Tremella aurantialba strain DZHSSJE001 and industrial cultivation method thereof

Abstract

The invention relates to the technical field of biology, in particular to a tremella aurantialba strain DZHSSJE001 and an industrial cultivation method thereof, the tremella aurantialba strain DZHSSJE001 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.41289, the ITS sequence is shown as SEQ NO.1, the tremella aurantialba strain is in a yeast state in a culture state, conidia are oval, most of the conidia are top bud reproduction, 1.6-2.7 * 3.4-4.1 [mu] m, and the preservation number is CGMCC NO.41289. Meanwhile, the invention further discloses an industrial cultivation method of the tremella aurantialba strain DZHSSJE001, and the problem that the industrial cultivation yield of the tremella aurantialba strain is poor is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Tremella fuciformis strain DZHSSJE001 and a factory cultivation method thereof. Background Art

[0002] Naematelia aurantialba (Bandoni & M.Zang) Millanes & Wedin) is a fungus of the family Naemateliae and the genus Naemateliae. The basidiocarp is large, hemispherical to amorphous, and is brain-shaped or split into several deep-grooved and thick lobes, 3-12 cm in diameter and 2-8 cm in height. The hymenium is all over the exposed surface, a broad and loose area containing the basidium; the protobasidium is initially elliptical to ovate; the mature lower basidium is spherical, subglobose, rarely pear-shaped to with a short-stalked base, 13-25 μm long and 11.5-19.5 picometers wide.

[0003] The fruiting bodies of golden ear are composed of hairy leather fungi and golden ear hyphae. Golden ear spores mostly grow in the form of budding on PDA. At present, the production of golden ear mostly uses the combination of golden ear solid species and leather fungi solid species, the combination of golden ear solid species and leather fungi liquid species, and the direct use of golden ear solid species as liquid species to inoculate mushroom sticks. The first two methods use a large amount of golden ear solid species, and the strain is unstable, which can easily cause batch contamination or poor fruiting body development. The direct use of golden ear solid species as liquid strains will result in inconsistent appearance of golden ear after inoculation, making mushroom management difficult, and the strain is prone to degeneration during generation, so the strain cannot be stored for a long time, and the quality of factory production batches is unstable.

[0004] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0005] The invention aims to solve the problem of poor quality of Tremella fuciformis strains in factory cultivation, and provides a Tremella fuciformis strain DZHSSJE001 and a factory cultivation method thereof.

[0006] In order to achieve the above purpose, the present invention discloses a Tremella fuciformis strain DZHSSJE001, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC NO.41289 and an ITS sequence as shown in SEQ NO.1.

[0007] The present invention also discloses a factory-based cultivation method of the Tremella fuciformis strain DZHSSJE001, comprising the following steps:

[0008] S1, medium preparation: add corn flour, potassium dihydrogen phosphate, and magnesium sulfate into water, heat with an induction cooker, stir until viscous, take 500 mL of each, put into a 1000 mL conical flask, seal with a silicone stopper, sterilize with high-pressure steam at 121°C for 30 min, and cool on a clean bench to below 25°C after sterilization;

[0009] S2, inoculation culture: take 1cm from the test tube or plate 2 The spores of golden ear of different sizes are inoculated into the culture medium in step S1 at a temperature below 25°C. After inoculation, the conical flask is placed on a shaker at a speed of 150 r / min, a temperature of 18-22°C, and cultured in the dark for 5 days. After the culture is completed, it can be used for production;

[0010] S3, preparation of conical flasks: peel potatoes and weigh 200g, boil them in water and add the potatoes, keep boiling for 20min, turn off the heat and add glucose, divide into 2 1000mL conical flasks, 500mL each, seal with silicone stoppers, sterilize with high pressure steam at 121℃ for 30min, and cool on a clean bench to below 25℃ after sterilization;

[0011] S4, take 4cm from the test tube or plate 2 The leather fungus of size is inoculated in the conical flask in step S3 at a temperature below 25°C, and after cultivation, the fermentation tank is inoculated;

[0012] S5, after sterilizing the fermentation tank, wait for the temperature to drop to 20℃, wait for 15 minutes for inoculation, take a cotton swab soaked in 95° alcohol and wrap it around the inoculation port, light it, quickly unscrew the inoculation port cover, sterilize the conical bottle mouth on the flame for 10 seconds, then pour the liquid bacterial strain of the leather fungus in the conical bottle into the fermentation tank, and test the remaining 10mL, pour the remaining bacterial strain on the sterilized plate to check whether there is contamination, sterilize the inoculation port cover on the flame for 10-15 seconds, quickly tighten it, and culture at 22℃ for 4 days;

[0013] S6, inject 4-5 mL of the liquid bacterial strain of the fungus into the bacterial stick, and then inject 0.1-0.3 mL of the golden ear spores into the same position;

[0014] S7. After inoculation, place the mushroom sticks in a room at 22℃, away from light, and control the carbon dioxide concentration below 1000ppm for cultivation. After 30-35 days, the primordium at the inoculation site begins to bulge. At this time, use a sharp knife to open it and harvest after the mushrooms have grown.

[0015] In step S1, the formula of the culture medium is: 5 g corn flour, 1 g potassium dihydrogen phosphate, 1 g magnesium sulfate, and 1 L water, and the corn flour is sieved through 80 mesh.

[0016] In step S3, the conical flask formula is: 200g potatoes, 20g glucose, and 1L water.

[0017] In step S5, the formula of the fermentation tank is: 0.5% potato flour, 0.5% yeast powder, 2% glucose, 0.15% potassium dihydrogen phosphate, and 0.15% magnesium sulfate.

[0018] In step S6, the formula of the mushroom stick is: 88% hardwood sawdust, 11% wheat bran, and 1% gypsum. The production process is as follows: add water to the materials according to the formula and mix well, adjust the humidity to 58%-60%, bag, sterilize, and cool to below 25°C before inoculation.

[0019] In step S7, the conditions for cultivating bacteria are as follows: the inoculated bacterial sticks are placed in a room at 22° C., away from light, and the carbon dioxide concentration is controlled to be less than 1000 ppm.

[0020] In step S7, the specific process of mushroom production management is as follows: the opened mushroom sticks are transferred to the mushroom production room, and the humidity is controlled at 88-95% and the carbon dioxide concentration is lower than 1000ppm in the first 15 days. The light intensity is increased to 600-900lx in the next 15 days to promote color change.

[0021] In step S7, the specific process of harvesting is as follows: when the golden ears are open for 25-30 days, the color is golden yellow, and the appearance is round and plump, they are cut off with a sharp knife and placed in a turnover basket.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The preparation of the golden ear spores in the present invention is simple, and the inoculation amount is easy to control, so that when the golden ear grows into a fruiting body on a mushroom stick, the time is consistent and the size is consistent, and the factory-based mushroom production management is more convenient. The golden ear solid seed is directly used as a liquid strain, and the liquid contains golden ear hyphae and leather mushroom hyphae. The amount of golden ear hyphae inoculated into the mushroom stick affects the time for the golden ear to grow into a fruiting body on the mushroom stick. By controlling the amount of the golden ear spores when inoculating the golden ear spores, the development time and size can be consistent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the operation diagram of inoculating bacteria sticks;

[0025] Figure 2 This is a diagram of the injection operation of golden ear spores;

[0026] Figure 3 This is a schematic diagram of mushroom opening;

[0027] Figure 4 Among them, (a) is the effect of inoculating Tremella fuciformis spores, and (b) is the effect of Tremella fuciformis solid directly as liquid strain;

[0028] Figure 5 It is the PDA culture medium morphology of Auricularia auricula spores;

[0029] Figure 6This is the harvesting form of golden ear spores DETAILED DESCRIPTION

[0030] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0031] The golden ear strain DZHSSJE001 involved in the following embodiments was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on April 19, 2024, with the deposit number CGMCC NO.41289, the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the ITS sequence is shown in SEQ NO.1.

[0032] Example

[0033] 1. Expansion of golden ear spores

[0034] (1) Culture medium preparation

[0035] Culture medium formula: 5g corn flour (sieved 80 mesh), 1g potassium dihydrogen phosphate, 1g magnesium sulfate, and 1L water.

[0036] Preparation method: Add corn flour, potassium dihydrogen phosphate and magnesium sulfate into water, heat with an induction cooker, and stir until viscous. Take 500 ml of each, put into a 1000 ml conical bottle, seal with a silicone plug, and sterilize with high-pressure steam at 121°C for 30 minutes. After sterilization, place on a clean bench to cool to below 25°C.

[0037] (2) Inoculate the spores of Auricularia auricula into a conical flask

[0038] Take 1 square centimeter of Auricularia aurantia spores from a test tube or plate and inoculate it into the above-mentioned culture medium at below 25°C.

[0039] (3) Culture of Auricularia auricula spores

[0040] After inoculation, place the conical flask on a shaker at a speed of 150 r / min, a temperature of 18-22°C, and incubate for 5 days in the dark. After the incubation, it can be used for production.

[0041] 2. Preparation of liquid strains of associated fungi

[0042] (1) Conical flask for inoculation of fungi

[0043] Conical flask formula: 200g potatoes, 20g glucose, 1L water

[0044] Preparation of conical flasks: Peel the potatoes and weigh 200g, boil them in water and add the potatoes, keep boiling for 20min, turn off the heat and add glucose, divide into 2 1000mL conical flasks, 500mL each, seal with silicone stoppers, sterilize with high pressure steam at 121℃ for 30 minutes. After sterilization, cool on a clean bench to below 25℃.

[0045] Take a 4 cm2 area of ​​fungus from a test tube or plate and inoculate it into the above-mentioned conical flask at a temperature below 25°C.

[0046] (2) Cultivation:

[0047] One day in advance, turn on the ultraviolet light for 1 hour to disinfect the magnetic stirrer and the workbench where the magnetic stirrer is placed. Place the inoculated conical flask on the magnetic stirrer for rotational culture at a speed of 600-800 r / min.

[0048] After culturing at 21°C for 4 days, let it stand for 30 minutes, observe the percentage of mycelium after sedimentation, and select conical flasks with orange-yellow bacterial solution, sedimentation ratio ≥ 80%, and clear upper liquid for fermentation tank inoculation;

[0049] (3) Preparation of fermentation tank culture medium:

[0050] Set the temperature to 123℃, open the inoculation port of the fermentation tank, add water to 2cm above the tank mirror, and when the temperature in the tank rises to 50℃, take out a small amount of water to dissolve the material. Then, when the temperature in the tank reaches 90℃, add the material from the inoculation port and add 50mL of edible oil at the same time;

[0051] Use a step-by-step heating method to heat from 90°C to 123°C, and sterilize during the heating process;

[0052] (4) Fermentation tank inoculation:

[0053] After sterilization, wait for 15 minutes for inoculation after the temperature drops to 20°C;

[0054] Take a cotton swab soaked in 95° alcohol and wrap it around the inoculation port and light it. Quickly unscrew the inoculation port cap, sterilize the conical flask mouth over the flame for 10 seconds, then pour the liquid bacterial strain of the leather fungus in the conical flask into the fermentation tank, and test the remaining 10mL. Pour the remaining bacterial strain onto the sterilized plate to test for contamination. Sterilize the inoculation port cap over the flame for 10-15 seconds and quickly tighten it.

[0055] (5) Fermentation tank culture:

[0056] After culturing at 22°C for 4 days, the preparation is complete.

[0057] Fermentation tank formula: potato flour 0.5%, yeast powder 0.5%, glucose 2%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.15%

[0058] 3. Making mushroom sticks

[0059] Formula: 88% broadleaf sawdust, 11% wheat bran, 1% gypsum.

[0060] Add water to the above materials, mix well, adjust the humidity to 58%-60%, and pack into bags.

[0061] After sterilization, the culture medium can be cooled to below 25°C before inoculation.

[0062] 4. Inoculate the bacteria sticks

[0063] First, inject 4-5 mL of liquid fungus into the fungus stick. Figure 1 As shown, 0.1-0.3 ml of Auricularia auriculariae spores were then injected into the same position, as Figure 2 shown.

[0064] 5. Cultivate bacteria

[0065] After inoculation, the bacterial sticks were placed in a room at 22°C, away from light, with the carbon dioxide concentration controlled below 1000ppm, for bacterial cultivation.

[0066] 6. Opening and fruiting

[0067] After 30-35 days of cultivation, the primordium at the inoculation site begins to bulge. At this time, use a sharp knife to cut it open and start fruiting management. Figure 3 shown.

[0068] 7. Mushroom production management

[0069] The opened mushroom sticks are transferred to the mushroom house.

[0070] In the first 15 days, the humidity was controlled at 88-95% and the carbon dioxide concentration was lower than 1000ppm.

[0071] Increase light intensity to 600-900lx in the last 15 days to promote color change.

[0072] 8. Harvest

[0073] The golden ears open for 25-30 days and are golden yellow in color. Figure 6 As shown, when the appearance is round and full, use a sharp knife to cut it off and put it in a turnover basket.

[0074] Comparative Example

[0075] The preparation method of golden ear liquid spawn is as follows: use golden ear fruiting body original species to prepare spawn liquid, culture the spawn liquid at 21°C for 4 days, then inoculate the spawn liquid into a fermentation tank, culture it at 21°C for 4 days, and use it for mushroom stick production.

[0076] The specific steps include:

[0077] 1. Preparation before vaccination:

[0078] (1) Prepare liquid culture medium:

[0079] The culture medium formula is: glucose 40g, corn flour 4g, soybean meal powder 6g, potato flour 6g, peptone 2g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g;

[0080] Put the material into a measuring cup, add 1.6L of tap water, stir to dissolve the culture medium, and finally adjust the volume to 2L;

[0081] (2) Liquid culture medium packaging and processing:

[0082] Place the funnel on the conical flask, stir the culture medium in the measuring cup, and then pour it into the conical flask. Stop when the liquid level reaches 500ml, and repeat this step to dispense;

[0083] (3) Sterilization: sterilize the conical flask at 123°C with high temperature and high humidity;

[0084] (4) Disinfection: Disinfect the workbench with ultraviolet light, spray the surface of the culture bottle, conical flask, test tube, and inoculation spoon with alcohol evenly for disinfection, and then place them on the workbench;

[0085] 2. Original seed inoculation:

[0086] Open the culture bottle in front of an alcohol lamp, use an inoculation spoon to dig an inoculation block on the surface of the golden ear fruiting body original seed, the size of which should not exceed 1 cubic centimeter, take 2 blocks and inoculate them into 2 test tubes for testing;

[0087] Scoop out a flat spoon, i.e. 15 inoculated blocks, and put them into a conical flask in front of an alcohol lamp, sterilize them on the flame of the alcohol lamp, and cover it;

[0088] After the inoculation spoon is sterilized on a flame, the next conical bottle is inoculated;

[0089] After inoculating the last conical flask, take 2 pieces of the original seed of the golden ear fruiting body and inoculate them into 2 test tubes;

[0090] 3. Culture of bacterial liquid:

[0091] One day in advance, turn on the ultraviolet light for 1 hour to disinfect the magnetic stirrer and the workbench where the magnetic stirrer is placed. Place the inoculated conical flask on the magnetic stirrer for rotational culture at a speed of 600 r / min.

[0092] After culturing at 21°C for 4 days, let it stand for 30 minutes, observe the percentage of mycelium after sedimentation, and select conical flasks with orange-yellow bacterial solution, sedimentation ratio ≥ 80%, and clear upper liquid for fermentation tank inoculation;

[0093] 4. Preparation of fermentation tank culture medium:

[0094] (1) Culture medium preparation:

[0095] The culture medium materials are: 200g yeast powder, 180g peptone, 50g soybean meal powder, 46g potassium dihydrogen phosphate, 46g magnesium sulfate, 522g glucose;

[0096] Set the temperature to 123℃, open the inoculation port of the fermentation tank, add 87 catties of water to 2 cm above the tank mirror, and when the temperature in the tank rises to 50℃, take out a small amount of water to dissolve the material. Then, when the temperature in the tank reaches 90℃, add the material from the inoculation port and add 50 ml of edible oil at the same time;

[0097] (2) Sterilization:

[0098] Use a step-by-step heating method to raise the temperature from 90°C to 123°C, and perform sterilization during the heating process. The specific method is: when the temperature rises from 90°C to 100°C, reduce the steam intake; when the temperature rises to 105°C, turn off the steam; when the temperature rises to 115°C, sterilize the filter, turn on the steam, and keep it for 30 minutes. After the filter sterilization is completed, drain the steam. During the steam draining period, the temperature will rise to 123°C. When the temperature reaches 123°C, keep this temperature for 70 minutes of sterilization.

[0099] 5. Fermentation tank inoculation:

[0100] After sterilization, wait for 15 minutes for inoculation after the temperature drops to 20°C;

[0101] Take a cotton swab soaked in 95° alcohol and wrap it around the inoculation port and light it. Quickly unscrew the inoculation port cover, sterilize the triangular bottle mouth on the flame for 10 seconds, then pour the golden ear liquid culture in the conical bottle into the fermentation tank, and test the remaining part. Sterilize the inoculation port cover on the flame for 10-15 seconds and quickly tighten it;

[0102] 6. Fermentation tank culture:

[0103] After culturing at 21°C for 4 days, the preparation is complete.

[0104] The inoculation effects in the embodiments and comparative examples are as follows Figure 4 As shown, (a) is the effect of inoculating Tremella fuciformis spores, and the occurrence time is consistent, and (b) is the effect of directly using Tremella fuciformis solid as liquid culture, and the occurrence time is inconsistent.

[0105] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. A Tremella fuciformis strain DZHSSJE001, characterized in that: The genes are deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number of CGMCC NO.41289 and the ITS sequence is shown in SEQ NO.

1.

2. A factory cultivation method of the Tremella fuciformis strain DZHSSJE001 as claimed in claim 1, characterized in that: The following steps are involved: S1, medium preparation: add corn flour, potassium dihydrogen phosphate, and magnesium sulfate into water, heat with an induction cooker, stir until viscous, take 500 mL of each, put into a 1000 mL conical flask, seal with a silicone stopper, sterilize with high-pressure steam at 121°C for 30 min, and cool on a clean bench to below 25°C after sterilization; S2, inoculation culture: take 1cm from the test tube or plate 2 The spores of golden ear of different sizes are inoculated into the culture medium in step S1 at a temperature below 25°C. After inoculation, the conical flask is placed on a shaker at a speed of 150 r / min, a temperature of 18-22°C, and cultured in the dark for 5 days. After the culture is completed, it can be used for production; S3, preparation of conical flasks: peel potatoes and weigh 200g, boil them in water and add the potatoes, keep boiling for 20min, turn off the heat and add glucose, divide into 2 1000mL conical flasks, 500mL each, seal with silicone stoppers, sterilize with high pressure steam at 121℃ for 30min, and cool on a clean bench to below 25℃ after sterilization; S4, take 4cm from the test tube or plate 2 The leather fungus of size is inoculated in the conical flask in step S3 at a temperature below 25°C, and after cultivation, the fermentation tank is inoculated; S5, after sterilizing the fermentation tank, wait for the temperature to drop to 20℃, wait for 15 minutes for inoculation, take a cotton swab soaked in 95° alcohol and wrap it around the inoculation port, light it, quickly unscrew the inoculation port cover, sterilize the conical bottle mouth on the flame for 10 seconds, then pour the liquid bacterial strain of the leather fungus in the conical bottle into the fermentation tank, and test the remaining 10mL, pour the remaining bacterial strain on the sterilized plate to check whether there is contamination, sterilize the inoculation port cover on the flame for 10-15 seconds, quickly tighten it, and culture at 22℃ for 4 days; S6, inject 4-5 mL of the liquid bacterial strain of the fungus into the bacterial stick, and then inject 0.1-0.3 mL of the golden ear spores into the same position; S7. After inoculation, place the mushroom sticks in a room at 22℃, away from light, and control the carbon dioxide concentration below 1000ppm for cultivation. After 30-35 days, the primordium at the inoculation site begins to bulge. At this time, use a sharp knife to open it and harvest after the mushrooms have grown.

3. The factory cultivation method of a Tremella fuciformis strain DZHSSJE001 as claimed in claim 2, characterized in that: In step S1, the formula of the culture medium is: 5 g corn flour, 1 g potassium dihydrogen phosphate, 1 g magnesium sulfate, and 1 L water, and the corn flour is sieved through 80 mesh.

4. The industrial cultivation method of the Tremella fuciformis strain DZHSSJE001 as claimed in claim 2, characterized in that: In step S3, the conical flask formula is: 200g potatoes, 20g glucose, and 1L water.

5. The industrial cultivation method of the Tremella fuciformis strain DZHSSJE001 as claimed in claim 2, characterized in that: In step S5, the formula of the fermentation tank is: 0.5% potato flour, 0.5% yeast powder, 2% glucose, 0.15% potassium dihydrogen phosphate, and 0.15% magnesium sulfate.

6. The industrial cultivation method of the Tremella fuciformis strain DZHSSJE001 according to claim 2, characterized in that: In step S6, the formula of the mushroom stick is: 88% hardwood sawdust, 11% wheat bran, and 1% gypsum. The production process is as follows: add water to the materials according to the formula and mix well, adjust the humidity to 58%-60%, bag, sterilize, and cool to below 25°C before inoculation.

7. The industrial cultivation method of the Tremella fuciformis strain DZHSSJE001 according to claim 2, characterized in that: In step S7, the conditions for cultivating bacteria are as follows: the inoculated bacterial sticks are placed in a room at 22° C., away from light, and the carbon dioxide concentration is controlled to be less than 1000 ppm.

8. The industrial cultivation method of the Tremella fuciformis strain DZHSSJE001 according to claim 2, characterized in that: In step S7, the specific process of mushroom production management is as follows: the opened mushroom sticks are transferred to the mushroom production room, and the humidity is controlled at 88-95% and the carbon dioxide concentration is lower than 1000ppm in the first 15 days. The light intensity is increased to 600-900lx in the next 15 days to promote color change.

9. The industrial cultivation method of the Tremella fuciformis strain DZHSSJE001 according to claim 2, characterized in that: In step S7, the specific process of harvesting is as follows: when the golden ears are open for 25-30 days, the color is golden yellow, and the appearance is round and full, they are cut off with a sharp knife and placed in a turnover basket.

Images