A method for cultivating Flammulina velutipes strains

Abstract

A method for cultivating Flammulina velutipes strains, by selecting well-growing fruiting bodies, performing tissue separation, activating and detoxifying on culture medium I, and then rejuvenating and verifying mushroom production on culture medium II, thereby realizing rapid activation and rejuvenation of the strains separated from the fruiting body tissues. The cultivation process of the invention is simple and convenient, the cultivation cost is low, and the cultivated Flammulina velutipes strains are of stable quality and strong vitality, meeting the actual needs of enterprises.

Description

Technical Field

[0001] The invention relates to the field of edible fungi, and in particular to a method for cultivating Flammulina velutipes strains. Background Art

[0002] Flammulina velutipes is an edible fungus cultivated in autumn, winter and early spring. It is famous for its smooth cap, crisp stem and delicious taste. It is especially a good ingredient for cold dishes and hot pot. It is nutritious, fragrant and delicious, and is deeply loved by the public.

[0003] Flammulina velutipes often uses tissue separation breeding technology to cultivate mycelium. Usually, the growth point at the junction of the mushroom cap and the mushroom stem is taken and placed on a PDA plate for cultivation. Mycelium will sprout from the growth point, and the performance of the fruiting body strain is verified through separation and purification, activation, and mushroom fruiting tests.

[0004] At present, this breeding technology has the following problems:

[0005] 1. The strains extracted from the fruiting bodies are basically purified and subcultured on PDA chemical culture medium, which makes it difficult to improve the activity of the isolated strains;

[0006] 2. The characteristics of the bacteria isolated and extracted from tissues are unstable and their use cycle is short.

[0007] Therefore, how to design a method for cultivating bacteria with stable quality and strong vitality is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0008] The purpose of the present invention is to provide a method for cultivating Flammulina velutipes strains in view of the deficiencies in the prior art. The method has a simple and convenient cultivation process, low cultivation cost, and the cultivated Flammulina velutipes strains have stable quality and strong vitality, thus meeting the actual needs of enterprises.

[0009] The technical solution of the present invention is: a method for cultivating Flammulina velutipes strains, comprising the following steps:

[0010] 1) Preparation of culture medium I

[0011] Take 35-40 parts by weight of agar medium and 5-10 parts by weight of agar powder, add them into water and mix well, sterilize to prepare a solid culture medium for later use;

[0012] 2) Preparation of culture medium II

[0013] Sawdust and rice bran are mixed, the moisture content is 60-65%, and the pH is ≥6.0, the mass ratio of sawdust to rice bran is 6-7:3-4, and the mixture is sterilized for standby use, wherein the sawdust is poplar sawdust with a particle size of ≤2 mm, obtained by fermentation;

[0014] 3) Take the fruiting body of Flammulina velutipes, disinfect it, cut the growth point at the junction of the mushroom cap and the mushroom stem, inoculate it on the solid culture medium I, and make sure the cut surface of the growth point is in contact with the culture medium I. Culture it in a closed environment at 18±0.5℃ for 10-13 days;

[0015] 4) scraping off 1-2 cm of the extended ends of the aerial hyphae, and cutting a plurality of first inoculation blocks along the edges of the aerial hyphae on the solid culture medium I;

[0016] 5) inoculating the first inoculum obtained in step 4) on another solid culture medium I, and culturing at 18±0.5°C for 8-10 days;

[0017] 6) Scrape off 1-2 cm of the extended ends of the aerial hyphae and cut multiple second inoculation blocks on the solid culture medium I;

[0018] 7) inoculating the second inoculum obtained in step 6) onto culture medium II, and culturing in a closed environment at 19° C. until the hyphae grow downward to 7-9 cm;

[0019] 8) cutting off the culture medium II at the upper part of the middle height of the hyphae, and cutting a plurality of third inoculation blocks on the culture medium II at the lower part;

[0020] 9) inoculating the third inoculum obtained in step 8) onto a PDA plate culture medium, and culturing in a closed environment at 18±0.5° C. for 10-15 days;

[0021] 10) Transfer the culture medium to another PDA plate and grow it to maturity to obtain the Flammulina velutipes strain.

[0022] Furthermore, in step 1), the weight volume ratio of agar culture medium to water is 35-45:1000.

[0023] Preferably, in step 1), the mass volume ratio of agar medium, agar powder and water is 35-45:3-6:1000.

[0024] Preferably, the thickness of the solid culture medium I obtained in step 1) is 4-6 cm.

[0025] Preferably, in step 2), the mass ratio of sawdust to rice bran is 6.2:3.8, and the moisture content is 64%.

[0026] Furthermore, in step 4), a first inoculation block is obtained by punching, and the diameter of the first inoculation block is 4 mm.

[0027] Furthermore, in step 6), a second inoculation block is obtained by punching, and the diameter of the second inoculation block is 4 mm.

[0028] Furthermore, in step 8), the size of the third inoculation block is 4 mm*4 mm*4 mm.

[0029] The above technical solution has the following beneficial effects:

[0030] 1. The cultivation method of the present invention is to activate the fungus in two-stage culture medium I, rejuvenate in culture medium II, and finally purify on PDA flat culture medium, so that the cultivated Flammulina velutipes strain has strong vitality. The hyphae generated on culture medium II and PDA flat culture medium are dense and white, with good vitality and few powder spores. The ball size of the liquid strain in the shake bottle is uniform, the length of the fungus thorn is moderate, the fungus liquid has no obvious stratification, and the use period can be extended by at least 3 months.

[0031] 2. The cultivation method of the present invention comprises the following steps: the culture medium I is used to activate the inoculated growth points of Flammulina velutipes, and the growth points of Flammulina velutipes quickly germinate and grow on the culture medium to obtain activated strains. The culture medium II is used to rejuvenate the activated strains, and finally the rejuvenated strains are purified by using a PDA plate culture medium. The culture medium I and the PDA culture medium are used as activation and purification culture mediums to make the strains germinate and grow quickly, and the culture medium II is used to rejuvenate the strains, and finally an activated, detoxified, purified, and vigorous strain is obtained.

[0032] 3. The present invention first utilizes the separated growth points of Flammulina velutipes to culture on culture medium I for 10-13 days, cuts off culture medium I containing mycelium, and inoculates it on another culture medium I, and cultures it at 18±0.5°C for 8-10 days, so that the strains grown on the other culture medium I are activated, purified, and detoxified to obtain a single strain. The activated strains are then inoculated into culture medium II, and the upper part of culture medium II is cut off, and the culture medium with moderate humidity and moderate mycelium age is taken from the middle part for transfer, so that the strains can grow and germinate quickly in the next step.

[0033] The applicant has verified through experiments that the Flammulina velutipes strains cultivated by the present invention have improved vitality, improved mycelium density and whiteness, significantly reduced powder spore count, uniform ball size and moderate thorn length of the liquid strain in the shake bottle, stable mycelium content of the production strain liquid at 1.1-1.3 quality inspection, and the mushroom cap of the Flammulina velutipes fruiting body obtained by cultivation is moderate in size and the mushroom body is firm, thus being a high-quality Flammulina velutipes product.

[0034] Further description is given below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The photos of the fruiting body cap, mushroom stem and mushroom root harvested in Example 2;

[0036] Figure 2 These are photos of the fruiting body cap, mushroom stem and mushroom root harvested from Control Example 2. DETAILED DESCRIPTION

[0037] Example 1

[0038] Prepare medium I:

[0039] Formula: 35 parts of agar culture medium, 5-10 parts of agar powder, 1L purified water, natural pH, mix well. Dispense into 1000mL triangular flasks, 500-600mL per triangular flask, wrap with cotton plugs and aluminum foil, sterilize at 121℃ for 20min, and cool naturally. After sterilization, cool to 50-60℃ and pour into a plate, ensuring that the culture medium thickness is 4-6mm, and set aside after solidification.

[0040] Preparation of culture medium II

[0041] Formula: 62% sawdust, 38% rice bran, 64% moisture content, natural pH. The sawdust is fine poplar sawdust that has been naturally fermented for 1 month, turned over twice a day, with an initial moisture of ≤65%, a pH of 6.0-7.4, and a particle size of ≤2mm, all passed through a 2mm round hole sieve. Use a 20mm*20cm glass test tube, fill each test tube with 10g, and the filling height is 10cm (from the bottom of the test tube upwards). Use a silicone test tube plug with a sand core to seal the solid culture medium test tube, sterilize it with high-pressure steam at 121℃ for 90min, and put it in a clean bench to cool it down to below 20℃.

[0042] Methods for separation of fruiting body tissue:

[0043] Select mushrooms with hard, straight, non-bending or cracked stems, unopened caps, and round, plump fruiting bodies. Spray an appropriate amount of alcohol for surface disinfection, gently remove the caps with tweezers, carefully cut off the conical growth point at the junction of the cap and stem with a scalpel, and use an inoculation needle to connect the growth point to the center of the flat plate culture dish of medium I, with the cut surface facing down, and gently press the growth point to make it completely contact with the surface of the culture medium. After sealing the plate with a sealing film, place it in a culture medium at 18±0.5℃ for 12 days, which is the first generation plate (P1).

[0044] The process of activation and rejuvenation of isolated strains from fruiting body tissue:

[0045] When the aerial hyphae on the surface of the P1 generation plate grows to 1-2 cm away from the wall of the culture dish, select the P1 plate with the best growth, scrape 1-2 cm of the aerial hyphae toward the center, and punch holes along the edge of the aerial hyphae with a 4 mm puncher. Then, the punched inoculum block is inoculated into the center of the plate culture dish of fresh culture medium I. After the plate is sealed with a sealing film, it is placed at 17.8°C and cultured for 9 days. This is the second generation plate (P2).

[0046] After two generations of plate culture, P1 and P2, the strains were purified and detoxified, and then entered the rejuvenation process.

[0047] Select the P2 plate strains with good growth for inoculation. Use a scalpel to scrape off 1-2 cm of aerial hyphae according to the growth of powder spores, and then use a 4mm puncher to punch holes. Use an inoculation needle to pick up the agar block with a diameter of 4 mm into the test tube containing the solid culture medium. Connect 5 inoculation blocks to each test tube, and make each inoculation block contact with the culture medium, and plug the test tube stopper. The inoculation operation is carried out in an ultra-clean workbench to prevent contamination. The inoculated solid test tubes are placed in a biochemical incubator at 19°C for culture until the mycelium grows down to about 8 cm. After the mycelium has grown well, the solid test tubes are selected according to the growth, mycelium state, and the number of powder spores, and the strains in the solid test tubes are taken out. Specifically:

[0048] For the strains in the solid culture medium, mark the center point of the culture medium height where the mycelium grows on the test tube, remove all the culture medium II on the top, pick out the solid culture medium from the middle of the test tube with an inoculation needle, cut it into small pieces of about 4mm*4mm*4mm, place it in the center of the PDA plate, and then gently press the culture medium block to make it better contact the surface of the culture medium, seal it with a sealing film, and place the plate in a 18±0.5℃ biochemical incubator for growth. The plate inoculated with the solid culture medium strain will grow to maturity in about 10-15 days, and then the most suitable plate strain will be selected according to the growth of the plate, and the plate strain will be transferred to another plate culture medium according to the normal transfer process, that is, the strain rejuvenation is completed, and this is the third generation plate (P3). After the P3 plate grows to maturity, the strain that has been separated, purified, activated, and rejuvenated is obtained.

[0049] Example 2

[0050] Take the strain cultivated in Example 1 and cultivate it according to the factory production process of Flammulina velutipes. The fruiting bodies of Flammulina velutipes obtained are shown in the following figure: Figure 1 shown.

[0051] Comparative Example 1

[0052] Take the same batch of Flammulina velutipes as in Example 1, take its growth point to the center of the PDA medium, culture at 17.8°C for 12 days to obtain a P1 plate, select the P1 plate strain with good growth for inoculation, scrape off 1-2 cm of aerial hyphae with a scalpel according to the growth of powder spores, and then use a 4mm puncher to perform a hole treatment. Place an agar block with a diameter of 4 mm on the PDA plate, culture at 17.8°C for 9 days to obtain a P2 plate, and cultivate the P2 plate strain according to the factory production process of Flammulina velutipes.

[0053] Comparative Example 2

[0054] Take the strain cultivated in Control Example 1 and cultivate it according to the factory production process of Flammulina velutipes. The photos of the mushroom cap, mushroom stem and mushroom root of the harvested Flammulina velutipes are as follows: Figure 2 shown.

[0055] The Flammulina velutipes fruiting bodies harvested from Example 2 and Comparative Example 2 were compared as follows:

[0056] As shown in the table above, compared with the original strain, the strain cultivated by the present invention has little difference in properties. The original strain has a tendency to degenerate, the cap is easy to enlarge, and the vitality begins to decline. The strain separated from the fruiting body tissue after activation and rejuvenation has improved vitality and the cap size is moderate. The average yield of the products compared with the two strains is not much different, and the quality is improved.

Claims

1. A method for cultivating Flammulina velutipes strains, characterized in that: The following steps are involved: 1) Preparation of culture medium I Take 35-40 parts by weight of agar medium and 5-10 parts by weight of agar powder, add them into water and mix well, sterilize to prepare a solid culture medium for later use; 2) Preparation of culture medium II Mix sawdust and rice bran, with a moisture content of 60-65%, a pH value of ≥6.0, and a mass ratio of sawdust to rice bran of 6-7:3-4, sterilize and set aside, wherein the sawdust is poplar sawdust with a particle size of ≤2 mm, obtained by fermentation; 3) Take the fruiting body of Flammulina velutipes, disinfect it, cut the growth point where the mushroom cap and the mushroom stem are connected, inoculate it on solid culture medium I, and make sure the cut surface of the growth point is in contact with culture medium I. Culture it in a closed environment at 18±0.5℃ for 10-13 days; 4) Scrape off 1-2 cm of the extended ends of the aerial hyphae, and cut multiple first inoculation blocks along the edge of the aerial hyphae on the solid culture medium I; 5) inoculating the first inoculum obtained in step 4) on another solid culture medium I and culturing for 8-10 days; 6) Scrape off 1-2 cm of the extended ends of the aerial hyphae and cut multiple second inoculation blocks on the solid medium I; 7) Inoculate the second inoculum obtained in step 6) onto culture medium II, and culture in a closed environment at 19° C. until the hyphae grow downward to 7-9 cm; 8) Cut off the culture medium II at the upper middle height of the mycelium, and cut multiple third inoculation blocks on the culture medium II at the lower part; 9) inoculating the third inoculum obtained in step 8) onto a PDA plate culture medium, and culturing in a closed environment at 18±0.5°C for 10-15 days; 10) Transfer to another PDA plate culture medium and grow to maturity to obtain Flammulina velutipes strains.

2. The cultivation method according to claim 1, characterized in that The thickness of the solid culture medium I obtained in step 1) is 4-6 cm.

3. The method for cultivating Flammulina velutipes strains according to claim 1, characterized in that: Step 2) The mass ratio of sawdust to rice bran is 6.2:3.8, and the moisture content is 64%.

4. The method for cultivating Flammulina velutipes strains according to claim 1, characterized in that: Step 4) A first inoculation block is obtained by punching, and the diameter of the first inoculation block is 4 mm.

5. The method for cultivating Flammulina velutipes strains according to claim 1, characterized in that: Step 6) A second inoculation block is obtained by punching, and the diameter of the second inoculation block is 4 mm.

6. The method for cultivating Flammulina velutipes strains according to claim 1, characterized in that: Step 8) The size of the third inoculation block is 4 mm*4 mm*4 mm.

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