Oriental small bridge fungus and artificial cultivation method thereof

Abstract

The invention belongs to the technical field of edible mushrooms, and particularly relates to a small-scale long-bridge fungus orientalis and an artificial cultivation method thereof. According to the invention, a strain of wild Ponicillium orientialis is obtained through collection, separation, identification and pure culture of wild fungi, is named as Kou mushroom CS12, is preserved in the China Center for Type Culture Collection (CCTCC), and has a preservation number of CCTCC NO: M20242547, and is named as Kou mushroom CS12, Kou mushroom CS12, Kou mushroom CS12, Kou mushroom CS12, Kou mushroom CS12, Kou mushroom CS12 and Kou mushroom CS12. The domestication and cultivation method of the Caou mushroom CS12 is formed through strain propagation, bag cultivation and fruiting light, temperature, water and gas condition control. The mushroom age of the kaiou mushroom CS12 is 40 to 85 days, and the biological conversion rate is 66.37 percent to 120.23 percent. The Caou mushroom CS12 sporocarp is rich in polysaccharide, protein, amino acid and other nutrient substances, delicious in taste, rich in nutrition, crisp and tender in stipe, fine and smooth in pileus, fresh in taste, rich in fragrance and excellent in mouthfeel.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi, and in particular relates to an oriental small long bridge mushroom and an artificial cultivation method thereof. Background Art

[0002] Ponticulomyces is a new genus proposed in Oudemansiella / Xerula in recent years. According to morphological and molecular data, it contains two species, namely Ponticulomyces kedrovayae from the Russian Far East and Ponticulumyces orientalis (Oudemansilla orientalis Zhu L. Yang) from China. The main characteristics of this genus are that the cap is sticky, the fruiting body grows on rotten wood, and there are two types of capsules. Ponticulomyces orientalis (Oudemansilla orientalis Zhu L. Yang) is one of the 966 taxa of edible fungi in my country, but there are no reports of artificial cultivation at home and abroad. Summary of the invention

[0003] The researchers of the present invention collected wild fruiting bodies and then isolated, identified, pure cultured and preserved the strain to obtain the CS12 strain of Trichoderma kaiopsis. A cultivation method of the CS12 strain of Trichoderma kaiopsis was formed by solid and liquid propagation, bag material cultivation and mushroom light, temperature, water and air condition control of the strain, which was specifically achieved by the following technical scheme:

[0004] A kind of oriental small long bridge mushroom was obtained by collecting, identifying, isolating and pure culturing wild mushrooms, and was named Kaio Mushroom CS12. It has been preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO:M 20242547 and the preservation date of November 13, 2024.

[0005] The method for obtaining the Oriental small long bridge mushroom comprises the following steps: collecting a wild mushroom from a broad-leaved tree stump in Qiandongnan, Guizhou, using 1‰ HgCl 2 After disinfection, take 1-3mm 3 The tissue block was cultured on a PDA medium plate at 25°C. When the mycelium grew to 2 / 3 of the plate area, the mycelium at the edge of the colony was picked for transfer culture to obtain a pure culture, i.e., a strain numbered as CS12. The PDA medium composition: 200g potato, 20g glucose, 15g agar, and 1000ml water.

[0006] Identification of strains: The fruiting bodies of the strains grow in clusters or singly, with a cap width of 20-70mm, hemispherical to conical protrusions, and the color of the fruiting bodies is "brown" to "dark brown" when immature, and "smoky gray" to "mouse gray" when mature. The color is darker in the middle and lighter at the edges. The gills are radial, with fine short soft hairs (when young) to slightly pubescent, and sticky when wet. The flesh of the strain is white. The gills are white, curved, sparse, and without edges. The stipe is 30-300×5-40mm, grayish white to brown from top to bottom, with small white scales, without rings and rhizomes. The 18S rDNA-ITS sequence analysis and comparison in the NCBI database (blast.ncbi.nlm.nih.gov) determined that the classification name is Ponticulomyces orientalis, and it is named Kai'ao Mushroom CS12.

[0007] 18S rDNA-ITS sequence analysis is:

[0008] -TTGACCTCAAATCAGGTAGACTAC

[0009] The artificial cultivation method of the oriental small long bridge mushroom comprises the following steps:

[0010] 1. Preparation of mother stock:

[0011] Select the preserved strains and pick a 5mm diameter bacterial block under sterile conditions to inoculate it into a culture medium plate or test tube, and culture it at 25°C until the mycelium covers the surface of the culture medium. The culture medium is made of the following raw materials: 200g / L potato (boiled liquid), 20g / L glucose, 15g / L agar, 2-15g / L peptone, 1-10g / L yeast powder, and natural pH.

[0012] 2. Stock seed preparation:

[0013] (1) Liquid stock preparation

[0014] Original seed liquid culture medium (g / L): soybean powder 10-100, corn powder 10-40, rapeseed meal 10-50, KH 2 PO 4 1-5, MgSO 4 1-5, peptone 5-20, yeast powder 1-10, pH 5.5-7.5. Accurately weigh the above materials or all of them according to the formula ratio to prepare the culture medium, sterilize at 121℃ for 30min; select the mother plate or test tube with white, dense, strong and uncontaminated mycelium, pick 2-6 pieces of mycelium with a diameter of 5mm and vigorous growth under sterile conditions, inoculate them into a 100-5000ml triangular flask with a liquid volume of 35-1500ml, let it stand for 12-36h after inoculation, and then place it on a shaker at 25℃ and 100-300r / min for 5-12 days.

[0015] (2) Solid stock preparation

[0016] Solid culture medium for original seed: 40-80% sawdust, 1-10% corn flour, 10-30% rice husk, 10-30% cottonseed hull, 10-35% wheat bran, 1-5% sucrose, 1-2% lime, 1-2% gypsum, natural pH. Accurately weigh the above materials or all of them according to the formula ratio to prepare the culture medium, mix well and add water to a water content of 55-65%, put it into a polypropylene plastic bag of 8cm×28cm×0.005cm, seal it with a ring, sterilize the original seed bag at 121℃ in a high-pressure steam sterilizer for 2h-4h, take it out when the temperature of the cultivation bag drops to 60-70℃, cool it to 28℃, and select a plate with white, dense, strong and uncontaminated mycelium. Under sterile conditions, pick 2-6 pieces of mycelium with a diameter of 1cm and grow vigorously and inoculate them into the bag, and place it in a 25℃ incubator away from light to cultivate until the bag is full.

[0017] 3. Preparation of cultivars:

[0018] (1) Liquid culture seed preparation

[0019] Liquid culture medium for cultivated species (g / L): wood powder (100 mesh) 20-200, bran 50-200, soybean powder 10-100, glucose 5-100, corn flour 10-40, peptone 1-10, potassium dihydrogen phosphate 1-5, magnesium sulfate 0.2-2, dapoxetine 0.1-2, VB10.002-0.5, pH 5.5-7.5. Accurately weigh several or all of the above materials according to the formula ratio to prepare the culture medium, add the above culture medium according to 1 / 2-2 / 3 of the liquid volume of the fermenter, sterilize at 121℃ for 30min, cool to 25℃, inoculate the liquid stock at an inoculation rate of 5%-20% of the volume of the culture medium in the fermenter, and culture for 5-20d. The culture condition is aeration of 20-40L / 1.0m 3 / h, the tank pressure is 0.03MPa, and the culture temperature is 25℃.

[0020] (2) Preparation of solid cultivars

[0021] Solid culture medium for cultivation: 40-80% sawdust, 1-10% corn flour, 10-30% rice husk, 10-30% cottonseed hull, 10-35% wheat bran, 1-5% sucrose, 1-2% lime, 1-2% gypsum, natural pH. Accurately weigh the above materials or all of them according to the formula ratio to prepare the culture medium, mix well and add water to a water content of 55-65%, put it into a 8cm×28cm×0.005cm polypropylene plastic bag, seal it with a ring, sterilize the packed original seed bag at 121℃ in a high-pressure steam sterilizer for 2h-4h, take it out when the temperature of the cultivation bag drops to 60-70℃, cool it to 28℃, inoculate 15-20ml liquid original seed or 1-10g solid original seed, and place it at 25℃ in the dark to cultivate until the bag is full.

[0022] 4. Preparation of cultivation bacteria bags:

[0023] Culture medium: wood chips 20-80%, camellia oleifera shell (powder) 10-40%, cottonseed shell 20-90%, bran 1-20%, sucrose 1-5%, lime 1-3%, gypsum 1-3%, natural pH.

[0024] Accurately weigh several or all of the above materials according to the formula ratio, mix well and add water to a water content of 55-65%, put them into a 8cm×28cm×0.005cm polypropylene plastic bag, seal it with a ring, sterilize the packed cultivation bag at 121°C in a high-pressure steam sterilizer for 2h-4h, take out the cultivation bag when the temperature drops to 60-70°C, cool it to 28°C, inoculate 20-35ml of liquid bacteria or 5-10g of solid bacteria, and place it at 25°C in the dark for cultivation until the bag is full.

[0025] 4. Mushroom production management:

[0026] (1) Cultivation without covering soil

[0027] Move the physiologically mature mushroom bags to the mushroom house, open the bags and place them neatly on the bed frame, keep the relative humidity of the room above 90%, light 100-500lx, control the temperature at 15-25℃, pay attention to ventilation, and primordium will appear in 10-20 days. Harvest when the fruiting body grows to 40-60% of the cap.

[0028] (2) Soil covering cultivation

[0029] Move the mushroom bags that have reached physiological maturity to the shelf frame of the mushroom house, cut off the upper plastic bag film at the top of the bag material, make 2-3 small holes on both sides of the mushroom bag to prevent water accumulation in the mushroom bag, cover the soil between the mushroom bags and on the material to 2-4cm above the material surface. After covering the soil, water it thoroughly once. The primordium will appear in 10-25 days. Harvest when the fruiting body grows to 40-60% of the cap.

[0030] The beneficial effects of the present invention are:

[0031] The invention obtains a wild oriental small long bridge mushroom by wild mushroom collection, separation, identification and pure culture, and is named as Kai'ao mushroom CS12. An artificial cultivation method of Kai'ao mushroom CS12 strain is formed by studying the solid and liquid propagation of the strain, bag material cultivation and mushroom light, temperature, water and air condition control. The invention can realize the commercial cultivation of Kai'ao mushroom CS12, add new species to the edible mushroom market, and provide an artificial cultivation method of wild edible strains. The age of Kai'ao mushroom CS12 is 40-85 days, and the bioconversion rate is 66.37%-120.23%. The fruiting body of Kai'ao mushroom CS12 is rich in nutrients such as polysaccharides, proteins, amino acids, etc., and is delicious and nutritious. The stem tastes crisp and tender, and the cap is smooth. It has a fresh taste, rich fragrance and excellent taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart for the artificial cultivation of Oriental Microporus;

[0033] Figure 2 This is a photo of the Oriental small long bridge mushroom harvested in Example 1;

[0034] Figure 3 It is a phylogenetic tree diagram. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0036] Example 1

[0037] 1. Preparation of mother stock

[0038] Culture medium (g / L): 200 potatoes (cooking liquid), 20 glucose, 2 peptone, 2 yeast powder, 15 agar, natural pH. Select the preserved mother strain and inoculate the 5 mm diameter bacteria block into the above culture medium plate under sterile conditions. Inoculate and culture at 25°C. After 7 days, the mycelium will cover the surface of the culture medium.

[0039] 2. Stock seed preparation

[0040] Solid medium: sawdust 40%, corn meal 2%, cottonseed hulls 30%, rice husks 10%, wheat bran 15%, sucrose 1%, lime 1%, gypsum 1%, pH natural.

[0041] Accurately weigh the materials according to the formula ratio, mix well and add water to 65% of the water content, put them into a 8cm×28cm×0.005cm polypropylene plastic bag, seal it with a ring, and sterilize the packed cultivation bag at 121°C in a high-pressure steam sterilizer for 2 hours. When the temperature of the cultivation bag drops to 60-70°C, take it out, cool it to 28°C, select the mother plate with white, dense, strong and uncontaminated mycelium, use a puncher with a diameter of 1 cm to take 5 mushroom blocks, inoculate them into the bag, and place it in a 26°C incubator away from light to cultivate until the bag is full.

[0042] 3. Cultivated seeds preparation

[0043] Solid medium: sawdust 40%, corn meal 2%, cottonseed hulls 30%, rice husks 10%, wheat bran 15%, sucrose 1%, lime 1%, gypsum 1%, pH natural.

[0044] Accurately weigh the materials according to the formula ratio, mix well and add water to 65% of the water content, put them into a 8cm×28cm×0.005cm polypropylene plastic bag, seal it with a ring, and sterilize the packed cultivation bag in a high-pressure steam sterilizer at 121°C for 2h. When the temperature of the cultivation bag drops to 60-70°C, take it out, cool it to 28°C, inoculate 5g of solid original seed, and culture it at 25°C in the dark until the bag is full.

[0045] 4. Preparation of cultivation bacteria bags

[0046] Culture medium: 33% sawdust, 50% cottonseed hull, 15% bran, 1% lime, 1% gypsum, natural pH.

[0047] Weigh the materials accurately according to the formula ratio, mix well, add water to 65% water content, put into 8cm×28cm×0.005cm polypropylene plastic bag, seal with a ring, sterilize the packed cultivation bag in a high pressure steam sterilizer at 121℃ for 4h, take out when the cultivation bag temperature drops to 60-70℃, cool to 28℃, inoculate about 5g solid bacteria, and culture at 25℃ away from light, and fill the bag in 42 days.

[0048] 5. Mushroom production management

[0049] Move the mushroom bags that have reached physiological maturity to the mushroom house, open the bags and place them neatly on the bed frame, keep the relative humidity of the room air above 90%, light 200-300lx, control the temperature at 18℃, ventilate, and primordium will appear in 16 days. Harvest when the fruiting body grows to 40-60% of the cap. The bioconversion rate is 103.25%.

[0050] Example 2

[0051] 1. Preparation of mother stock

[0052] Culture medium (g / L): 200 potatoes (boiled liquid), 20 glucose, 15 agar, natural pH. Select the preserved mother strain and inoculate 5 mm diameter bacteria blocks into the above culture medium plate under sterile conditions. Inoculate and culture at 25°C. After 7 days, the mycelium will cover the surface of the culture medium.

[0053] 2. Stock seed preparation

[0054] Liquid culture medium (g / L): soybean powder 10, corn powder 20, KH 2 PO 4 1. MgSO 4 1. Peptone 5, pH 6.5. Add according to the proportion and sterilize at 121℃ for 30min.

[0055] Accurately weigh several or all of the above materials according to the formula ratio to prepare the culture medium, sterilize at 121℃ for 30min; use a 5mm diameter puncher to take 5 white, dense, strong and uncontaminated mother culture plate blocks under sterile conditions, inoculate them into a 250ml triangular flask with a liquid volume of 90ml. After inoculation, let it stand for 12h, and then place it on a shaker at 25℃ and 180r / min for 7 days.

[0056] 3. Cultivated seeds preparation

[0057] Liquid culture medium (g / L): wood flour (100 mesh) 20, corn flour 10, KH 2 PO 4 1. MgSO 4 1. Peptone 3, Bubble 0.5, pH 6.5.

[0058] Add 30L of the above culture medium to a 50L fermenter and sterilize at 121℃ for 30min. Inoculate the liquid stock at 10% of the volume of the culture medium and culture for 10 days. The culture conditions are ventilation volume of 1.0m 3 / h, the tank pressure is 0.03MPa, and the culture temperature is 25℃.

[0059] 4. Preparation of cultivation bacteria bags

[0060] Culture medium: 33% sawdust, 50% cottonseed hull, 15% bran, 1% lime, 1% gypsum, natural pH.

[0061] Weigh the materials accurately according to the formula ratio, mix well, add water to 65% water content, put into 8cm×28cm×0.005cm polypropylene plastic bags, seal with a ring, sterilize the packed cultivation bags in a high pressure steam sterilizer at 121℃ for 4h, take out when the cultivation bag temperature drops to 60-70℃, cool to 28℃, inoculate 15ml liquid bacteria per bag, place at 25℃ and culture in dark, and fill the bag in 51d.

[0062] 5. Mushroom production management

[0063] Move the physiologically mature mushroom bags to the mushroom house, open the bags and place them neatly on the bed frame, keep the relative humidity of the room above 90%, light 200-300lx, control the temperature at 20℃, ventilate, and primordium will appear in 16 days. Harvest when the fruiting body grows to 40-60% of the cap. The bioconversion rate is 66.37%.

[0064] Morphology of fruiting body after domestication

[0065] The cap of Kaio Mushroom No. CS12 is 20-60mm wide, hemispherical to conical, and the color of the fruiting body is "brown" to "dark brown" when immature, and "smoky gray" to "mouse gray" when mature. The color is darker in the middle and lighter at the edges. The gills are radial, fine and short (when young) to slightly pubescent, and sticky when wet. The flesh is white. The gills are white, curved, sparse, and without edges. The stipe is 30-300×5-40mm, grayish white to brown from top to bottom, with small white scales, without rings and rhizomes. The bioconversion rate is 66.37%-120.23%.

[0066] The relevant nutritional components of the oriental small long bridge mushroom cultivated by the method of Example 1 were determined, and the results are shown in Table 1.

[0067] Table 1 Example 1 Determination of relevant nutritional components in fresh fruiting bodies

[0068]

[0069] It is necessary to point out here that the above embodiments and comparative examples are limited to further elaborating and understanding the technical solution of the present invention, and cannot be understood as further limiting the technical solution of the present invention. Inventions and creations with non-outstanding essential features and significant progress made by those skilled in the art still fall within the protection scope of the present invention.

Claims

1. An oriental small long bridge mushroom, characterized in that It was obtained through collection, isolation, identification and pure culture of wild mushrooms, and named as Kaio Mushroom CS12. It has been preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO:M 20242547 and the preservation date of November 13, 2024.

2. The method for obtaining the oriental small long bridge mushroom according to claim 1, characterized in that: The following steps are involved: A wild mushroom was collected from a broad-leaved tree stump in Qiandongnan Prefecture, Guizhou Province. After disinfection with 1‰ HgCl2, 1-3 mm 3 The tissue blocks were cultured on PDA medium plates at 25°C. When the mycelium grew to 2 / 3 of the plate area, the mycelium at the edge of the colony was picked up for transfer culture to obtain a pure culture, which was a strain numbered CS12.

3. The artificial cultivation method of an oriental small long bridge mushroom as claimed in claim 1, characterized in that: The following steps are involved: (1) Preparation of mother stock: Select the preserved strains and pick out the 5mm diameter bacteria blocks under sterile conditions and inoculate them into the culture medium plate or test tube, and culture them at 25°C until the mycelium covers the surface of the culture medium; the culture medium is made of the following raw materials: 200g / L potato (boiled liquid), 20g / L glucose, 15g / L agar, 2-15g / L peptone, 1-10g / L yeast powder, and the pH is natural; (2) Stock seed preparation: (a) Liquid stock preparation Original seed liquid culture medium (g / L): soybean powder 10-100, corn flour 10-40, rapeseed meal 10-50, KH2PO4 1-5, MgSO4 1-5, peptone 5-20, yeast powder 1-10, pH 5.5-7.5; accurately weigh the above several or all materials according to the formula ratio to prepare the culture medium, and sterilize at 121°C for 30 minutes; select the mother seed plate or test tube with white, dense, strong and pollution-free mycelium, pick 2-6 pieces of mycelium with a diameter of 5mm and vigorous growth under sterile conditions, inoculate them into a 100-5000ml triangular flask with a liquid volume of 35-1500ml, let it stand for 12-36h after inoculation, and then place it on a shaking table at 25°C and 100-300r / min for 5-12 days; (b) Solid stock preparation Solid culture medium for original seeds: 40-80% sawdust, 1-10% corn flour, 10-30% rice husk, 10-30% cottonseed hull, 10-35% wheat bran, 1-5% sucrose, 1-2% lime, 1-2% gypsum, natural pH; accurately weigh several or all of the above materials according to the formula ratio to prepare the culture medium, mix well and add water to a water content of 55-65%, put it into a polypropylene plastic bag of 8cm×28cm×0.005cm, seal it with a ring, sterilize the packed original seed bag at 121℃ in a high-pressure steam sterilizer for 2h-4h, take it out when the temperature of the cultivation bag drops to 60-70℃, cool it to 28℃, select a flat plate with white, dense, strong and uncontaminated mycelium, pick 2-6 pieces of mycelium with a diameter of 1cm and vigorous growth under sterile conditions and inoculate them into the bag, place it in a 25℃ incubator away from light and culture until the bag is full. (3) Cultivated seeds preparation: (a) Liquid culture preparation Cultivated species liquid culture medium (g / L): wood powder (100 mesh) 20-200, bran 50-200, soybean powder 10-100, glucose 5-100, corn flour 10-40, peptone 1-10, potassium dihydrogen phosphate 1-5, magnesium sulfate 0.2-2, dapoxetine 0.1-2, VB10.002-0.5, pH 5.5-7.5; accurately weigh the above materials or all of them according to the formula ratio to prepare the culture medium, add the above culture medium according to 1 / 2-2 / 3 of the liquid volume of the fermenter, sterilize at 121°C for 30 minutes, cool to 25°C, inoculate the liquid stock at an inoculation amount of 5%-20% of the volume of the culture medium in the fermenter, and culture for 5-20 days; the culture condition is that the ventilation volume is 20-40L / 1.0m 3 / h, tank pressure is 0.03MPa, and culture temperature is 25℃; (b) Preparation of solid culture seeds Solid culture medium for cultivated species: 40-80% sawdust, 1-10% corn flour, 10-30% rice husk, 10-30% cottonseed hull, 10-35% wheat bran, 1-5% sucrose, 1-2% lime, 1-2% gypsum, natural pH; accurately weigh several or all of the above materials according to the formula ratio to prepare the culture medium, mix well and add water to a water content of 55-65%, put it into a polypropylene plastic bag of 8cm×28cm×0.005cm, seal it with a ring, sterilize the packed original seed bag at 121℃ in a high-pressure steam sterilizer for 2h-4h, take it out when the temperature of the cultivation bag drops to 60-70℃, cool it to 28℃, inoculate 15-20ml liquid original seed or 1-10g solid original seed, and place it at 25℃ in the dark to cultivate until the bag is full; (4) Preparation of cultivation bacteria bag: Culture medium: sawdust 20-80%, camellia hull (powder) 10-40%, cottonseed hull 20-90%, bran 1-20%, sucrose 1-5%, lime 1-3%, gypsum 1-3%, natural pH; Accurately weigh several or all of the above materials according to the formula ratio, mix well, add water to a water content of 55-65%, put them into a 8cm×28cm×0.005cm polypropylene plastic bag, seal it with a ring, sterilize the packed cultivation bag in a high-pressure steam sterilizer at 121°C for 2h-4h, take it out when the temperature of the cultivation bag drops to 60-70°C, cool it to 28°C, inoculate 20-35ml of liquid bacteria or 5-10g of solid bacteria, and place it at 25°C in the dark to cultivate until the bag is full; (5) Mushroom production management: (a) Cultivation without soil covering Move the physiologically mature mushroom bags to the mushroom house, open the bags and place them neatly on the bed frame, keep the relative humidity of the room above 90%, light intensity 100-500lx, temperature 15-25℃, pay attention to ventilation, primordium will appear in 10-20 days, and harvest when the fruiting body grows to 40-60% of the cap; (b) Soil covering cultivation Move the physiologically mature mushroom bags to the shelf frame of the mushroom house, cut off the upper plastic bag film at the same level as the upper edge of the bag material, make 2-3 small holes on both sides of the mushroom bag to prevent water accumulation in the bag, cover the mushroom bags and the material with soil to 2-4cm above the material surface, and water it thoroughly once after covering the soil. Primordia will appear in 10-25 days, and the fruiting body will be harvested when the cap opens 40-60%.

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