High-brittleness white flammulina velutipes HJ120 and application thereof

Abstract

The invention belongs to the technical field of edible mushroom cultivation, and particularly relates to a high-brittleness white flammulina velutipes HJ120 strain and application thereof. The technical problem to be solved by the invention is to cultivate a flammulina velutipes variety with better quality. According to the technical scheme of the invention, the high-brittleness white flammulina velutipes HJ120 is obtained, and the preservation number of the flammulina velutipes HJ120 is CCTCC (China Center for Type Culture Collection) NO: M 20242218. The invention also provides a food which comprises the white flammulina velutipes, spores of the white flammulina velutipes or / and an extract of the white flammulina velutipes.

Description

Technical Field

[0001] The present invention belongs to the technical field of edible mushroom cultivation, and particularly relates to a high-brittleness white Flammulina velutipes HJ120 and its application. Background Art

[0002] Flammulina velutipes, a fungus belonging to the genus Flammulina of the Tricholomataceae family, is an aerobic and light-averse fungus. Its cap is spherical, with a thin edge, yellowish-brown, smooth on the surface, connected at the base, and grows in clusters. The dried product resembles daylily, so it is named Flammulina velutipes. The surface of Flammulina velutipes is yellow, dark yellowish-brown or cinnamon-colored, slightly darker in the middle, and creamy-yellow at the edge. The filaments of Flammulina velutipes are white to milky white or slightly pinkish, with varying lengths. Flammulina velutipes is distributed in China, Japan, Russia, Europe, North America, Australia and other places. It grows in clusters on the rotten tree stumps or roots of broad-leaved trees such as elm and willow, and occasionally also grows on the living standing trees of various broad-leaved trees. Flammulina velutipes is a low-temperature edible mushroom, and the optimum temperature is about 8°C; it is sensitive to water requirements and needs more water. To prevent the occurrence of pests and diseases, the humidity should be low when the temperature is high.

[0003] Flammulina velutipes is divided into three categories: yellow species, light-colored species and white species according to color. White Flammulina velutipes is a white variant (F. var. velutipes) of Flammulina velutipes, with a pure white body. Because the fruiting body of Flammulina velutipes occurs at a relatively low temperature, it is also known as winter mushroom. It also has common names such as Flammulina velutipes, Pucao mushroom, Collybia velutipes, and frozen mushroom. Flammulina velutipes belongs to the Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, Flammulina or Collybia. In the late 1980s, white varieties selected in Japan entered the Chinese market. With their high appearance value and high yield, they quickly replaced the yellow varieties. For more than 30 years, Japanese white Flammulina velutipes varieties such as M50 and T022 have dominated the Chinese Flammulina velutipes market, and domestic production enterprises need to spend a lot of money to purchase mother strains externally every year. Through cross-breeding means, China has successively selected white Flammulina velutipes varieties such as Chuanjin No. 7, Chuanjin No. 8, and Chuanjin No. 9. Their stability and commercial appearance are excellent, but their taste is soft and easy to get stuck between teeth. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to cultivate a Flammulina velutipes variety with better quality.

[0005] The technical solution of the present invention is a high-brittleness white Flammulina velutipes HJ120, and its deposit number is CCTCC NO: M20242218.

[0006] The fruiting body of HJ120 has a white and hemispherical cap with a diameter of 0.48 cm to 0.88 cm; the gills are white, slightly dense, and of unequal length; the stipe is white, 8.5 cm to 14.7 cm long, 0.13 cm to 0.39 cm in diameter, cylindrical, and without villi at the base.

[0007] The present invention also provides a food product, which comprises the white Flammulina velutipes HJ120, the spores of the white Flammulina velutipes HJ120, and / or the extract of the white Flammulina velutipes HJ120.

[0008] Furthermore, the above-mentioned food product is at least one of the following: fresh food, air-dried food, freeze-dried food, canned food, or salted food.

[0009] Even further, the food product is a health food or a functional food.

[0010] The present invention also provides a cultivation method for the white Flammulina velutipes HJ120, which includes the following steps:

[0011] S1. Prepare materials according to the weight ratio of 9% cottonseed hull, 30% corn cob, 12% broad bean shell, 11% sawdust, 17% wheat bran, 9% rice bran, 5% corn flour, 4% soybean hull, 1% soybean meal, 1% lime, and 1% calcium carbonate. Add water and mix evenly. Adjust the pH to 5.8 - 6.2 and control the water content at 63 - 65%. Pack into mushroom bottles and perform high-pressure sterilization.

[0012] S2. Inoculate the mushroom bottles with the spawn and cultivate at 18 - 20°C and a humidity of 65 - 70% for 20 - 25 days until the mycelium fills the mushroom bottles.

[0013] S3. Bud induction stage: Scratch the surface of the mycelium using the flat scratching method. After scratching, transfer the bottles to the fruiting room for temperature-controlled bud induction. During this process, control the relative air humidity at 90 - 95%, the temperature at 12 - 13°C, increase ventilation, and primordia will appear in about 10 - 14 days.

[0014] S4. Mushroom bud differentiation stage: Control the relative air humidity at 85 - 90%, the temperature at 8°C, the CO2 concentration below 0.1%, and provide 200 Lx of light for 2 hours every day.

[0015] S5. Mushroom stalk elongation stage: When the mushroom buds grow to 2 - 3 cm, cover the bottle mouth with mushroom wrapping sheets. Keep the relative air humidity at 75%, the temperature at 8 - 13°C, and the light dark.

[0016] S6. Harvesting: When the fruiting body grows to 12 - 15 cm and the diameter of the mushroom cap is 0.5 - 1.0 cm, and the edge of the mushroom cap is curled inwards and not yet opened, harvest in a timely manner.

[0017] Furthermore, in step S1, the high-pressure sterilization is carried out at 121°C for 2 hours.

[0018] Specifically, in step S1, each mushroom bottle is filled with 350 ± 50 g.

[0019] Preferably, in step S2, the inoculation standards are as follows: For solid spawn in 750 mL bottles, inoculate 22 - 26 mushroom bottles; for liquid spawn, the inoculation amount for each mushroom bottle is 20 - 30 mL.

[0020] More preferably, 25 culture bottles are inoculated with the solid spawn of 750 mL per bottle.

[0021] Advantages of the present invention: The present invention provides a strain of white Flammulina velutipes with excellent appearance and high brittleness. While retaining the advantages of high yield, high nutritional value, and easy cultivation, this Flammulina velutipes strain also has the characteristics of tender and crispy taste and not being prone to getting stuck between teeth; it is more easily accepted by consumers and has good commercial prospects. This application has also explored an industrial production mode suitable for this strain, ensuring the stability of the production cycle, the quality and yield of the mushrooms.

[0022] The white Flammulina velutipes HJ120 of the present invention was deposited at the China Center for Type Culture Collection on October 15, 2024. Deposit address: Wuhan University, Wuhan, China; Zip code 430072, Deposit number: CCTCC NO: M20242218. Description of the Drawings

[0023] Figure 1 Appearance diagram of Rongjin 567.

[0024] Figure 2 Appearance diagram of Chuanjin No. 7.

[0025] Figure 3 Pairing and hybridization of mononuclears in pairs.

[0026] Figure 4 Hyphal clamp connections, as shown by the red circles in the figure.

[0027] Figure 5 ISSR clustering diagram.

[0028] Figure 6 State of HJ120 fruiting body during the growth period.

[0029] Figure 7 Cap traits of HJ120.

[0030] Figure 8 Stipe traits of HJ120.

[0031] Figure 9 Comparison diagram of HJ120 and Chuanjin No. 7 (labeled as D in the figure). Detailed Embodiments

[0032] The present invention involves breeding the high-brittleness yellow Flammulina velutipes variety Rongjin 567 ( Figure 1 ) and the white Flammulina velutipes variety Chuanjin No. 7 ( Figure 2)Perform single-spore hybridization breeding, and use chewing texture, commercial traits, yield, etc. as the main indicators to screen and obtain excellent new strains of white Flammulina velutipes for industrial production. The screened strains were also subjected to DUS determination and ISSR analysis to confirm that they are new varieties.

[0033] Meanwhile, this application has also explored an industrial production mode suitable for this strain, ensuring the stability of the production cycle, mushroom quality and yield. The specific production method is as follows:

[0034] 1 Culture medium formula

[0035] Cottonseed hull 9%, corn cob 30%, broad bean hull 12%, wood chips 11%, wheat bran 17%, rice bran 9%, corn flour 5%, soybean skin 4%, soybean meal 1%, lime 1% and calcium carbonate 1%, pH 5.8 - 6.2, water content 63 - 65%.

[0036] 2 Mixing materials and filling bottles

[0037] Weigh the materials according to the production volume and the culture medium formula, mechanically stir evenly, and gradually add water to 63 - 65% and pH 5.8 - 6.2. The cultivation bottles use 1100 mL plastic bottles, with a dry material weight of 350 ± 50 g per bottle, and punch holes in the middle.

[0038] 3 Sterilization and inoculation

[0039] Sterilize the prepared mushroom bottles in time, with a pressure value of 1.5 kg / cm 3 and a temperature of 121 °C for 2 - 2.5 h. After sterilization, send them into a sterile cooling room, and inoculate when the mushroom bottles cool down to 20 - 23 °C. One bottle (750 mL) of solid spawn is inoculated into 22 - 26 bottles, and the inoculation amount of liquid spawn per bottle is 20 - 30 mL. The spawn is inoculated at the bottom of the culture material hole and the bottleneck part.

[0040] 4 Mycelium growth management

[0041] During the mycelium growth period, control the temperature at 18 - 20 °C, humidity at 65 - 70%, and cultivate in the dark for 20 - 25 d.

[0042] 5 Mushroom fruiting management

[0043] Bud primordium formation stage: Use the flat scratching method to scratch the mycelium, and then transfer it to the fruiting room to control the temperature for bud primordium formation. Control the relative air humidity at 90 - 95%, temperature at 12 - 13 °C, increase ventilation, and primordia will appear in about 10 - 14 d;

[0044] Mushroom bud differentiation stage: Control the relative air humidity at 85 - 90%, temperature at 8 °C, CO2 concentration below 0.1%, and give 200 Lx light for 2 h every day;

[0045] Stem elongation stage: When the mushroom buds grow to 2 - 3 cm, wrap the bottle mouth with mushroom wrapping slices. The relative air humidity is 75%, the temperature is 8 - 13 °C, and the light is dark.

[0046] 6 Harvesting

[0047] When the fruiting body grows to 12 - 15 cm and the diameter of the mushroom cap is 0.5 - 1.0 cm, and the edge of the mushroom cap is involuted and not yet opened, harvest it in time.

[0048] 7 Grading and packaging

[0049] After harvesting, cut off the mushroom head, classify it according to product quality and market demand, weigh and package it, then evacuate and seal it. Pre - cool it in a warehouse at 3 - 5 °C and sell it on the same day.

[0050] Breeding of Example HJ120

[0051] 1. Isolation of spores and identification of monokaryotic hyphae

[0052] Cut off the mushroom stem of the mature fresh Flammulina velutipes fruiting body in a sterile environment, place the side with gills flat on a sterilized petri dish, put it in a sterile room at 18 - 20 °C, keep the environmental humidity, and collect the spore print overnight. Dilute the spore liquid concentration to about 10 3 cells / mL by gradient dilution method. Take 100 μL of spore liquid from each of the two parents and evenly coat it on a PDA plate culture dish, then invert it and place it in an incubator at 22 - 24 °C for 3 - 5 d. Observe the spore germination situation. When tiny colonies visible to the naked eye appear on the culture medium, pick a single colony with an inoculation needle and transfer it to a PDA plate. Obliquely insert a sterilized cover glass into the culture medium at the edge of the colony, and culture it in an incubator at 22 - 24 °C for 2 - 3 d. After the hyphae climb onto the cover glass, take out the cover glass, stain it with lactophenol cotton blue reagent, and observe with a 100 - fold optical microscope. Those without clamp connections are monokaryons. A total of 42 monokaryons of Rongjin 567 and 49 monokaryons of Chuanjin No. 7 are obtained.

[0053] 2. Pairing and hybridization of monokaryotic hyphae

[0054] Pair the monokaryons that have passed the microscope inspection in pairs. Under sterile conditions, cut a mycelial block with a diameter of about 0.8 cm with a sterilized pipette and culture it on a PDA plate. The distance between the two mycelial blocks is 1 cm, and culture it in an incubator at 22 - 24 °C for 3 - 5 d ( Figure 3 ). When a protruding fusion line forms between two fusible single colonies, pick the hyphae at the fusion site to another PDA medium for culture in a sterile environment. Obliquely insert a sterilized cover glass into the culture medium at the edge of the colony, and culture it in an incubator at 22 - 24 °C for 2 - 3 d. After the hyphae climb onto the cover glass, take out the cover glass and use the monokaryotic hypha inspection method to check whether the fusion is successful. Those with clamp connections ( Figure 4) Transfer the mycelium to a PDA slant medium. Among 2058 hybridization combinations, clamp connections were observed in 300 pairs, and the pairing success rate was 14.58%.

[0055] 3. Primary screening in bag cultivation test

[0056] Adopt the culture medium formula: cottonseed hull 9%, corncob 30%, broad bean hull 12%, sawdust 11%, wheat bran 17%, rice bran 9%, corn flour 5%, soybean skin 4%, soybean meal 1%, lime 1%, calcium carbonate 1%, water content 63 - 65%, pH 5.8 - 6.2. Use 14 cm × 33 cm polypropylene mushroom bags, each bag filled with 350 g ± 50 g of dry material, sterilize at 121 °C under high pressure for 2 h, directly inoculate 5 bags of each hybrid with a mother culture test tube, and culture at a constant temperature of 18 - 20 °C in the dark in the culture room. When the mycelium fills the bag and reaches physiological maturity, scrape off the surface seed blocks and old mycelium, and transfer them to the fruiting room. During December to January of the following year, the mushroom bags can fruit naturally in the fruiting room, keep the fruiting room in the dark condition, and the air humidity above 85%. When the mushroom buds grow to 2 - 3 cm, put on bags to increase the CO2 concentration and promote the elongation of the mushroom stalk. Harvest when the mushroom stalk grows to 12 - 15 cm and the mushroom cap diameter is 0.5 - 1.0 cm.

[0057] Among the 300 hybrid offspring obtained, 261 plants fruited normally in the bag cultivation test. Based on comprehensive data such as the fruiting time, yield, mushroom cap diameter, mushroom stalk diameter and length, 15 relatively excellent white flammulina velutipes strains were initially screened out, namely HJ57, HJ66, HJ69, HJ100, HJ120, HJ141, HJ162, HJ163, HJ186, HJ221, HJ230, HJ253, HJ278, HJ298, HJ311. Among them, the average yield of strain HJ278 was the highest, reaching 261.5 g, and the yields of HJ120 and HJ100 were the second highest, being 246.8 g and 246.7 g respectively; based on comprehensive indexes such as the color and shape of the mushroom cap and stalk, and the degree of involution of the mushroom stalk edge, strains HJ66, HJ120, HJ141 and HJ230 showed better performance.

[0058] Observation data table of fruiting of 15 white flammulina velutipes strains

[0059]

[0060] 4. DUS test and evaluation

[0061] Carry out ISSR molecular markers and cluster analysis on the above 15 white flammulina velutipes strains. The results show that when classifying 25 strains at 75% similarity, they can be divided into 9 large groups, among which group I is the largest, containing 8 strains. From the clustering diagram ( Figure 5)It seems that HJ108 and HJ206, HJ69 and HJ162, HJ163 and HJ311, HJ120 and HJ230 are the same strains respectively, and only one of them needs to be selected for application.

[0062] Meanwhile, continuous two-round cultivation tests were carried out on 15 hybrid strains. According to the requirements in the "Testing Guidelines for Specificity (Distinguishability), Uniformity and Stability of Plant Varieties - Flammulina velutipes", for the liquid spawn formula, sucrose is 2%, soybean meal powder is 0.4%, magnesium sulfate is 0.05%, and potassium dihydrogen phosphate is 0.05%. For the cultivation substrate formula, corncob is 42%, rice bran is 35%, wheat bran is 10%, cottonseed hull is 12%, and calcium carbonate is 1%. The pH is 5.8 - 6.2, the water content is 63 - 65%, and 1100 mL plastic cultivation bottles are used, with inoculation by punching in the center. There are 100 bottles for each variety, and 3 replicates are set. For each strain, 3 fresh fruiting body samples of the first flush are randomly collected in each round, and multiple points are sampled from each for brittleness measurement. The instrument used is the TA.XTC-18 texture analyzer of Baosheng Technology. The average brittleness of the parent control Rongjin 567 in the measurement results is 347 g, and the average brittleness value of Chuanjin No. 7 is 211 g. Among the white Flammulina velutipes hybrid strains, HJ120 has the highest average brittleness value, which is 370 g.

[0063] Table 2 Brittleness measurement values of cultivated strains

[0064]

[0065]

[0066] Specificity, uniformity and stability tests were carried out on the HJ120 strain for 2 consecutive growth cycles. The population traits were observed from the whole plot and the mixed sample observation population with a maturity of 70%. 60 fruiting bodies of each strain were sampled for individual part observation.

[0067] The mycelium culture time of HJ120 is 20 - 25 d, the primordium formation time is 10 - 14 d, and the fruiting body Figure 6 ) growth time is 14 - 18 d, and the number of fruiting bodies is medium to many. The surface color of the mushroom cap is white, the degree of edge involution is strong, and the Figure 7 ) diameter of the mushroom cap is 4.8 - 8.8 mm, the height of the mushroom cap is 2.7 - 6.2 mm, and the thickness of the mushroom cap is 2.0 - 3.9 mm. The Figure 8 ) color uniformity of the HJ120 stipe is high, the adhesion degree is low, the length of the stipe is 8.5 - 14.7 cm, and the diameter of the stipe is 1.3 - 3.9 mm. It can be seen that HJ120 Figure 9 ) has good commercial application prospects and is sent to the China Center for Type Culture Collection (CCTCC) for preservation.

[0068] 5. Supporting factory cultivation technology

[0069] 5.1 Cultivation material formula

[0070] Cottonseed hull 9%, corncob 30%, broad bean hull 12%, sawdust 11%, wheat bran 17%, rice bran 9%, corn flour 5%, soybean hull 4%, soybean meal 1%, lime 1% and calcium carbonate 1%, pH 5.8 - 6.2, water content 63 - 65%.

[0071] 5.2 Mixing the materials and filling the bottles

[0072] Weigh the materials according to the production volume and the culture medium formula, mix them evenly by machine, and gradually add water to 63 - 65% and pH 5.8 - 6.2. The cultivation bottles use 1100 mL plastic bottles, with each bag of dry materials weighing 350 ± 50 g, and make a hole in the middle.

[0073] 5.3 Sterilization and inoculation

[0074] Sterilize the prepared fungus bottles in time, with the pressure value at 1.5 kg / cm 3 and keep the temperature at 121 °C for 2 - 2.5 h. After sterilization, send them into the aseptic cooling room, and inoculate when the fungus bottles cool down to 20 - 23 °C. One bottle (750 mL) of solid strain can inoculate about 25 bottles, and the inoculation amount of each bottle of liquid strain is 20 - 30 mL. The strain is inoculated at the bottom of the culture material hole and the bottleneck part.

[0075] 5.4 Mycelium growth management

[0076] During the mycelium growth period, control the temperature at 18 - 20 °C, the humidity at 65 - 70%, and culture in the dark for 20 - 25 d.

[0077] 5.5 Mushroom fruiting management

[0078] Bud induction period: Use the flat scratching method to scratch the mycelium, and then transfer it to the mushroom fruiting room to control the temperature for bud induction. Control the relative air humidity at 90 - 95%, the temperature at 12 - 13 °C, increase ventilation, and primordia will appear in about 10 - 14 d;

[0079] Mushroom bud differentiation period: Control the relative air humidity at 85 - 90%, the temperature at 8 °C, the CO2 concentration below 0.1%, and give 200 Lx of light for 2 h every day;

[0080] Mushroom stalk elongation period: When the mushroom buds grow to about 2 cm, wrap the bottle mouth with mushroom wrapping sheets, the relative air humidity is 75%, the temperature is 8 - 13 °C, and the light is dark.

[0081] 5.6 Harvesting

[0082] When the fruiting body grows to 12 - 15 cm and the diameter of the mushroom cap is 0.5 - 1.0 cm, and the edge of the mushroom cap is involuted and not yet opened, harvest it in time.

[0083] 5.7 Grading and packaging

[0084] After harvesting, cut off the mushroom heads, classify them according to product quality and market demand, weigh and package them, and then evacuate and seal. Pre-cool in a warehouse at 3-5°C and sell them on the same day.

Claims

1. A highly brittle white enoki mushroom, Flammulina velutipes HJ120, whose accession number is CCTCC No: M20242218.

2. A food, characterized in that: The invention comprises the white Flammulina velutipes HJ120, spores of the white Flammulina velutipes HJ120 and / or an extract of the white Flammulina velutipes HJ120 as claimed in claim 1.

3. The food according to claim 2, characterized in that: At least one of fresh food, air-dried food, freeze-dried food, canned food or salted food.

4. The food according to claim 2, characterized in that: The food is a health food or a functional food.

5. The cultivation method of white Flammulina velutipes HJ120 according to claim 1, characterized in that: The steps include: S1. Prepare materials according to the weight ratio of 9% cottonseed hulls, 30% corn cobs, 12% broad bean hulls, 11% sawdust, 17% bran, 9% rice bran, 5% corn flour, 4% soybean hulls, 1% soybean meal, 1% lime and 1% calcium carbonate, add water and mix evenly, adjust pH to 5.8-6.2, and control water content to 63-65%; pack into bacteria bottles; and sterilize by high pressure; S2. Inoculate the bacteria into a bacterial bottle and culture it at 18-20°C and 65-70% humidity for 20-25 days until the mycelium fills the bacterial bottle. S3, bud-inducing period: Use flat scratching method to scratch the fungi, and then move them into the fruiting room to control the temperature and induce bud-inducing. During the process, the relative humidity of the air is controlled at 90-95%, the temperature is 12-13℃, and ventilation is increased. Buds begin to appear in about 10-14 days; S4, Mushroom bud differentiation period: control the relative humidity of the air to 85-90%, the temperature to 8°C, the CO2 concentration to below 0.1%, and provide 200Lx light for 2h every day; S5, stem elongation period: when the mushroom buds grow to 2-3cm, cover the bottle mouth with mushroom wrapping sheet, the relative humidity of the air is 75%, the temperature is 8-13℃, and the light is dark; S6. Harvesting: When the fruiting bodies grow to 12-15 cm and the cap diameter is 0.5-1.0 cm, harvest in time when the cap edges are rolled inward and have not yet opened.

6. The cultivation method according to claim 5, characterized in that: In step S1, high pressure sterilization is performed at 121° C. for 2 hours.

7. The cultivation method according to claim 5, characterized in that: In step S1, each bacteria bottle is filled with 350±50g.

8. The cultivation method according to claim 7, characterized in that: In step S2, the inoculation standard is as follows: 750 mL / bottle of solid bacteria is inoculated into 22 to 26 bacteria bottles; the inoculation volume of each bacteria bottle of liquid bacteria is 20 to 30 mL.

9. The cultivation method according to claim 8, characterized in that: 750mL / bottle of solid bacteria were used to inoculate 25 bacteria bottles.

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