High-activity edible mushroom seed high-efficiency preparation process

Abstract

The application relates to the technical field of biological bacteria, and discloses a high-efficiency preparation process of high-activity edible mushroom seeds, which comprises the following steps: S1, primary strain cultivation and screening: inoculating the strain on a culture medium, performing segmented culture, and obtaining a primary F1 strain; S2, adaptability domestication of the second to third generations of strains: 50% culture medium raw materials and 50% target strain cultivation medium are used, a temperature difference is set for day-night alternation, and an F2 generation strain is screened; 20% culture medium raw materials and 80% target strain cultivation medium are used, and the water content is reduced, and an F3 generation strain is screened; S3, intensive domestication of the fourth to sixth generations of strains: the carbon-nitrogen ratio of the primary culture medium is adjusted, variable temperature control oxygen culture is adopted, and an F4 generation strain is screened; the F5 generation strain is screened on the basis of the culture medium; the culture medium adopts the target strain cultivation medium, and the F6 generation strain is screened; and S4, stable subculture cultivation of the strain: the F6 generation strain is continuously subcultured for two times, and finally, a stable high-activity strain is obtained.

Description

Technical Field

[0001] This invention relates to the field of biological technology, specifically to an efficient process for preparing highly active edible fungi seeds. Background Technology

[0002] Edible fungi, as a nutritious and delicious food, are widely popular worldwide. With increasing market demand, large-scale, factory-style production of edible fungi is becoming a trend. In the production process, the quality of the fungal strain and the efficiency of preparation are key factors affecting yield and quality.

[0003] Traditional methods for preparing edible fungi spawn have many drawbacks. For example, when using cotton hulls or sawdust as raw materials, bottling is time-consuming, production efficiency is low, and the spawn, once fully grown with mycelium, clumps together, making it difficult to break up during use. This results in slow inoculation, slow mycelial germination and substrate consumption, and a high contamination rate. While wheat or corn kernels are nutritious, once fully grown with mycelium, the spawn is also difficult to break into individual particles, affecting inoculation speed. Furthermore, they are prone to exposure, rotting, and pest infestation later on, and the raw material cost is relatively high. In addition, traditional methods result in slow germination and substrate consumption of the inoculated spawn, requiring a long time to fully grow the spawn in the bottle. The mycelium within the same bottle (bag) is uneven, with significant differences in age, greatly affecting the quality of the spawn.

[0004] To meet the growing market demand for edible fungi and improve the economic benefits and quality stability of edible fungi production, developing a high-activity, low-cost, and high-efficiency edible fungi seed preparation process is of great practical significance. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides an efficient process for preparing highly active edible fungi seeds.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution: a highly efficient process for preparing highly active edible fungi seeds, comprising the following steps:

[0009] S1. Primary strain cultivation and screening

[0010] The culture medium raw materials are cleaned, impurities removed, soaked, nutrient supplemented and sterilized. 5% of the culture medium mass of the strain is inoculated with the strain and cultured in segments. The fastest growing and healthiest strain is selected and cultured repeatedly 2 to 3 times to obtain the first generation F1 strain.

[0011] S2, second- to third-generation strains' adaptation and domestication

[0012] Retain 50% of the above-mentioned culture medium raw materials, add 50% of the target strain culture substrate, inoculate the first generation F1 strain, set a day-night alternation with a temperature difference of 15℃ / 12 h and 30℃ / 12 h, and screen for stable F2 generation strains; retain 20% of the above-mentioned culture medium raw materials, add 80% of the target strain culture substrate, inoculate the F2 generation strain, reduce the moisture content of the culture medium by 10-15%, and screen for stable F3 generation strains.

[0013] S3, fourth to sixth generation strains intensive domestication

[0014] Based on the initial culture medium, the carbon-to-nitrogen ratio was adjusted to 25-35:1, and 0.3% of compound amino acids and 0.02% of humic acid were added by weight of the culture medium. Temperature-controlled and oxygen-controlled culture was employed, and F3 generation strains were inoculated. Stable F4 generation strains were then screened. Based on the above culture medium, the carbon source supply was reduced by 10%, and the ratio of organic to inorganic nitrogen in the nitrogen source was adjusted to 1:1. F4 generation strains were inoculated, and stable F5 generation strains were screened. Using the target strain's culture substrate, F5 generation strains were inoculated, and fast-growing, high-yielding F6 generation strains were screened.

[0015] S4. Stable subculturing of bacterial strains

[0016] The selected F6 generation strain was passaged twice to observe whether its characteristics were stable, ensuring that high activity, strong resistance, and stability did not degenerate during the passage process, and finally obtaining a stable, highly active strain.

[0017] Further, the initial strain cultivation and screening:

[0018] A1. The culture medium raw materials are selected from 40-45 parts of oat grains, 25-30 parts of millet, 15-20 parts of buckwheat hulls, and 10-15 parts of brewer's grains. The oat grains and millet are rinsed with clean water to remove surface impurities. The buckwheat hulls are crushed and passed through a 40-mesh sieve. The brewer's grains are dried at 60-80℃ for 3-5 hours, crushed, and set aside. The pretreated raw materials are mixed evenly and frozen at -10 to -20℃ for 12-24 hours.

[0019] A2. Immerse the frozen composite raw materials in water with a temperature gradient. The initial water temperature is 20-30℃, and the soaking time is 6-8 hours. Then, the temperature is raised to 40-45℃, and the soaking time is continued for 3-6 hours. Finally, the temperature is raised to 60-65℃, and the soaking time is continued for 3-6 hours.

[0020] A3. After extraction, remove the raw materials and drain the water. Add yeast extract at 2% of the dry weight of the mixed raw materials and vitamin C at 1%. Stir well.

[0021] A4. Using an automatic bagging machine, the treated composite raw materials are filled into high-temperature resistant polypropylene bags, with the filling volume being 60% of the bag volume. The mixture is then stored at 121℃ and 1.5 kg / cm³. 2 Maintain pressure for 20-30 minutes;

[0022] A5. Under sterile conditions, inoculate the bacterial culture at 5% of the weight of the raw material, spread it evenly on the surface of the culture medium, and place the bacterial bag on a three-dimensional shaker and shake it at a speed of 15~20 r / min for 10~15 min.

[0023] A6. First stage of cultivation for 1-3 days: The cultivation temperature is controlled at 28-30℃, the relative humidity is 70-75%, the carbon dioxide concentration is controlled below 0.3%, and ventilation is carried out twice a day for 30 minutes each time;

[0024] The second stage of cultivation lasts 4-6 days: the temperature is reduced to 23-25℃, the relative humidity is 60-65%, the carbon dioxide concentration is controlled below 0.2%, the ventilation is carried out 3 times a day for 40 minutes each time, and at the same time, the diffused light with an intensity of 100-200 lx is provided for 1-2 hours a day.

[0025] The third stage of cultivation lasts 7-8 days: Maintain the temperature and humidity of the second stage of cultivation, stop the light, and ventilate 4 times a day for 30 minutes each time.

[0026] Furthermore, in step A2, the mixture is stirred every 6 hours during the extraction process, and the extraction water is a mixed solution containing 0.02% potassium dihydrogen phosphate and 0.01% magnesium sulfate.

[0027] Furthermore, in step A5, the fungal strain is one of the following: wood ear fungus, oyster mushroom, or enoki mushroom.

[0028] Furthermore, the cultivation substrate for the target strain is the substrate in the cultivation environment of the target strain; Auricularia auricula cultivation substrate: 70-75 parts hardwood sawdust, 15-20 parts wheat bran, 3-5 parts corn flour, 1-2 parts gypsum powder, 1-1.5 parts sucrose, and 120-140 parts water; Oyster mushroom cultivation substrate: 80-85 parts cottonseed hulls, 10-15 parts wheat bran, 10-20 parts crushed corn cobs, 2-3 parts lime powder, 1-2 parts gypsum powder, and 130-150 parts water; Enoki mushroom cultivation substrate: 30-35 parts broadleaf sawdust, 30-35 parts cottonseed hulls, 20-25 parts crushed corn cobs, 5-8 parts wheat bran, 1-1.5 parts gypsum powder, 0.1-0.2 parts potassium dihydrogen phosphate, and 110-130 parts water.

[0029] Furthermore, the compound amino acids are lysine, methionine, and phenylalanine mixed in a ratio of 2:1:1.

[0030] Furthermore, the temperature and oxygen control parameters for the F4 generation bacterial column were maintained at 20℃ for 8 hours and 32℃ for 16 hours per day, with the oxygen concentration controlled at 15-17%.

[0031] Furthermore, the F5 generation strain was cultured with the following parameters: humidity reduced by 5-10%, pH adjusted to 5.0-5.5, and subjected to strong light irradiation of 300-350 lx for 3-4 hours per day.

[0032] Furthermore, the F6 generation strain was cultured with a temperature range of 12-25℃, a relative humidity range of 50-80%, a day-night cycle, and ventilated 6 times a day for 30 minutes each time, creating a complex environment close to nature.

[0033] (iii) Beneficial technical effects

[0034] This invention's process, through multiple generations of targeted domestication and precise cultivation, exhibits significant advantages. Regarding strain activity, mycelial growth rate is increased by 50-80%, with daily growth rates of Auricularia auricula-judae, Pleurotus ostreatus, and Enoki mushrooms reaching 4.5-6.0 mm respectively, and the time to full bag filling is shortened by 30%-40%, effectively improving production efficiency. Enzyme activity is significantly enhanced, improving the ability to decompose the substrate, laying the foundation for rapid growth and high yield. The fruiting cycle is shortened by 15-20 days, and the yield per unit area is increased by 40-60%, with uniform fruiting body morphology and excellent quality, resulting in a significant improvement in overall benefits and providing a highly efficient and stable strain guarantee for large-scale cultivation.

[0035] The process of this invention provides an efficient and reliable technical means for the edible fungi industry, which helps to develop more edible fungi varieties that adapt to different environments and market demands, promotes the development of the edible fungi industry towards diversification and efficiency, and is of great significance for promoting the adjustment of agricultural industrial structure and increasing farmers' income. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, all components of the culture medium formulation of this invention are commercially available.

[0038] All parts used in this invention are by weight.

[0039] Example 1

[0040] The fungus strain is wood ear fungus.

[0041] The substrate for cultivating the target strain is the substrate in the cultivation environment of the target strain; the substrate for cultivating black fungus consists of 70 parts hardwood sawdust, 15 parts wheat bran, 3 parts corn flour, 1 part gypsum powder, 1 part sucrose, and 120 parts water.

[0042] The compound amino acid is a mixture of lysine, methionine, and phenylalanine in a ratio of 2:1:1.

[0043] A highly efficient process for preparing highly active edible fungi seeds includes the following steps:

[0044] S1. Primary strain cultivation and screening

[0045] The culture medium raw materials were cleaned, impurities removed, soaked, nutrient supplemented and sterilized. 5% of the culture medium mass of the strain was inoculated and cultured in segments. The fastest growing and healthiest strain was selected and cultured twice to obtain the first generation F1 strain.

[0046] S2, second- to third-generation strains' adaptation and domestication

[0047] Retain 50% of the above-mentioned culture medium raw materials, add 50% of the target strain culture substrate, inoculate the first generation F1 strain, set the temperature difference of 15℃ / 12 h and 30℃ / 12 h day and night alternation, and screen the F2 generation strain that can grow stably.

[0048] Retain 20% of the above-mentioned culture medium raw materials, add 80% of the target strain culture substrate, inoculate the F2 generation strain, reduce the water content of the culture medium by 10%, and screen for stable F3 generation strains.

[0049] S3, fourth to sixth generation strains intensive domestication

[0050] Based on the initial culture medium, the carbon-nitrogen ratio was adjusted to 25:1, and 0.3% of compound amino acids and 0.02% of humic acid by weight of the culture medium were added. The culture was carried out using temperature-controlled oxygenation, maintaining 20℃ for 8 hours and 32℃ for 16 hours every day, with the oxygen concentration controlled at 15%. The F3 generation strain was inoculated, and the F4 generation strain that could grow stably was screened out.

[0051] Based on the above culture medium, the carbon source supply was reduced by 10%, and the ratio of organic nitrogen to inorganic nitrogen in the nitrogen source was adjusted to 1:1. The humidity was reduced by 5%, the pH was adjusted to 5.0, and strong light with an intensity of 300 lx was applied for 3 hours a day. The F4 generation strain was inoculated, and the stable F5 generation strain was screened out.

[0052] The culture medium used the target strain cultivation substrate, inoculated with F5 generation strains, with temperature ranging from 12 to 25℃, relative humidity ranging from 50 to 80%, alternating between day and night, and ventilation 6 times a day for 30 minutes each time, to create a complex environment close to nature, and screen out F6 generation strains with fast growth and high yield.

[0053] S4. Stable subculturing of bacterial strains

[0054] The selected F6 generation strain was passaged twice to observe whether its characteristics were stable, ensuring that high activity, strong resistance, and stability did not degenerate during the passage process, and finally obtaining a stable, highly active strain.

[0055] Primary strain cultivation and screening:

[0056] A1. The culture medium raw materials are 40 parts oat grains, 25 parts millet, 15 parts buckwheat hulls, and 10 parts brewer's grains. The oat grains and millet are rinsed with clean water to remove surface impurities. The buckwheat hulls are crushed and passed through a 40-mesh sieve. The brewer's grains are dried at 60℃ for 3 hours, crushed, and set aside. The pretreated raw materials are mixed evenly and frozen at -10℃ for 12 hours.

[0057] A2. The frozen composite raw material was placed in water with a temperature gradient for extraction. The mixture was stirred every 6 hours during the extraction process. The extraction water was a mixed solution with 0.02% potassium dihydrogen phosphate and 0.01% magnesium sulfate. The initial water temperature was 20℃, and the mixture was soaked for 6 hours. Then the temperature was raised to 40℃ and the mixture was soaked for another 3 hours. Finally, the temperature was raised to 60℃ and the mixture was soaked for another 3 hours.

[0058] A3. After extraction, remove the raw materials and drain the water. Add yeast extract at 2% of the dry weight of the mixed raw materials and vitamin C at 1%. Stir well.

[0059] A4. Using an automatic bagging machine, the treated composite raw materials are filled into high-temperature resistant polypropylene bags, with the filling volume being 60% of the bag volume. The mixture is then stored at 121℃ and 1.5 kg / cm³. 2 Maintain pressure for 20 minutes;

[0060] A5. Under sterile conditions, inoculate the bacterial culture at 5% of the weight of the raw material, spread it evenly on the surface of the culture medium, and place the bacterial bag on a three-dimensional shaker and shake it at 15 r / min for 10 min.

[0061] A6. First stage of cultivation for 1 day: The cultivation temperature is controlled at 28℃, the relative humidity is 70%, the carbon dioxide concentration is controlled below 0.3%, and ventilation is carried out twice a day for 30 minutes each time;

[0062] The second stage of cultivation lasted for 4 days: the temperature was lowered to 23℃, the relative humidity was 60%, the carbon dioxide concentration was controlled below 0.2%, the ventilation was carried out 3 times a day for 40 minutes each time, and at the same time, the diffused light with an intensity of 100 lx was provided for 1 hour a day.

[0063] The third stage of cultivation lasts for 7 days: Maintain the temperature and humidity of the second stage of cultivation, stop the light, and ventilate 4 times a day for 30 minutes each time.

[0064] Example 2

[0065] The fungus is oyster mushroom.

[0066] The cultivation substrate for the target strain is the substrate in the cultivation environment of the target strain; oyster mushroom cultivation substrate: 80 parts cottonseed hulls, 15 parts wheat bran, 10 parts crushed corn cobs, 3 parts lime powder, 1 part gypsum powder, and 150 parts water.

[0067] The compound amino acid is a mixture of lysine, methionine, and phenylalanine in a ratio of 2:1:1.

[0068] A highly efficient process for preparing highly active edible fungi seeds includes the following steps:

[0069] S1. Primary strain cultivation and screening

[0070] The culture medium raw materials were cleaned, impurities removed, soaked, nutrient supplemented and sterilized. 5% of the culture medium mass of the strain was inoculated and cultured in segments. The fastest growing and healthiest strain was selected and cultured three times to obtain the first generation F1 strain.

[0071] S2, second- to third-generation strains' adaptation and domestication

[0072] Retain 50% of the above-mentioned culture medium raw materials, add 50% of the target strain culture substrate, inoculate the first generation F1 strain, set the temperature difference of 15℃ / 12 h and 30℃ / 12 h day and night alternation, and screen the F2 generation strain that can grow stably.

[0073] Retain 20% of the above-mentioned culture medium raw materials, add 80% of the target strain culture substrate, inoculate the F2 generation strain, reduce the water content of the culture medium by 12%, and screen for stable F3 generation strains.

[0074] S3, fourth to sixth generation strains intensive domestication

[0075] Based on the initial culture medium, the carbon-nitrogen ratio was adjusted to 30:1, and 0.3% of compound amino acids and 0.02% of humic acid by weight of the culture medium were added. The culture was carried out using temperature-controlled oxygenation, maintaining 20℃ for 8 hours and 32℃ for 16 hours every day, with the oxygen concentration controlled at 16%. The F3 generation strain was inoculated, and the F4 generation strain that could grow stably was screened out.

[0076] Based on the above culture medium, the carbon source supply was reduced by 10%, and the ratio of organic nitrogen to inorganic nitrogen in the nitrogen source was adjusted to 1:1. The humidity was reduced by 8%, the pH was adjusted to 5.2, and strong light with an intensity of 320 lx was applied for 4 hours a day. The F4 generation strain was inoculated, and the stable F5 generation strain was screened out.

[0077] The culture medium used the target strain cultivation substrate, inoculated with F5 generation strains, with temperature ranging from 12 to 25℃, relative humidity ranging from 50 to 80%, alternating between day and night, and ventilation 6 times a day for 30 minutes each time, to create a complex environment close to nature, and screen out F6 generation strains with fast growth and high yield.

[0078] S4. Stable subculturing of bacterial strains

[0079] The selected F6 generation strain was passaged twice to observe whether its characteristics were stable, ensuring that high activity, strong resistance, and stability did not degenerate during the passage process, and finally obtaining a stable, highly active strain.

[0080] Primary strain cultivation and screening:

[0081] A1. The culture medium raw materials are 45 parts oat grains, 30 parts millet, 20 parts buckwheat hulls, and 10 parts brewer's grains. The oat grains and millet are rinsed with clean water to remove surface impurities. The buckwheat hulls are crushed and passed through a 40-mesh sieve. The brewer's grains are dried at 60℃ for 5 hours, crushed, and set aside. The pretreated raw materials are mixed evenly and frozen at -15℃ for 15 hours.

[0082] A2. The frozen composite raw material was placed in water with a temperature gradient for extraction. The mixture was stirred every 6 hours during the extraction process. The water used for extraction was a mixed solution of 0.02% potassium dihydrogen phosphate and 0.01% magnesium sulfate. The initial water temperature was 25℃, and the mixture was soaked for 8 hours. Then the temperature was raised to 40℃ and the mixture was soaked for another 4 hours. Finally, the temperature was raised to 65℃ and the mixture was soaked for 5 hours.

[0083] A3. After extraction, remove the raw materials and drain the water. Add yeast extract at 2% of the dry weight of the mixed raw materials and vitamin C at 1%. Stir well.

[0084] A4. Using an automatic bagging machine, the treated composite raw materials are filled into high-temperature resistant polypropylene bags, with the filling volume being 60% of the bag volume. The mixture is then stored at 121℃ and 1.5 kg / cm³. 2 Maintain pressure for 25 minutes;

[0085] A5. Under sterile conditions, inoculate the bacterial culture at 5% of the weight of the raw material, spread it evenly on the surface of the culture medium, and place the bacterial bag on a three-dimensional shaker and shake it at 18 r / min for 10 min.

[0086] A6. First stage of cultivation for 2 days: The cultivation temperature is controlled at 30℃, the relative humidity is 75%, the carbon dioxide concentration is controlled below 0.3%, and ventilation is carried out twice a day for 30 minutes each time;

[0087] The second stage of cultivation lasted for 5 days: the temperature was lowered to 24℃, the relative humidity was 62%, the carbon dioxide concentration was controlled below 0.2%, the ventilation was carried out 3 times a day for 40 minutes each time, and at the same time, the diffused light with an intensity of 150 lx was provided for 2 hours a day.

[0088] The third stage of cultivation lasts for 7 days: Maintain the temperature and humidity of the second stage of cultivation, stop the light, and ventilate 4 times a day for 30 minutes each time.

[0089] Example 3

[0090] The fungus strain is enoki mushroom.

[0091] The cultivation substrate for the target strain is the substrate in the cultivation environment of the target strain; the cultivation substrate for enoki mushrooms consists of: 35 parts broadleaf tree wood chips, 35 parts cottonseed hulls, 25 parts crushed corn cobs, 8 parts wheat bran, 1.5 parts gypsum powder, 0.2 parts potassium dihydrogen phosphate, and 130 parts water.

[0092] The compound amino acid is a mixture of lysine, methionine, and phenylalanine in a ratio of 2:1:1.

[0093] A highly efficient process for preparing highly active edible fungi seeds includes the following steps:

[0094] S1. Primary strain cultivation and screening

[0095] The culture medium raw materials were cleaned, impurities removed, soaked, nutrient supplemented and sterilized. 5% of the culture medium mass of the strain was inoculated and cultured in segments. The fastest growing and healthiest strain was selected and cultured three times to obtain the first generation F1 strain.

[0096] S2, second- to third-generation strains' adaptation and domestication

[0097] Retain 50% of the above-mentioned culture medium raw materials, add 50% of the target strain culture substrate, inoculate the first generation F1 strain, set the temperature difference of 15℃ / 12 h and 30℃ / 12 h day and night alternation, and screen the F2 generation strain that can grow stably.

[0098] Retain 20% of the above-mentioned culture medium raw materials, add 80% of the target strain culture substrate, inoculate the F2 generation strain, reduce the water content of the culture medium by 15%, and screen for stable F3 generation strains.

[0099] S3, fourth to sixth generation strains intensive domestication

[0100] Based on the initial culture medium, the carbon-nitrogen ratio was adjusted to 35:1, and 0.3% of compound amino acids and 0.02% of humic acid by weight of the culture medium were added. The culture was carried out using temperature-controlled oxygenation, maintaining 20℃ for 8 hours and 32℃ for 16 hours every day, with the oxygen concentration controlled at 17%. The F3 generation strain was inoculated, and the F4 generation strain that could grow stably was screened out.

[0101] Based on the above culture medium, the carbon source supply was reduced by 10%, and the ratio of organic nitrogen to inorganic nitrogen in the nitrogen source was adjusted to 1:1. The humidity was reduced by 10%, the pH was adjusted to 5.5, and strong light with an intensity of 350 lx was applied for 4 hours a day. The F4 generation strain was inoculated, and the stable F5 generation strain was screened out.

[0102] The culture medium used the target strain cultivation substrate, inoculated with F5 generation strains, with temperature ranging from 12 to 25℃, relative humidity ranging from 50 to 80%, alternating between day and night, and ventilation 6 times a day for 30 minutes each time, to create a complex environment close to nature, and screen out F6 generation strains with fast growth and high yield.

[0103] S4. Stable subculturing of bacterial strains

[0104] The selected F6 generation strain was passaged twice to observe whether its characteristics were stable, ensuring that high activity, strong resistance, and stability did not degenerate during the passage process, and finally obtaining a stable, highly active strain.

[0105] Primary strain cultivation and screening:

[0106] A1. The culture medium raw materials are 45 parts oat grains, 30 parts millet, 20 parts buckwheat hulls, and 15 parts brewer's grains. The oat grains and millet are rinsed with clean water to remove surface impurities. The buckwheat hulls are crushed and passed through a 40-mesh sieve. The brewer's grains are dried at 80℃ for 5 hours, crushed, and set aside. The pretreated raw materials are mixed evenly and frozen at -20℃ for 24 hours.

[0107] A2. The frozen composite raw material was placed in water with a temperature gradient for extraction. During the extraction process, the mixture was stirred once every 6 hours. The extraction water was a mixed solution with 0.02% potassium dihydrogen phosphate and 0.01% magnesium sulfate. The initial water temperature was 30℃, and the mixture was soaked for 8 hours. Then the temperature was raised to 45℃ and the mixture was soaked for another 6 hours. Finally, the temperature was raised to 65℃ and the mixture was soaked for another 6 hours.

[0108] A3. After extraction, remove the raw materials and drain the water. Add yeast extract at 2% of the dry weight of the mixed raw materials and vitamin C at 1%. Stir well.

[0109] A4. Using an automatic bagging machine, the treated composite raw materials are filled into high-temperature resistant polypropylene bags, with the filling volume being 60% of the bag volume. The mixture is then stored at 121℃ and 1.5 kg / cm³. 2 Maintain pressure for 30 minutes;

[0110] A5. Under sterile conditions, inoculate the bacterial culture at 5% of the weight of the raw material, spread it evenly on the surface of the culture medium, and place the bacterial bag on a three-dimensional shaker and shake it at 20 r / min for 15 min.

[0111] A6. First stage of cultivation for 3 days: The cultivation temperature is controlled at 30℃, the relative humidity is 75%, the carbon dioxide concentration is controlled below 0.3%, and ventilation is carried out twice a day for 30 minutes each time;

[0112] The second stage of cultivation lasted for 6 days: the temperature was lowered to 25℃, the relative humidity was 65%, the carbon dioxide concentration was controlled below 0.2%, the ventilation was carried out 3 times a day for 40 minutes each time, and at the same time, the diffused light with an intensity of 200 lx was provided for 2 hours a day.

[0113] The third stage of cultivation lasts for 8 days: Maintain the temperature and humidity of the second stage of cultivation, stop the light, and ventilate 4 times a day for 30 minutes each time.

[0114] Comparative Example 1

[0115] Culture medium formula: 75 parts hardwood sawdust, 20 parts wheat bran, 2 parts gypsum powder, 2 parts sucrose, and 130 parts water.

[0116] Preparation steps:

[0117] Mix hardwood sawdust, bran, and other raw materials, add water and stir, pack into polypropylene bags, and autoclave at 121℃ for 2 hours.

[0118] After cooling, inoculate the culture medium with 5% of the culture medium mass under sterile conditions and spread it evenly on the surface of the culture medium.

[0119] The culture temperature is controlled at 25℃ and the relative humidity at 75%. Ventilate once a day for 30 minutes each time and culture in the dark until the mycelium has fully grown in the bag.

[0120] Select vigorous strains for subculture, repeat twice, to obtain wood ear fungus strains.

[0121] Comparative Example 2

[0122] Culture medium formula: 85 parts cottonseed hulls, 10 parts wheat bran, 2 parts lime, 1 part gypsum, and 140 parts water.

[0123] Preparation steps:

[0124] Cottonseed hulls were soaked for 24 hours in advance, mixed evenly with other raw materials, packed into polypropylene bags, and sterilized at normal pressure at 100°C for 10 hours.

[0125] After cooling, inoculate with 8% of the culture medium by weight of the inoculum. After inoculation, place in a 28°C incubation room with a relative humidity of 65% and ventilate once a day for 20 minutes each time.

[0126] Cultivate until the mycelium fully covers the bag, select healthy strains and subculture twice to obtain oyster mushroom spawn.

[0127] Comparative Example 3

[0128] Culture medium formula: 40 parts sawdust, 30 parts cottonseed hulls, 20 parts wheat bran, 5 parts corn flour, 1 part gypsum, 0.3 parts potassium dihydrogen phosphate, and 120 parts water.

[0129] Preparation steps:

[0130] After mixing the raw materials, add water to adjust the moisture content to 58%, then put them into glass bottles and autoclave at 121℃ for 1.5 h.

[0131] Under aseptic conditions, inoculate the culture medium with 6% of the bacterial culture and place it in a 23°C incubation room with 60% relative humidity. Incubate in the dark and ventilate once a day for 25 minutes each time.

[0132] After the mycelium has fully colonized the bottle, select superior strains and subculture them twice to obtain conventional enoki mushroom strains.

[0133] Microbial strain testing:

[0134] Black fungus: Use 70 parts hardwood sawdust, 15 parts wheat bran, 3 parts corn flour, 1 part gypsum powder, 1 part sucrose, and 120 parts water. Mix them evenly and pack them into 17 cm × 33 cm polypropylene cultivation bags. Each bag weighs about 500 g of dry material and is sterilized at 121℃ for 2 hours.

[0135] Oyster mushrooms: Use 80 parts cottonseed hulls, 15 parts wheat bran, 10 parts crushed corn cobs, 3 parts lime powder, 1 part gypsum powder, and 150 parts water. Mix them together and pack them into bags (same specifications as above). Sterilize at 100℃ under normal pressure for 10 hours.

[0136] Enoki mushrooms: The mixture consists of 35 parts broadleaf tree wood chips, 35 parts cottonseed hulls, 25 parts crushed corn cobs, 8 parts wheat bran, 1.5 parts gypsum powder, 0.2 parts potassium dihydrogen phosphate, and 130 parts water. After packaging, the mixture is sterilized at 121℃ for 1.5 hours.

[0137] Examples 1-3 and Comparative Examples 1-3 were inoculated under sterile conditions. The inoculation amount was uniformly 8% of the dry weight of the cultivation substrate. After inoculation, the inoculation was sealed with sterile cotton plugs. Each group was set up with 3 replicates, with 30 bags per replicate.

[0138] Mycelium growth stage: Temperature 25℃, relative humidity 65%, ventilate twice a day for 30 minutes each time, incubate in the dark until the mycelium has fully grown in the bag.

[0139] Mushroom-growing stage:

[0140] Black fungus: Temperature 20℃, relative humidity 85%, light exposure 10 hours a day, intensity 500 lx, ventilation 3 times a day, 40 minutes each time.

[0141] Oyster mushrooms: Temperature 22℃, relative humidity 80%, natural diffused light, ventilate 4 times a day for 30 minutes each time.

[0142] Enoki mushrooms: Cultivate at 10℃ and 85-90% relative humidity in the dark, and ventilate twice a day for 20 minutes each time.

[0143] 1. Mycelial growth rate: Measure the length of mycelial spread every day after inoculation and calculate the average daily growth rate (mm / day).

[0144] 2. Time for mycelium to fully colonize the culture medium: Record the time (in days) required from inoculation to when the mycelium has fully colonized the culture medium.

[0145] 3. Extracellular enzyme activity: Cellulase and ligninase activities (U / g) of mycelia were determined by spectrophotometry, and samples were taken for testing when the mycelia filled the bag.

[0146] 4. Fruiting time: Inoculate the spawn into the cultivation substrate and record the time (in days) from inoculation to the appearance of the first flush of mushrooms.

[0147] 5. Yield per unit area: Calculate the average yield per unit area by counting the fresh weight (kg) of each bag (bottle) of edible fungi.

[0148] Table 1. Results of the test for wood ear fungus

[0149] parameter Example 1 Comparative Example 1 Mycelial growth rate (mm / day) 4.5-5.0 2.0-2.5 Time to full mycelial colonization of the bag (days) 15-18 25-30 Cellulase activity (U / g) 35-40 20-25 Ligninase activity (U / g) 25-30 15-18 Mushroom harvesting time (days) 40-45 55-60 Yield per unit (kg / bag) 0.8-0.9 0.5-0.6

[0150] Table 2. Test results of oyster mushrooms

[0151] parameter Example 2 Comparative Example 2 Mycelial growth rate (mm / day) 5.5-6.0 3.0-3.5 Time to full mycelial colonization of the bag (days) 10-12 15-20 Cellulase activity (U / g) 40-45 25-30 Ligninase activity (U / g) 30-35 20-23 Mushroom harvesting time (days) 25-30 35-40 Yield per unit (kg / bag) 1.2-1.3 0.8-0.9

[0152] Table 3. Test results for Enoki mushrooms

[0153] parameter Example 3 Comparative Example 3 Mycelial growth rate (mm / day) 4.0-4.5 2.5-3.0 Time to full mycelial colonization of the bag (days) 18-22 20-25 Cellulase activity (U / g) 30-35 18-22 Ligninase activity (U / g) 20-25 12-15 Mushroom harvesting time (days) 35-40 45-50 Yield per unit (kg / bag) 0.7-0.8 0.4-0.5

[0154] As shown in the table above, the mycelial growth rate of the examples was significantly faster than that of the comparative examples, and the average time for mycelium to fully fill the bag was shortened by 30-40%. The cellulase and ligninase activities of the mycelium in the examples were higher than those in the comparative examples, indicating that the strains cultivated by the process in the examples had a stronger ability to decompose the culture medium and could utilize nutrients more efficiently. The fruiting time of the examples was shortened by an average of 10-15 days, and the yield per unit area was increased by 40-60%. Through comparison, it can be seen that the high-activity edible fungus seed preparation process of the examples can effectively improve the activity of the strains and significantly improve the quality and production efficiency of edible fungus strains.

[0155] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly efficient preparation process for highly active edible fungi seeds, characterized in that, Includes the following steps: S1. Primary strain cultivation and screening The culture medium raw materials are cleaned, impurities removed, soaked, nutrient supplemented and sterilized. 5% of the culture medium mass of the strain is inoculated with the strain and cultured in segments. The fastest growing and healthiest strain is selected and cultured repeatedly 2 to 3 times to obtain the first generation F1 strain. S2, second- to third-generation strains' adaptation and domestication Retain 50% of the above-mentioned culture medium raw materials, add 50% of the target strain culture substrate, inoculate the first generation F1 strain, set a day-night alternation with a temperature difference of 15℃ / 12 h and 30℃ / 12 h, and screen for stable F2 generation strains; retain 20% of the above-mentioned culture medium raw materials, add 80% of the target strain culture substrate, inoculate the F2 generation strain, reduce the moisture content of the culture medium by 10-15%, and screen for stable F3 generation strains. S3, fourth to sixth generation strains intensive domestication Based on the initial culture medium, the carbon-to-nitrogen ratio was adjusted to 25-35:1, and 0.3% of compound amino acids and 0.02% of humic acid were added by weight of the culture medium. Temperature-controlled aerobic culture was employed, and F3 generation strains were inoculated. Stable F4 generation strains were then screened. Based on the above culture medium, the carbon source supply was reduced by 10%, and the ratio of organic to inorganic nitrogen in the nitrogen source was adjusted to 1:

1. F4 generation strains were inoculated, and stable F5 generation strains were screened. The target strain culture substrate was used as the culture medium, and F5 generation strains were inoculated. Fast-growing and high-yielding F6 generation strains were screened. S4, stable subculturing of the strain. The selected F6 generation strain was passaged twice to observe whether its traits were stable, ensuring that high activity, strong resistance, and stability did not degenerate during the passage process, and finally obtaining a stable, highly active strain. The initial strain cultivation and screening: A1. The culture medium raw materials are 40-45 parts of oat grains, 25-30 parts of millet, 15-20 parts of buckwheat hulls, and 10-15 parts of brewer's grains; rinse the oat grains and millet separately with clean water to remove surface impurities; Buckwheat hulls are crushed and passed through a 40-mesh sieve; brewer's grains are dried at 60-80℃ for 3-5 hours, crushed, and set aside; the pre-treated raw materials are mixed evenly and frozen at -10--20℃ for 12-24 hours; A2, the frozen composite raw materials are extracted in water with a temperature gradient, with an initial water temperature of 20-30℃ for 6-8 hours, then the temperature is raised to 40-45℃ and the soaking continues for 3-6 hours; finally, the temperature is raised to 60-65℃ and the soaking continues for 3-6 hours. A3. After extraction, remove the raw materials and drain the water. Add yeast extract at 2% of the dry weight of the mixed raw materials and vitamin C at 1%. Stir well. A4. Using an automatic bagging machine, the treated composite raw materials are filled into high-temperature resistant polypropylene bags, with the filling volume being 60% of the bag volume. The mixture is then stored at 121℃ and 1.5 kg / cm³. 2 Maintain pressure for 20-30 minutes; A5. Under sterile conditions, inoculate the bacterial culture at 5% of the weight of the raw material, spread it evenly on the surface of the culture medium, and place the bacterial bag on a three-dimensional shaker and shake it at a speed of 15~20 r / min for 10~15 min. A6. First stage of cultivation for 1-3 days: The cultivation temperature is controlled at 28-30℃, the relative humidity is 70-75%, the carbon dioxide concentration is controlled below 0.3%, and ventilation is carried out twice a day for 30 minutes each time; The second stage of cultivation lasts 4-6 days: the temperature is reduced to 23-25℃, the relative humidity is 60-65%, the carbon dioxide concentration is controlled below 0.2%, the ventilation is carried out 3 times a day for 40 minutes each time, and at the same time, the diffused light with an intensity of 100-200 lx is provided for 1-2 hours a day. The third stage of cultivation lasts 7-8 days: Maintain the temperature and humidity of the second stage of cultivation, stop the light, and ventilate 4 times a day for 30 minutes each time; In step A2, the mixture is stirred every 6 hours during the extraction process, and the water used for extraction is a mixed solution containing 0.02% potassium dihydrogen phosphate and 0.01% magnesium sulfate. The target spore cultivation substrate is the substrate in the cultivation environment of the target spore; Auricularia auricula-judae cultivation substrate: 70-75 parts hardwood sawdust, 15-20 parts wheat bran, 3-5 parts corn flour, 1-2 parts gypsum powder, 1-1.5 parts sucrose, 120-140 parts water; Oyster mushroom cultivation substrate: 80-85 parts cottonseed hulls, 10-15 parts wheat bran, 10-20 parts crushed corn cobs, 2-3 parts lime powder, 1-2 parts gypsum powder, 130-150 parts water; Enoki mushroom cultivation substrate: 30-35 parts broadleaf wood sawdust, 30-35 parts cottonseed hulls, 20-25 parts crushed corn cobs, 5-8 parts wheat bran, 1-1.5 parts gypsum powder, 0.1-0.2 parts potassium dihydrogen phosphate, 110-130 parts water; The temperature and oxygen control parameters for the F4 generation strain were: maintaining 20℃ for 8 hours and 32℃ for 16 hours per day, with the oxygen concentration controlled at 15-17%. The F5 generation strain was cultured with the following parameters: humidity reduced by 5-10%, pH adjusted to 5.0-5.5, and subjected to strong light irradiation of 300-350 lx for 3-4 hours per day. The F6 generation strain was cultured with a temperature range of 12-25℃, a relative humidity range of 50-80%, alternating day and night, and ventilated 6 times a day for 30 minutes each time to create a complex environment close to nature.

2. The efficient preparation process for highly active edible fungi seeds according to claim 1, characterized in that, The fungal strain in step A5 is one of the following: wood ear fungus, oyster mushroom, or enoki mushroom.

3. The efficient preparation process for highly active edible fungi seeds according to claim 1, characterized in that, The compound amino acids are lysine, methionine, and phenylalanine mixed in a mass ratio of 2:1:1.