelium;
[0011] The third mycelium is inoculated into a PDA culture medium and second activated culture is carried out at 22-25℃ to obtain a fourth mycelium;
[0012] The fourth mycelium is inoculated into a conventional sawdust culture medium and third cultivation culture is carried out at 25-28℃ to obtain a fifth mycelium;
[0013] The fifth mycelium is inoculated into a cPDA culture medium and third activated culture is carried out at 25-28℃ to obtain a strain of Auricularia auricula-judae with restored characteristics.
[0014] Preferably, the barren sawdust culture medium comprises the following components in percentage by mass: 93.5%-94.5% sawdust, 2.8%-3.2% wheat bran, 0.9%-1.1% soybean meal, 0.9%-1.1% lime and 0.9%-1.1% gypsum;
[0015] The sawdust culture medium comprises the following components in percentage by mass: 91.5%-92.5% sawdust, 4.8%-5.2% wheat bran, 0.9%-1.1% soybean meal, 0.9%-1.1% lime and 0.9%-1.1% gypsum;
[0016] The conventional sawdust culture medium comprises the following components in percentage by mass: 84.5%-85.5% sawdust, 9.5%-10.5% wheat bran, 2.8%-3.2% soybean meal, 0.9%-1.1% lime and 0.9%-1.1% gypsum.
[0017] Preferably, the sawdust comprises coarse sawdust and fine sawdust;
[0018] In the barren sawdust culture medium, the mass ratio of coarse sawdust to fine sawdust is 2:1;
[0019] In the sawdust culture medium, the mass ratio of coarse sawdust to fine sawdust is 3:2;
[0020] In the conventional sawdust culture medium, the mass ratio of coarse sawdust to fine sawdust is 1:1.
[0021] Preferably, the sawdust comprises oak sawdust.
[0022] Preferably, the barren PDA culture medium comprises the following components: 200g / L potatoes, 10g / L glucose and 20g / L agar powder;
[0023] The PDA culture medium comprises the following components: potato 200 g / L, glucose 15 g / L and agar powder 20 g / L;
[0024] The cPDA culture medium comprises the following components: potato 200 g / L, glucose 15 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 1 g / L and agar powder 20 g / L.
[0025] Preferably, the humidity of the first cultivation culture or the second cultivation culture is 25-35%;
[0026] The humidity of the third cultivation culture is 30%-40%;
[0027] The humidity of the first activation culture, the second activation culture or the third activation culture is 30%-40%.
[0028] Preferably, the time of the first activation culture, the second activation culture or the third activation culture is 10-12 d;
[0029] The time of the first cultivation culture or the second cultivation culture is 30-35 d;
[0030] The time of the third cultivation culture is 25-30 d.
[0031] Preferably, the first activation culture, the second activation culture, the third activation culture, the first cultivation culture, the second cultivation culture or the third cultivation culture is light-avoiding culture.
[0032] Preferably, the Auricularia auricula-judae comprises an Auricularia auricula-judae black 29 strain.
[0033] The application provides application of the method for recovering species of the subculture degenerated Auricularia auricula-judae strain in Auricularia auricula-judae breeding and / or preservation.
[0034] The application provides a method for recovering species of a subculture degenerated Auricularia auricula-judae strain. BRIEF DESCRIPTION OF DRAWINGS
[0035] Figure 1 Technical roadmap for strain property recovery of the degenerative Auricularia auricula-judae strain;
[0036] Figure 2 Comparison results of mycelium growth of black 29G1, black 29G10 and black 29G10 recovery strains;
[0037] Figure 3 Comparison results of mycelium growth rate of black 29G1, black 29G10 and black 29G10 recovery strains;
[0038] Figure 4 Comparison results of Auricularia auricula-judae mycelium amylase activity of black 29G1, black 29G10 and black 29G10 recovery strains
[0039] Figure 5 Comparison results of Auricularia auricula-judae mycelium laccase activity of black 29G1, black 29G10 and black 29G10 recovery strains
[0040] Figure 6 Comparison results of Auricularia auricula-judae mycelium cellulase activity of black 29G1, black 29G10 and black 29G10 recovery strains
[0041] Figure 7 Comparison results of Auricularia auricula-judae mycelium trypsin activity of black 29G1, black 29G10 and black 29G10 recovery strains;
[0042] Figure 8 Comparison results of single bag yield of black 29G1, black 29G10 and black 29G10 recovery strains
[0043] Figure 9 Comparison results of Auricularia auricula-judae fruiting body sensory evaluation score of black 29G1, black 29G10 and black 29G10 recovery strains. DETAILED DESCRIPTION
[0044] The application provides a method for recovering the strain property of a degenerative Auricularia auricula-judae strain, comprising the following steps: inoculating an activated degenerative Auricularia auricula-judae strain into a poor sawdust culture medium, and carrying out first cultivation at 18-20°C to obtain first mycelium;
[0045] Inoculating the first mycelium into a poor PDA culture medium, and carrying out first activation culture at 20-22°C to obtain second mycelium;
[0046] Inoculating the second mycelium into a sawdust culture medium, and carrying out second cultivation at 22-25°C to obtain third mycelium;
[0047] Inoculating the third mycelium into a PDA culture medium, and carrying out second activation culture at 22-25°C to obtain fourth mycelium;
[0048] The fourth mycelium is inoculated into a conventional sawdust culture medium, and third cultivation culture is carried out at 25-28℃ to obtain a fifth mycelium;
[0049] The fifth mycelium is inoculated into a cPDA culture medium, and third activation culture is carried out at 25-28℃ to obtain a strain of Auricularia auricula-judae with restored species.
[0050] The present application inoculates the activated subculture degenerative Auricularia auricula-judae strain into a poor sawdust culture medium, and first cultivation culture is carried out at 18-20℃ to obtain a first mycelium.
[0051] The present application does not make special restrictions on the species of Auricularia auricula-judae, and the species of Auricularia auricula-judae known in the art can be used. In order to illustrate the implementation method of the method provided by the present application, Auricularia auricula-judae black 29 strain is taken as an example for illustration. The subculture degenerative Auricularia auricula-judae strain is Auricularia auricula-judae black 29 strain after 10 generations of subculture, and Auricularia auricula-judae black 29 subculture 10 strain (black 29G10) is obtained.
[0052] In the present application, the activation method of the subculture degenerative Auricularia auricula-judae strain preferably activates the subculture degenerative Auricularia auricula-judae strain for 10d by inoculating it into a PDA culture medium. The inoculation amount of the activated subculture degenerative Auricularia auricula-judae strain is 10 pieces of fungus cake per bag. The diameter of the fungus cake is preferably 0.3-0.5cm, and more preferably 0.4cm. The sampling site of the fungus cake is preferably the fungus block taken at a distance of 0.3-0.8cm from the mycelium tip.
[0053] In the present application, the poor sawdust culture medium preferably comprises the following mass percentage components: 93.5%-94.5% sawdust, 2.8%-3.2% wheat bran, 0.9%-1.1% soybean meal, 0.9%-1.1% lime and 0.9%-1.1% gypsum, and more preferably 94% sawdust, 3% wheat bran, 1% soybean meal, 1% lime and 1% gypsum. The sawdust preferably comprises coarse sawdust and fine sawdust. The particle size of the coarse sawdust is preferably 0.4-0.7cm, and more preferably 0.5-0.6cm. The particle size of the fine sawdust is preferably 0.1-0.4cm, and more preferably 0.2-0.3cm. In the poor sawdust culture medium, the mass ratio of coarse sawdust to fine sawdust is preferably 2:1. Different mass ratios of coarse sawdust and fine sawdust make the substrate water holding capacity and water retention characteristics different, and the aeration performance also different, thereby affecting the growth rate and growth of Auricularia auricula-judae mycelium. The optimal carbon source for Auricularia auricula-judae mycelium growth is mainly hard wood sawdust, of which tsubo sawdust is the best. Therefore, the sawdust preferably comprises tsubo sawdust. The temperature of the first cultivation culture is preferably 19℃. The humidity of the first cultivation culture is preferably 25%-35%, and more preferably 30%. The first cultivation culture is preferably light-free culture. The time of the first cultivation culture is preferably 30-35d, and more preferably 32d.
[0054] After the first mycelium is obtained, the first mycelium is inoculated into a poor PDA culture medium, and first activation culture is carried out at 20-22℃ to obtain a second mycelium.
[0055] In the present application, the poor PDA culture medium preferably comprises the following components: potato 200g / L, glucose 10g / L and agar powder 20g / L. The inoculation amount of the first mycelium is 1 plug per dish. The plug is preferably sawdust spawn at a distance of 0.3-0.8cm from the tip of the mycelium, and the diameter is preferably 0.2-0.5cm. The temperature of the first activation culture is preferably 21℃. The humidity of the first activation culture is preferably 30%-40%, more preferably 35%. The time of the first activation culture is preferably 10-12d, more preferably 11d. The first activation culture is preferably carried out in the dark.
[0056] After the second mycelium is obtained, the second mycelium is inoculated into a sawdust culture medium, and second cultivation culture is carried out at 22-25℃ to obtain a third mycelium.
[0057] In the present application, the sawdust culture medium preferably comprises the following components by mass percentage: 91.5%-92.5% sawdust, 4.8%-5.2% wheat bran, 0.9%-1.1% soybean meal, 0.9%-1.1% lime and 0.9%-1.1% gypsum, more preferably 92% sawdust, 5% wheat bran, 1% soybean meal, 1% lime and 1% gypsum. In the sawdust culture medium, the mass ratio of coarse sawdust to fine sawdust is preferably 3:2. The temperature of the second cultivation culture is preferably 23-24℃. The humidity of the second cultivation culture is preferably 25%-35%, more preferably 30%. The time of the second cultivation culture is preferably 30-35d, more preferably 32-34d, most preferably 33d. The second cultivation culture is preferably carried out in the dark. The inoculation method of the second mycelium is consistent with that of the first mycelium.
[0058] After the third mycelium is obtained, the third mycelium is inoculated into a PDA culture medium, and second activation culture is carried out at 22-25℃ to obtain a fourth mycelium.
[0059] In the present application, the PDA culture medium preferably comprises the following components: potato 200g / L, glucose 15g / L and agar powder 20g / L. The humidity of the second activation culture is preferably 30%-40%, more preferably 35%. The temperature of the second activation culture is preferably 23-24℃. The time of the second activation culture is preferably 10-12d, more preferably 11d. The second activation culture is preferably carried out in the dark.
[0060] After the fourth mycelium is obtained, the fourth mycelium is inoculated into a conventional sawdust culture medium, and third cultivation culture is carried out at 25-28℃ to obtain a fifth mycelium.
[0061] In the present application, the conventional sawdust culture medium preferably comprises the following mass percentage components: 84.5% to 85.5% sawdust, 9.5% to 10.5% wheat bran, 2.8% to 3.2% soybean meal, 0.9% to 1.1% lime and 0.9% to 1.1% gypsum, more preferably 85% sawdust, 10% wheat bran, 3% soybean meal, 1% lime and 1% gypsum. In the conventional sawdust culture medium, the mass ratio of coarse sawdust and fine sawdust is preferably 1:1. The temperature of the third cultivation preferably ranges from 26 to 27°C. The humidity of the third cultivation preferably ranges from 30% to 40%, more preferably 35%. The time of the third cultivation preferably ranges from 25 to 30 days, more preferably 28 days. The third cultivation is preferably carried out in the dark. The inoculation of the fourth mycelium is the same as the second mycelium inoculation method.
[0062] After obtaining the fifth mycelium, the present application inoculates the fifth mycelium into a cPDA culture medium and carries out a third activation culture at 25 to 28°C to obtain a subculture degenerative Auricularia auricula strain.
[0063] In the present application, the cPDA culture medium preferably comprises the following components: 200 g / L potato, 15 g / L glucose, 2 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate and 20 g / L agar powder. The humidity of the third activation culture preferably ranges from 30% to 40%, more preferably 35%. The temperature of the third activation culture preferably ranges from 26 to 27°C. The time of the third activation culture preferably ranges from 10 to 12 days, more preferably 11 days. The humidity of the third activation culture preferably ranges from 30% to 40%, more preferably 35%. The inoculation method of the fifth mycelium is the same as the inoculation method of the first mycelium.
[0064] In the present application, the Auricularia auricula strain recovered by the above method has a significant increase in mycelium growth, mycelium growth rate, mycelium amylase activity, mycelium laccase activity, mycelium cellulase activity, mycelium trypsin activity, single bag yield and Auricularia auricula fruit body sensory score compared with before recovery, and each index is restored to the level of the Auricularia auricula strain before subculture.
[0065] The present application provides an application of the method for restoring the strain properties of a subculture degenerative Auricularia auricula strain in Auricularia auricula breeding and / or preservation.
[0066] The method for restoring the strain properties of a subculture degenerative Auricularia auricula strain and the application thereof provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.
[0067] Example 1
[0068] The Auricularia auricula-judae black 29 strain is activated regularly to obtain a G1 generation (denoted as black 29G1), and after the mycelium of the G1 generation fills the culture dish, it is punched with a 1 cm puncher and transferred to a cPDA culture medium, and after being cultured at 25°C for 15 days in a constant temperature incubator in the dark, a G2 generation (denoted as black 29G2) is obtained. Subsequent generations are uniformly transferred according to this method to a G10 generation (denoted as black 29G10). The Auricularia auricula-judae strain (black 29G10) after 10 generations appears to have degenerated, mainly manifested as weak mycelial growth, slow growth, reduced enzyme activity, and reduced single-bag fruiting body yield and sensory evaluation score.
[0069] Example 2
[0070] A method for recovering the species of a subcultured degenerated Auricularia auricula-judae strain, comprising the following steps:
[0071] (1) The Auricularia auricula-judae strain black 29G10 is inoculated in a PDA culture medium for 10 days, and a mycelial block 0.3 cm away from the tip of the mycelium is picked and transferred to a poor nutrient wood chip culture medium. The culture medium formula is: 94% mixed wood chips, 3% wheat bran, 1% soybean cake powder, 1% lime and 1% gypsum, wherein the mixed wood chips are oak wood chips, the mass ratio of coarse wood chips to fine wood chips is 2:1, the particle size of the coarse wood chips is 0.4 cm, and the particle size of the fine wood chips is 0.1 cm.
[0072] (2) After inoculation, low-temperature culture is carried out in the dark at 18°C for 35 days, and the humidity is 25%. The culture is stopped when the mycelium grows to 0.5 cm before the bottom of the culture medium.
[0073] (3) The wood chip inoculum 0.3 cm away from the tip of the mycelium is picked and transferred to a poor PDA culture medium for activation. The poor PDA culture medium formula is: 200 g / L potatoes, 10 g / L glucose and 20 g / L agar powder.
[0074] (4) After inoculation, low-temperature culture is carried out in the dark at 20°C for 12 days, and the humidity is 30%. The culture is stopped when the mycelium grows to 0.5 cm before the edge of the culture dish.
[0075] (5) The mycelial block 0.5 cm away from the tip of the mycelium is picked and transferred to a wood chip culture medium. The wood chip culture medium formula is: 92% mixed wood chips, 5% wheat bran, 1% soybean cake powder, 1% lime and 1% gypsum. The mixed wood chips are oak wood chips, and the mass ratio of coarse wood chips to fine wood chips is 3:2.
[0076] (6) After inoculation, low-temperature culture is carried out in the dark at 22°C for 25 days, and the humidity is 25%. The culture is stopped when the mycelium grows to 0.5 cm before the bottom of the culture medium.
[0077] (7) picking up the wood chip inoculum at 0.3 cm from the hyphal tip, with a diameter of 0.2 cm, and transferring it to PDA medium for activation, wherein the PDA medium is composed of 200 g / L potato, 15 g / L glucose and 20 g / L agar powder.
[0078] (8) after inoculation, incubating at 22 °C in the dark for 10 days, with a humidity of 30%, until the hyphae grow to 0.5 cm from the edge of the culture dish.
[0079] (9) picking up the agar block at 0.3 cm from the hyphal tip, with a diameter of 0.3 cm, and transferring 10 agar blocks to the conventional wood chip culture medium, wherein the conventional wood chip culture medium is composed of 85% mixed wood chips, 10% wheat bran, 3% soybean cake powder, 1% lime and 1% gypsum, and the mixed wood chips are composed of oak wood chips, with a mass ratio of coarse wood chips to fine wood chips of 1:1.
[0080] (10) after inoculation, incubating at 25 °C in the dark for 25 days, with a humidity of 30%, until the hyphae grow to 0.5 cm from the bottom of the culture medium.
[0081] (11) picking up the wood chip inoculum at 0.3 cm from the hyphal tip, with a diameter of 0.2 cm, and transferring it to cPDA medium for activation, wherein the PDA medium is composed of 200 g / L potato, 15 g / L glucose, 2 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate and 20 g / L agar powder.
[0082] (12) after inoculation, incubating at 25 °C in the dark for 10 days, with a humidity of 30%, until the hyphae grow to 0.5 cm from the edge of the culture dish. The obtained Auricularia auricula-judae inoculum is preserved in a 4 °C refrigerator for standby use.
[0083] Example 3
[0084] A method for recovering the strain characteristics of a degenerated Auricularia auricula-judae strain, comprising the following steps:
[0085] (1) inoculating the Auricularia auricula-judae strain Hei 29G10 in PDA medium for activation for 10 days, picking up the agar block at 0.8 cm from the hyphal tip, with a diameter of 0.5 cm, and transferring 10 agar blocks to the nutrient-poor wood chip culture medium, wherein the culture medium is composed of 94% mixed wood chips, 3% wheat bran, 1% soybean cake powder, 1% lime and 1% gypsum, and the mixed wood chips are composed of oak wood chips, with a mass ratio of coarse wood chips to fine wood chips of 2:1, a particle size of the coarse wood chips of 0.7 cm and a particle size of the fine wood chips of 0.4 cm.
[0086] (2) after inoculation, incubating at 18 °C in the dark for 30-35 days, with a humidity of 35%, until the hyphae grow to 0.5 cm from the bottom of the culture medium.
[0087] (3) Pick the wood chip inoculum at 0.3-0.8 cm from the tip of the mycelium, with a diameter of 0.5 cm, and transfer to a poor PDA medium for activation. The poor PDA medium is composed of 200 g / L potato, 10 g / L glucose, and 20 g / L agar powder.
[0088] (4) After inoculation, incubate at 22°C in the dark for 10 days, with a humidity of 40%. Stop the incubation when the mycelium grows to 0.5 cm from the edge of the culture dish.
[0089] (5) Pick the mycelial block at 0.8 cm from the tip of the mycelium, with a diameter of 0.5 cm, and transfer 10 pieces to the wood chip culture medium. The wood chip culture medium is composed of 92% mixed wood chips, 5% wheat bran, 1% soybean cake powder, 1% lime, and 1% gypsum. The mixed wood chips are oak wood chips, with a mass ratio of coarse wood chips to fine wood chips of 3:2.
[0090] (6) After inoculation, incubate at 25°C in the dark for 25 days, with a humidity of 35%. Stop the incubation when the mycelium grows to 0.5 cm from the bottom of the culture medium.
[0091] (7) Pick the wood chip inoculum at 0.8 cm from the tip of the mycelium, with a diameter of 0.5 cm, and transfer to a PDA medium for activation. The PDA medium is composed of 200 g / L potato, 15 g / L glucose, and 20 g / L agar powder.
[0092] (8) After inoculation, incubate at 25°C in the dark for 10 days, with a humidity of 40%. Stop the incubation when the mycelium grows to 0.5 cm from the edge of the culture dish.
[0093] (9) Pick the mycelial block at 0.8 cm from the tip of the mycelium, with a diameter of 0.5 cm, and transfer 10 pieces to the regular wood chip culture medium. The regular wood chip culture medium is composed of 85% mixed wood chips, 10% wheat bran, 3% soybean cake powder, 1% lime, and 1% gypsum. The mixed wood chips are oak wood chips, with a mass ratio of coarse wood chips to fine wood chips of 1:1.
[0094] (10) After inoculation, incubate at 28°C in the dark for 25 days, with a humidity of 40%. Stop the incubation when the mycelium grows to 0.5 cm from the bottom of the culture medium.
[0095] (11) Pick the wood chip inoculum at 0.8 cm from the tip of the mycelium, with a diameter of 0.5 cm, and transfer to a cPDA medium for activation. The cPDA medium is composed of 200 g / L potato, 15 g / L glucose, 2 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, and 20 g / L agar powder.
[0096] (12) After inoculation, cultivate at 28℃ for 10 days in dark with humidity of 40%, stop cultivation when mycelium grows to 0.5 cm before the edge of the culture dish. The obtained Auricularia auricula-judae strain is preserved in 4℃ refrigerator for standby use.
[0097] Example 4
[0098] A method for recovering the strain character of a subcultured degenerated Auricularia auricula-judae strain, comprising the following steps:
[0099] (1) Inoculate Auricularia auricula-judae strain Hei 29G10 in PDA medium for 10 days, pick up the mycelium block 0.5 cm from the tip of the mycelium, the mycelium block is a round cake with a diameter of 0.4 cm, transfer 10 round cakes to the wood chip culture medium, the culture medium formula is: 94% miscellaneous wood chips, 3% wheat bran, 1% soybean cake powder, 1% lime and 1% gypsum, wherein the miscellaneous wood chips are oak wood chips, the mass ratio of coarse wood chips to fine wood chips is 2:1, the particle size of coarse wood chips is 0.5 cm, and the particle size of fine wood chips is 0.3 cm.
[0100] (2) After inoculation, cultivate at 19℃ for 32 days in dark with humidity of 30%, stop cultivation when mycelium grows to 0.5 cm before the bottom of the culture medium.
[0101] (3) Pick up the wood chip strain 0.5 cm from the tip of the mycelium, the mycelium block is a round cake with a diameter of 0.3 cm, transfer to the poor PDA medium for activation, the poor PDA medium formula is: 200 g / L potatoes, 10 g / L glucose and 20 g / L agar powder.
[0102] (4) After inoculation, cultivate at 21℃ for 11 days in dark with humidity of 35%, stop cultivation when mycelium grows to 0.5 cm before the edge of the culture dish.
[0103] (5) Pick up the mycelium block 0.5 cm from the tip of the mycelium, the mycelium block is a round cake with a diameter of 0.4 cm, transfer 10 round cakes to the wood chip culture medium, the wood chip culture medium formula is: 92% miscellaneous wood chips, 5% wheat bran, 1% soybean cake powder, 1% lime and 1% gypsum, wherein the miscellaneous wood chips are oak wood chips, and the mass ratio of coarse wood chips to fine wood chips is 3:2.
[0104] (6) After inoculation, cultivate at 23℃ for 28 days in dark with humidity of 30%, stop cultivation when mycelium grows to 0.5 cm before the bottom of the culture medium.
[0105] (7) Pick up the wood chip strain 0.5 cm from the tip of the mycelium, the mycelium block is a round cake with a diameter of 0.4 cm, transfer to the PDA medium for activation, the PDA medium formula is: 200 g / L potatoes, 15 g / L glucose and 20 g / L agar powder.
[0106] (8) Inoculation at 23°C for 11 days in low temperature and dark, humidity 30%-40%, stop culture when mycelium grows to 0.5 cm before the edge of the culture dish.
[0107] (9) Pick up the mycelium block 0.5 cm from the tip of the mycelium, the block is a round cake with a diameter of 0.4 cm, transfer 10 round cakes to the conventional wood chip culture medium, the formula of the conventional wood chip culture medium is: 85% mixed wood chip, 10% wheat bran, 3% soybean cake powder, 1% lime, 1% gypsum. The mixed wood chip is oak wood chip, the mass ratio of coarse wood chip to fine wood chip is 1:1.
[0108] (10) Inoculation at 27°C for 28 days in low temperature and dark, humidity 35%, stop culture when mycelium grows to 0.5 cm before the bottom of the culture medium.
[0109] (11) Pick up the wood chip mycelium 0.5 cm from the tip of the mycelium, the block is a round cake with a diameter of 0.3 cm, transfer to cPDA culture medium for activation, the formula of PDA culture medium is: potato 200 g / L, glucose 15 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 1 g / L, agar powder 20 g / L.
[0110] (12) Inoculation at 26°C for 11 days in dark, humidity 35%, stop culture when mycelium grows to 0.5 cm before the edge of the culture dish. Store the obtained Auricularia auricula-judae mycelium in a 4°C refrigerator for standby use.
[0111] Example 5
[0112] Strain identification
[0113] The Auricularia auricula-judae 29G1, Auricularia auricula-judae 29G10 and the strains cultured in Examples 2-4 (Auricularia auricula-judae 29G10 after strain restoration) are respectively detected for the following indexes: mycelial growth, mycelial growth rate, Auricularia auricula-judae mycelial amylase activity, Auricularia auricula-judae mycelial laccase activity, Auricularia auricula-judae mycelial cellulase activity, Auricularia auricula-judae mycelial trypsin activity, single bag yield and Auricularia auricula-judae fruit body sensory evaluation score. The mycelial growth rate is determined by cross-line method. The Auricularia auricula-judae mycelial amylase activity, laccase activity, cellulase activity and trypsin activity are detected by using enzyme activity detection kit of Suzhou Gexisi Biological Technology Co., Ltd. according to the operation steps of the kit. The standard of Auricularia auricula-judae fruit body sensory evaluation score is shown in Table 1. The detection samples are set in triplicate, and the detection results are calculated as average value.
[0114] Table 1 Standard table of Auricularia auricula-judae fruit body sensory evaluation
[0115]
[0116]
[0117] The mycelium growth form and growth vigor of the subculture were observed as shown in Fig. 1. Figure 2 After 10 times of subculture, the mycelium of A. aegyptiacus gradually became sparse and the growth vigor became weak (Fig. 1B). After recovery, the mycelium of A. aegyptiacus became dense and the growth vigor became strong (Fig. 1C). Figure 2 The mycelium growth vigor of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 1C vs. Fig. 1A). Therefore, subculture affected the mycelium growth vigor of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium growth vigor. Figure 2 Figure 2 The mycelium growth rate of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 2C vs. Fig. 2A). Therefore, subculture affected the mycelium growth rate of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium growth rate.
[0118] The mycelium amylase activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 3C vs. Fig. 3A). Therefore, subculture affected the mycelium amylase activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium amylase activity. Figure 3 The mycelium laccase activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 4C vs. Fig. 4A). Therefore, subculture affected the mycelium laccase activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium laccase activity.
[0119] Figure 4 The mycelium cellulase activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 5C vs. Fig. 5A). Therefore, subculture affected the mycelium cellulase activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium cellulase activity.
[0120] The mycelium trypsin activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 6C vs. Fig. 6A). Therefore, subculture affected the mycelium trypsin activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium trypsin activity. Figure 5 The mycelium amylase activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 3C vs. Fig. 3A). Therefore, subculture affected the mycelium amylase activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium amylase activity.
[0121] Figure 6 The mycelium laccase activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 4C vs. Fig. 4A). Therefore, subculture affected the mycelium laccase activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium laccase activity.
[0122] The mycelium cellulase activity of A. aegyptiacus after recovery was not significantly different from that of A. aegyptiacus G1 (Fig. 5C vs. Fig. 5A). Therefore, subculture affected the mycelium cellulase activity of A. aegyptiacus, and recovery was beneficial to the recovery of the mycelium cellulase activity. Figure 7 The trypsin activity of the mycelium of Hei 29G10- after recovery was not significantly different from that of Hei 29G1 (P>0.05). Therefore, the subculture affected the trypsin activity of the mycelium of Auricularia auricula-judae, and recovery was beneficial to the recovery of the trypsin activity of the mycelium.
[0123] The results of the single-bag yield after subculture are shown in Table 4. Figure 8 The single-bag yield of Hei 29 after 10 times of subculture was significantly lower than that of Hei 29G1 (P<0.05). Therefore, the subculture significantly affected the single-bag yield of Auricularia auricula-judae. The single-bag yield of Hei 29G10- after recovery was not significantly different from that of Hei 29G1 (P>0.05). Therefore, the subculture affected the single-bag yield of Auricularia auricula-judae, and recovery was beneficial to the recovery of the single-bag yield.
[0124] The sensory evaluation scores of the fruiting bodies of Auricularia auricula-judae are shown in Table 5. Figure 9 The sensory evaluation scores of Auricularia auricula-judae after 10 times of subculture were significantly lower than those of Hei 29G1 (P<0.05). Therefore, the subculture significantly affected the sensory evaluation scores of Auricularia auricula-judae. The sensory evaluation scores of Hei 29G10- after recovery were not significantly different from those of Hei 29G1 (P>0.05). Therefore, the subculture affected the sensory evaluation scores of Auricularia auricula-judae, and recovery was beneficial to the recovery of the sensory evaluation scores.
[0125] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for restoring the strain identity of a subcultured degenerative Auricularia auricula-judae strain, characterized by, Includes the following steps: The activated subgenerated degenerated black fungus strain was inoculated into a barren sawdust culture medium and cultured for the first time at 18-20℃ to obtain the first mycelium; The barren sawdust culture medium consists of the following components by weight percentage: 93.5%–94.5% sawdust, 2.8%–3.2% wheat bran, 0.9%–1.1% soybean meal, 0.9%–1.1% lime, and 0.9%–1.1% gypsum; the sawdust in the barren sawdust culture medium is coarse sawdust and fine sawdust; the mass ratio of coarse sawdust to fine sawdust is 2:1; the humidity of the first cultivation culture is 25%–35%. The first mycelium was inoculated into a poor PDA medium and activated at 20–22°C to obtain the second mycelium; The depleted PDA culture medium consists of the following components: 200 g / L potato, 10 g / L glucose, and 20 g / L agar powder; the humidity of the first activation culture is 30%–40%. The second mycelium was inoculated into a sawdust culture medium and cultured at 22–25°C to obtain the third mycelium; The sawdust culture medium comprises the following components by weight percentage: 91.5%–92.5% sawdust, 4.8%–5.2% wheat bran, 0.9%–1.1% soybean meal, 0.9%–1.1% lime, and 0.9%–1.1% gypsum; the sawdust in the culture medium is coarse sawdust and fine sawdust; the mass ratio of coarse sawdust to fine sawdust is 3:2; the humidity of the second cultivation culture is 25%–35%. The third hyphae were inoculated into PDA medium and subjected to a second activation culture at 22–25°C to obtain the fourth hyphae; The PDA culture medium consists of the following components: 200 g / L potato, 15 g / L glucose, and 20 g / L agar powder; the humidity of the second activation culture is 30%–40%. The fourth mycelium was inoculated into a conventional sawdust culture medium and cultured for the third time at 25-28°C to obtain the fifth mycelium; The conventional sawdust culture medium consists of the following components by weight percentage: 84.5%–85.5% sawdust, 9.5%–10.5% wheat bran, 2.8%–3.2% soybean meal, 0.9%–1.1% lime, and 0.9%–1.1% gypsum; the sawdust in the conventional sawdust culture medium is coarse sawdust and fine sawdust, and the mass ratio of coarse sawdust to fine sawdust is 1:1; the humidity of the third cultivation culture is 30%–40%; The fifth mycelium was inoculated into cPDA medium and activated in a third culture at 25-28℃ to obtain a black fungus strain with restored characteristics. The cPDA culture medium package consists of the following components: 200 g / L potato, 15 g / L glucose, 2 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, and 20 g / L agar powder; the humidity of the third activation culture is 30%–40%.
2. The method of claim 1, wherein the strain of Auricularia auricula-judae is a subcultured degenerative strain. The wood chips include oak wood chips.
3. The method of claim 1, wherein the strain of Auricularia auricula-judae is a subcultured degenerative strain of Auricularia auricula-judae. The duration of the first, second, or third activation culture is 10–12 days. The cultivation period for either the first or second cultivation is 30–35 days. The third cultivation period is 25–30 days.
4. The method of claim 1, wherein the strain of Auricularia auricula-judae is a subcultured degenerative strain of Auricularia auricula-judae. The first activation culture, the second activation culture, the third activation culture, the first cultivation culture, the second cultivation culture, or the third cultivation culture is a light-avoiding culture.
5. The method for restoring the fruiting body characteristics of a subcultured degenerative Auricularia auricular-judae strain according to any one of claims 1 to 4, wherein the degenerative strain is a strain of Auricularia auricular-judae that has been subcultured for more than 10 generations. The Auricularia auricular-judae includes an Auricularia auricular-judae black 29 strain.
6. Use of the method for recovering the species character of the subcultured degenerative Auricularia auricular-judae strain in Auricularia auricular-judae breeding and / or preservation.
Citation Information
Patent Citations
Simple black fungus strain rejuvenation method
CN113711842A