CN109220530B - Black fungus strain and application thereof - Google Patents
Black fungus strain and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a black fungus strain, which is a black fungus strain Guiyun No. 3, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has a preservation number of CGMCC No.15367, and has a preservation date of 2018, 3 months and 22 days. The soaking rate of the fruiting bodies of the black fungus reaches more than 20.0, which is far higher than that of the black fungus strains which are popularized and cultivated at present; the iron content of the black fungus fruiting body is not lower than 166mg/Kg, the polysaccharide content is not lower than 4.15g/100g, and the iron content and the polysaccharide content of the black fungus fruiting body are higher than those of the black fungus strains which are popularized and cultivated in the Guangxi region. The black fungus of the invention has single sheet shape, no vein or little vein, soft and elastic ear, smooth and smooth mouth feel and is deeply loved by consumers. The fresh product is yellow brown, bright and transparent; the dried product has black brown abdominal surface, gray black back surface, excellent appearance and wide market prospect.
Description
Technical Field
The invention relates to the field of edible fungi, and particularly relates to a black fungus strain and application thereof.
Background
Auricularia auricula belongs to the order of Auriculariales, the family of Auriculariaceae, the class of Heterobasidiomycetes, and is one of four edible fungus cultivars in China. Black fungus belongs to medium-temperature edible fungi, has the characteristics of cold resistance and heat intolerance, and is generally distributed in regions which are cooler in summer in main production areas. Guangxi is also one of the main production areas of black fungus in China, the cultivated black fungus variety takes 916 as the leading factor, the new family, the black fungus. The Guangxi black fungus has abundant resources, and especially the black fungus (local common name is hundred-color black fungus) in Bai-color areas of Guangxi has the most distribution and the best quality. In recent years, the protection and utilization of the Pleurotus citrinopileatus resource are very important to relevant departments of the Guangxi government, and the research strength of domesticating and cultivating the native Auricularia auricular species in Guangxi by using the Pleurotus citrinopileatus resource is also enhanced by local scientific research departments, but the excellent Auricularia auricular species screened, domesticated and cultivated from the Pleurotus citrinopileatus wild resource is few at present.
The soaking rate of the fruiting body of the existing black fungus is generally 9-15, the sugar content is generally 2.0-3.0 g/100g, and the iron content is 30-80 mg/Kg. The content of nutrient substances and the quality of the existing black fungus are not outstanding.
Disclosure of Invention
The invention overcomes the technical problems of the black fungus and provides a black fungus strain.
In order to solve the problems, the invention provides a black fungus strain:
the classification name of the black fungus strain provided by the invention is as follows: auricularia (Auricularia auricular-judae) belongs to the class Auricularia species of Auricularia of the class Heterobasidiomycota of the class Auriculariales. The black fungus strain is preserved in China general microbiological culture Collection center, and the addresses are as follows: the No. 3 Xilu Beijing, Chaoyang, is No.1 Hospital, with the preservation number of CGMCC No.15367 and the preservation date of 2018, 3 months and 12 days.
The genetic difference analysis of the black fungus strain of the invention comprises the following steps: adopts Guiyun No. 3, 916, Xinke No. 10, Xinke No. 4, black three, Baiyun No. 6, Au8129, Au149, YE2, black 29, HME2, Ty013, Ty041, Ty051, Ty063, YEDay 3The results of ISSR analysis of genetic differences of the black fungus strains of the invention are shown in FIGS. 3-15, and the 13 primers of the invention can be used for 16 black fungus strainsThe strain can amplify stable polymorphism band. The DNA (ISSR) fingerprint polymorphism analysis result of the strain shows that the difference between the banding pattern and the quantity of the Guiyun No. 3 strain and other strains is large. ISSR cluster analysis (fig. 2) shows that: the genetic similarity coefficient of Guiyun No. 3 and other strains is below 0.68, the strain genetic relationship is far, which shows that the Guiyun No. 3 strain has larger genetic difference with other black fungus strains, and the antagonistic experiment results (as shown in figure 16-figure 18) of the black fungus Guiyun No. 3 strain and the New family No. 4, 916 and Baiyun No. 6 strains show that the Guiyun No. 3 strain and the New family No. 4, 916 and Baiyun No. 6 strains of the black fungus mainly planted in Guangxi have obvious hypha antagonistic reaction, and the strains are incompatible. The ISSR analysis of the genetic difference of the strains and the antagonistic experiment result of the strains show that the black fungus strain is a new black fungus strain.
The morphological characteristics of the black fungus are as follows: ear size (length x width) about 4.21cm x 5.28 cm; the ear base is smaller; ear thickness: 1.07 mm; the fruit body is in the shape of: single raw, sheet; the ear vessels are few, and the basal part of the fruiting body has a few big tendons. The color of the sporocarp is yellow brown, the colloid content is high, the elasticity of the ear is good, the ear has no handle, the edge of the sporocarp is not cracked, and the sporocarp body is smooth or slightly wrinkled; the infertile villi is obvious; marrow layer is arranged on the cross section; the period of emergence is 55-70 days, which belongs to medium-early maturity.
The colony morphology characteristics of the black fungus on the PDA solid culture medium are as follows: hypha morphology: villiform; colony morphology: a circular shape; the density of hyphae is medium; the aerial hyphae are abundant and developed; the edges of the bacterial colonies are neat; the pigment is yellow brown; hypha growth rate: 9.84 mm/d; the color of the hypha is pure white.
The black fungus cultivation characters of the invention are as follows: the anti-impurity capacity is middle; the high temperature resistance is strong; the biological conversion rate is 120-140%; the soaking rate of the fruiting body is not less than 20.0, and the fruiting body is soft.
The black fungus strain is obtained by screening, domesticating and cultivating a hundred-color wild black fungus, and the screening and domesticating method comprises the following steps:
(1) carrying out tissue separation and purification on the collected hundred-color wild black fungus to obtain a mother strain Ty 026;
(2) cultivating, domesticating and screening the parent strain Ty026 to obtain 60 first generation tissue isolates of the Ty026 strain, which are marked as F1Generation; the screening method comprises the following steps: selecting fruiting body with thick and solid ear, moderate size and less vein from parent strain Ty026, and performing tissue separation and purification;
(3) to F1Cultivating, domesticating and screening the generation strains to obtain 30 second generation tissue isolates, which are marked as F2Generation; the screening method comprises the following steps: from F1Selecting fruiting body with stable performance, high yield, thick and solid ear, moderate size and less vein from the substitute strain, and performing tissue separation and purification;
(4) to F2Cultivating, domesticating and screening the substitute strain to obtain 30 third generation tissue isolates, which are marked as F3Generation; the screening method comprises the following steps: from F2Selecting fruiting body with stable performance, high yield, thick and solid ear, moderate size and less vein from the substitute strain, and performing tissue separation and purification;
(5) to F3Cultivating, domesticating and screening the substitute strains to obtain the black fungus strains; the screening method comprises the following steps: from F3Selecting strains with thick and solid lugs, moderate size, less veins, high yield and uniform and stable characters from the substitute strains;
wherein, in the steps (1) to (5), the tissue separation and purification method of the fruiting body is as follows: disinfecting the surface of a black fungus entity for 1-3 min by adopting an ethanol solution with the volume percentage of 70%, cutting tissue blocks of 2cm multiplied by 2cm, continuing to disinfect for 1min by adopting the ethanol solution with the volume percentage of 70%, washing for 2-3 times by adopting sterile water, naturally airing, inoculating into a PAD culture medium, and culturing at constant temperature of 25-28 ℃ until hyphae grows to fill the test tube; selecting mycelium at the front section of the test tube, transferring the mycelium to a test tube slant culture medium for culture, and repeatedly selecting and transplanting for 2-3 times according to the method to obtain a purified strain; the test tube slant culture medium comprises the following components: 200g of potato, 20g of glucose, 20g of agar and 1000ml of water;
in the steps (1) to (5), the black fungus cultivation and domestication method adopts bag-type clinker cultivation, and the cultivation steps are as follows:
(1) preparing a fungus bag: selecting a short polyethylene angle-folded bag of 17cm multiplied by 33cm multiplied by 0.004cm or a long polyethylene angle-folded bag of 15cm multiplied by 55cm multiplied by 0.004cm, bagging the cultivation material, placing the cultivation material into a sterilization pot for sterilization, raising the temperature to 100 ℃ within 4 hours after the cultivation material is put into the pot, and sterilizing the cultivation material for 12 to 16 hours at constant temperature, wherein the fire can not be stopped in the period; naturally cooling the temperature in the pot to 60 ℃ after sterilization, taking out the material rod when the temperature is hot, moving the material rod into a cooling chamber or an inoculation chamber, and inoculating according to the aseptic operation requirement when the temperature of the material rod is reduced to below 28 ℃; during inoculation, the short bag is directly inoculated at the bag opening, the long bag is inoculated by punching 3-4 holes on the bag body, the diameter of each hole is 1.5-2.0 cm, and the depth of each hole is 2-2.5 cm; the cultivation material comprises the following components in parts by weight: 40-80 parts of miscellaneous tree dust, 10-15 parts of wheat bran, 0.5-1 part of gypsum, 0.5-1 part of lime, 0-1 part of cane sugar, 0-25 parts of cottonseed hulls and 0-42 parts of mulberry branch crumbs;
(2) hypha culture: after inoculation, placing the fungus sticks in a dark, dry and well ventilated environment for hypha culture, wherein the environment temperature of a culture site is 25-28 ℃, the air humidity is below 70%, the hypha culture time is 2 months, and pile turning, pile scattering and ventilation are carried out according to the environment temperature and the hypha growth condition;
(3) puncturing holes: when hyphae grow over the fungus bags and a small amount of black fungus base is formed, pricking holes are formed when the hyphae are physiologically mature; the ear is pierced by adopting a bag-removing-free piercing manner, the number of the piercing holes is 80-100 per bag, the number of the piercing holes is 150 per bag, the aperture is 0.4cm, and the hole depth is 0.5 cm. Piling up the fungus sticks in a shape like a Chinese character 'jing' after pricking, opening doors and windows at the same time, and culturing the fungus for 7-10 days;
(4) arranging the fields: when the black fungus bases are formed at most of the openings of the fungus sticks, the fungus sticks are arranged in a greenhouse or in the open air under the sunny or cloudy weather condition that the temperature is stable below 22 ℃; furrowing is well done before the yard, the east-west furrowing trend is carried out, the furrowing width is 1-1.2m, the length is not limited, the furrowing ditch width is 40cm, and lime is scattered on the furrowing surface; the short bag mushroom sticks can be directly vertically placed on the furrowing surface, and the long bag mushroom sticks need to be erected on the furrowing surface to form a frame, so that the mushroom sticks lean against the frame obliquely; when the fungus sticks are placed, the distance between the two fungus sticks is 10cm, so that ventilation and light receiving are facilitated;
(5) and (3) ear emergence management: after 2-3 days of discharge, starting to manage the water content; spraying water for 3-5 times every day by using a water mist spraying belt; after the ear slices grow to be more than 1cm, the water spraying frequency can be reduced, and the water spraying amount per time is increased; when the temperature is higher than 25 ℃, water is sprayed in the morning and evening, so that water spraying at high temperature is avoided; stopping water spraying 1-2 days before harvesting, and stopping water spraying 5-10 days after harvesting; when raining for a plurality of continuous days, a thin film is adopted for rain sheltering;
(6) harvesting: when the ear is unfolded and the ear root is contracted and thinned, the agaric is mature and can be harvested; stopping water for 1-2 days or collecting the ear after the ear is closed;
(7) and (3) pest control: the prevention is mainly used for preventing the pollution of other bacteria such as neurospora, trichoderma and the like from the production and preparation of the bacteria sticks, and a sterilization closing, an inoculation closing and a hypha culture closing are well performed; preventing the nematode from being infected by the spray water or soil.
The fruiting body soaking rate of the black fungus cultured by the black fungus strain is not less than 20.0, the iron content is not less than 166mg/Kg, and the polysaccharide content is not less than 4.15g/100 g; the fruiting body of Auricularia is in single piece shape, has no vein or little vein, and has soft and elastic ear, yellow brown color before drying, bright color and transparency, and dark brown abdomen and gray black back after drying.
The invention also aims to protect the application of the black fungus strain, and the application method comprises the following steps: preparing the black fungus fruiting body obtained by cultivating the black fungus strain into instant black fungus powder, wherein the instant black fungus powder is prepared by the following method: and (3) cleaning the black fungus, steaming at high temperature and high pressure for 20-30 min, drying at constant temperature of 60 ℃ until the water content of the black fungus is 12-15%, and performing ultrafine powder crushed to obtain the instant black fungus powder. The instant black fungus powder prepared by the invention is prepared from the following components in a solid-to-liquid ratio of 1 g: 5ml of the mixture is mixed with water and stirred into paste, and then the paste is kept stand for 1-3 min, and the volume of the paste is expanded by more than 200%.
The instant black fungus powder can be prepared into instant brewing food, such as: instant brewing foods such as instant brewing rice paste, instant brewing flour paste, instant brewing milk tea, instant brewing beverage, instant brewing instant noodles and the like. The instant brewing food containing the instant black fungus powder has high iron content and high nutritional value, when the mass percentage of the instant black fungus powder in the instant brewing food is higher than 5%, the instant brewing food has obvious swelling effect during brewing, paste such as rice paste obtained by brewing has high adhesiveness and high stability, and compared with the instant brewing food containing the instant black fungus powder in the common aspect, more instant noodles are obtained, and the instant brewing food containing the instant black fungus powder has the effects of compressing cooked wheaten food and being convenient to carry.
Compared with the prior art, the invention has the following beneficial effects:
(1) the Guiyun No. 3 black fungus is a new black fungus strain, the black fungus fruiting body is in a single-piece shape, has no vein or few vein, is soft and elastic, has smooth and smooth mouthfeel, and is popular with consumers. The black fungus fruiting body is yellow brown before being dried, bright and transparent, has black brown belly and gray black back after being dried, has excellent appearance and wide market prospect.
(2) The soaking rate of the fruiting body of the black fungus provided by the invention is higher than that of the black fungus of other strains and is not lower than 20.0; the iron content of the black fungus fruiting body is not lower than 166mg/Kg and is far higher than that of the black fungus strain which is popularized and cultivated in the Guangxi region; the polysaccharide content of the black fungus fruiting body is higher than that of the black fungus strains which are popularized and cultivated in the Guangxi region, and the polysaccharide content of the black fungus fruiting body is not lower than 4.15g/100 g.
Drawings
FIG. 1 is a breeding line spectrogram of Auricularia auricula;
FIG. 2 is a diagram of cluster analysis of Auricularia auricula strains based on ISSR analysis according to the present invention;
FIG. 3 is a PCR electrophoretogram of primer number 1 in Table 2;
FIG. 4 is a PCR electrophoretogram of primer number 2 in Table 2;
FIG. 5 is a PCR electrophoretogram of primer number 3 in Table 2;
FIG. 6 is a PCR electrophoretogram of primer number 4 in Table 2;
FIG. 7 is a PCR electrophoretogram of primer No. 5 in Table 2;
FIG. 8 is a PCR electrophoretogram of primer number 6 in Table 2;
FIG. 9 is a PCR electrophoretogram of primer No. 7 in Table 2;
FIG. 10 is a PCR electrophoretogram of primer number 8 in Table 2;
FIG. 11 is a PCR electrophoretogram of primer number 9 in Table 2;
FIG. 12 is a PCR electrophoretogram of primer number 10 in Table 2;
FIG. 13 is a PCR electrophoretogram of primer No. 11 in Table 2;
FIG. 14 is a PCR electrophoretogram of primer number 12 in Table 2;
FIG. 15 is a PCR electrophoretogram of primer number 13 in Table 2;
in FIGS. 3-15, the lane order is from left to right: DNA Marker, Guiyun No. 3, 916, New Ke No. 10, New Ke No. 4, Heishan, Baiyun No. 6, Au8129, Au149, YE2, black 29, HME2, TY013, TY041, TY051, TY063, YE day 3.
FIG. 16 is an antagonistic diagram of Guiyun No. 3 and New family No. 4 strains on a culture dish, wherein the Guiyun No. 3 strain is arranged below the graph, and the New family No. 4 strain is arranged below the graph;
FIG. 17 is an antagonistic diagram of Guiyun No. 3 and 916 strains on a culture dish, wherein the Guiyun No. 3 strain is arranged below the antagonistic diagram, and the 916 strain is arranged below the antagonistic diagram;
FIG. 18 is an antagonistic diagram of Guiyun No. 3 and Baiyun No. 6 strains, wherein the Guiyun No. 3 strain is arranged below the antagonistic diagram, and the Baiyun No. 6 strain is arranged below the antagonistic diagram;
FIG. 19 is a diagram showing the fruiting bodies of Guiyun No. 3 and Xinke No. 4, wherein the left side of the diagram shows the fruiting body of Guiyun No. 3, and the right side shows the fruiting body of Xinke No. 4;
FIG. 20 shows the sub-entity diagrams of 916 and black 3, wherein the left side of the diagram shows the sub-entity diagram of 916, and the right side shows the sub-entity diagram of black 3;
FIG. 21 is a diagram of the bacteria sticks of Guiyun No. 3 and New family No. 4, wherein the left side of the diagram is the bacteria stick of Guiyun No. 3, and the right side is the bacteria stick of New family No. 4;
Detailed Description
The present invention will be further described with reference to examples and tests.
Example 1
The black fungus (Auricularia auricular-judae) of the invention belongs to the species Auricularia auricula (L.) Underw of Auricularia of the family Auriculariaceae of the class Heterobasidiomycotina of the class Auriculariales. The black fungus strain is preserved in China general microbiological culture Collection center, and the addresses are as follows: the No. 3 Xilu Beijing, Chaoyang, is No.1 Hospital, with the preservation number of CGMCC No.15367 and the preservation date of 2018, 3 months and 12 days.
The black fungus strain is obtained by screening, domesticating and cultivating Bai-color wild black fungus, and the screening and domesticating method comprises the following steps:
(1) carrying out tissue separation and purification on the collected hundred-color wild black fungus to obtain a mother strain Ty 026;
(2) cultivating, domesticating and screening the parent strain Ty026 to obtain 60 first generation tissue isolates of the Ty026 strain, which are marked as F1Generation; the screening method comprises the following steps: selecting fruiting body with thick and solid ear, moderate size and less vein from parent strain Ty026, and performing tissue separation and purification;
(3) to F1Cultivating, domesticating and screening the generation strains to obtain 30 second generation tissue isolates, which are marked as F2Generation; the screening method comprises the following steps: from F1Selecting fruiting body with stable performance, high yield, thick and solid ear, moderate size and less vein from the substitute strain, and performing tissue separation and purification;
(4) to F2Cultivating, domesticating and screening the substitute strain to obtain 30 third generation tissue isolates, which are marked as F3Generation; the screening method comprises the following steps: from F2Selecting fruiting body with stable performance, high yield, thick and solid ear, moderate size and less vein from the substitute strain, and performing tissue separation and purification;
(5) to F3Cultivating, domesticating and screening the substitute strains to obtain the black fungus strains; the screening method comprises the following steps: from F3Selecting strains with thick and solid lugs, moderate size, less veins, high yield and uniform and stable characters from the substitute strains;
wherein, in the steps (1) to (5), the tissue separation and purification method of the fruiting body is as follows: sterilizing the surface of the black fungus by adopting an ethanol solution with the volume percentage of 70% for 3min, cutting tissue blocks with the size of 2cm multiplied by 2cm, continuously sterilizing by adopting the ethanol solution with the volume percentage of 70% for 1min, washing by adopting sterile water for 2 times, naturally airing, inoculating into a PAD culture medium, and culturing at constant temperature of 28 ℃ until hypha grows to fill a test tube; selecting mycelium at the front section of the test tube, transferring the mycelium to a test tube slant culture medium for culture, and repeatedly selecting and transplanting for 2 times according to the method to obtain a purified strain; the test tube slant culture medium comprises the following components: 200g of potato, 20g of glucose, 20g of agar and 1000ml of water;
in the steps (1) to (5), the black fungus cultivation and domestication method adopts bag-type clinker cultivation, and the cultivation steps are as follows:
(1) preparing a fungus bag: selecting a short polyethylene angle-folded bag of 17cm multiplied by 33cm multiplied by 0.004cm, bagging the cultivation material, placing the bagged cultivation material into a sterilization pot for sterilization, raising the temperature to 100 ℃ within 4 hours after the cultivation material is put into the pot, and sterilizing the bag for 16 hours at constant temperature, wherein the fire can not be stopped in the period; naturally cooling the temperature in the pot to 60 ℃ after sterilization, taking out the material rod when the temperature is hot, moving the material rod into a cooling chamber or an inoculation chamber, and inoculating according to the aseptic operation requirement when the temperature of the material rod is reduced to below 28 ℃; when inoculating, the short bag is directly inoculated at the bag opening; the cultivation material comprises the following components in parts by mass: 80 parts of mixed wood chips, 10 parts of wheat bran, 1 part of gypsum, 0.5 part of lime, 1 part of cane sugar and 42 parts of mulberry branch chips;
(2) hypha culture: after inoculation, placing the fungus sticks in a dark, dry and well ventilated environment for hypha culture, wherein the environmental temperature of a culture site is 28 ℃, the air humidity is below 70%, the hypha culture time is 2 months, and pile turning, pile scattering and ventilation are carried out according to the environmental temperature and the hypha growth condition;
(3) puncturing holes: when hyphae grow over the fungus bags and a small amount of black fungus base is formed, pricking holes are formed when the hyphae are physiologically mature; the bag is not taken off, the bag is punctured, the number of punctured holes is 80, the diameter of the punctured holes is 0.4cm, and the depth of the punctured holes is 0.5 cm. Stacking the punctured fungus sticks in a Chinese character 'jing' shape, opening doors and windows at the same time, and culturing the fungi for 10 days;
(4) arranging the fields: when the black fungus bases are formed at most of the openings of the fungus sticks, the fungus sticks are arranged in a greenhouse under the sunny or cloudy weather condition that the temperature is stable below 22 ℃; furrowing is well done before the discharge, the east-west furrowing trend is carried out, the furrowing width is 1.2m, the length is not limited, the furrowing ditch width is 40cm, and lime is scattered on furrowing surfaces; the short bag mushroom sticks can be directly vertically placed on the furrowing surface; when the fungus sticks are placed, the distance between the two fungus sticks is 10cm, so that ventilation and light receiving are facilitated;
(5) and (3) ear emergence management: after 3 days of discharge, starting to manage the water content; spraying water with water mist spraying belt for 5 times every day; after the ear slices grow to be more than 1cm, the water spraying frequency can be reduced, and the water spraying amount per time is increased; when the temperature is higher than 25 ℃, water is sprayed in the morning and evening, so that water spraying at high temperature is avoided; stopping water spraying 2 days before harvesting, and stopping water spraying 10 days after harvesting; when raining for a plurality of continuous days, a thin film is adopted for rain sheltering;
(6) harvesting: when the ear is unfolded and the ear root is contracted and thinned, the agaric is mature and can be harvested; stopping water for 2 days or getting clear after rain, collecting the ear after the ear is closed;
(7) and (3) pest control: the prevention is mainly used for preventing the pollution of other bacteria such as neurospora, trichoderma and the like from the production and preparation of the bacteria sticks, and a sterilization closing, an inoculation closing and a hypha culture closing are well performed; preventing the nematode from being infected by the spray water or soil.
Example 2
The black fungus (Auricularia auricular-judae) of the invention belongs to the species Auricularia auricula (L.) Underw of Auricularia of the family Auriculariaceae of the class Heterobasidiomycotina of the class Auriculariales. The black fungus strain is preserved in China general microbiological culture Collection center, and the addresses are as follows: the No. 3 Xilu Beijing, Chaoyang, is No.1 Hospital, with the preservation number of CGMCC No.15367 and the preservation date of 2018, 3 months and 12 days.
The black fungus strain is obtained by screening, domesticating and cultivating Bai-color wild black fungus, and the screening and domesticating method is different from that of the embodiment 1 in that:
in the steps (1) to (5), the tissue separation and purification method of the fruiting body is as follows: sterilizing the surface of the black fungus by adopting an ethanol solution with the volume percentage of 70% for 1min, cutting tissue blocks with the size of 2cm multiplied by 2cm, continuously sterilizing by adopting the ethanol solution with the volume percentage of 70% for 1min, washing by adopting sterile water for 3 times, naturally airing, inoculating into a PAD culture medium, and culturing at constant temperature of 25 ℃ until hyphae grow to fill the test tube; selecting mycelium at the front section of the test tube, transferring the mycelium to a test tube slant culture medium for culture, and repeatedly selecting and transplanting for 3 times according to the method to obtain a purified strain; the test tube slant culture medium comprises the following components: 200g of potato, 20g of glucose, 20g of agar and 1000ml of water;
in the steps (1) to (5), the black fungus cultivation and domestication method adopts bag type clinker cultivation, and the cultivation steps are as follows:
(1) preparing a fungus bag: selecting a polyethylene angle-folded bag long bag of 15cm multiplied by 55cm multiplied by 0.004cm, bagging the cultivation material, placing the bag in a sterilization pot for sterilization, raising the temperature to 100 ℃ within 4 hours after the cultivation material is put in the pot, and sterilizing the bag for 12 hours at constant temperature, wherein the fire can not be stopped in the period; naturally cooling the temperature in the pot to 60 ℃ after sterilization, taking out the material rod when the temperature is hot, moving the material rod into a cooling chamber or an inoculation chamber, and inoculating according to the aseptic operation requirement when the temperature of the material rod is reduced to below 28 ℃; when inoculating, 4 holes are drilled on the bag body for inoculation, the diameter of each hole is 2.0cm, and the depth of each hole is 2.5 cm; the cultivation material comprises the following components in parts by mass: 40 parts of mixed wood chips, 15 parts of wheat bran, 0.5 part of gypsum, 1 part of lime and 25 parts of cottonseed hulls;
(2) hypha culture: after inoculation, placing the fungus sticks in a dark, dry and well ventilated environment for hypha culture, wherein the environment temperature of a culture site is 25 ℃, the air humidity is below 70%, the hypha culture time is 2 months, and pile turning, pile scattering and ventilation are carried out according to the environment temperature and the hypha growth condition;
(3) puncturing holes: when hyphae grow over the fungus bags and a small amount of black fungus base is formed, pricking holes are formed when the hyphae are physiologically mature; the bag-removing-free pricking is adopted to produce the ears, the number of the pricking holes is 200 per bag, the aperture is 0.4cm, and the hole depth is 0.5 cm. Stacking the punctured fungus sticks in a shape like a Chinese character 'jing', opening doors and windows at the same time, and culturing the fungi for 7 days;
(4) arranging the fields: when the black fungus bases are formed at most of the openings of the fungus sticks, the fungus sticks are arranged in the open air under the sunny or cloudy weather condition that the temperature is stable below 22 ℃; furrowing is well done before the discharge, the east-west furrowing trend is carried out, the furrowing width is 1m, the length is not limited, the furrowing ditch width is 40cm, and lime is scattered on the furrowing surface; erecting a frame on the furrowing surface of the fungus sticks, and enabling the fungus sticks to lean against the frame obliquely; when the fungus sticks are placed, the distance between the two fungus sticks is 10cm, so that ventilation and light receiving are facilitated;
(5) and (3) ear emergence management: after 2 days of discharge, starting to manage the water content; spraying water for 3 times per day by using a water mist spraying belt; after the ear slices grow to be more than 1cm, the water spraying frequency can be reduced, and the water spraying amount per time is increased; when the temperature is higher than 25 ℃, water is sprayed in the morning and evening, so that water spraying at high temperature is avoided; stopping water spraying 1 day before harvesting, and stopping water spraying 5 days after harvesting; when raining for a plurality of continuous days, a thin film is adopted for rain sheltering;
(6) harvesting: when the ear is unfolded and the ear root is contracted and thinned, the agaric is mature and can be harvested; stopping water for 1-2 days or collecting the ear after the ear is closed;
(7) and (3) pest control: the prevention is mainly used for preventing the pollution of other bacteria such as neurospora, trichoderma and the like from the production and preparation of the bacteria sticks, and a sterilization closing, an inoculation closing and a hypha culture closing are well performed; preventing the nematode from being infected by the spray water or soil.
Example 3
The black fungus (Auricularia auricular-judae) of the invention belongs to the species Auricularia auricula (L.) Underw of Auricularia of the family Auriculariaceae of the class Heterobasidiomycotina of the class Auriculariales. The black fungus strain is preserved in China general microbiological culture Collection center, and the addresses are as follows: the No. 3 Xilu Beijing, Chaoyang, is No.1 Hospital, with the preservation number of CGMCC No.15367 and the preservation date of 2018, 3 months and 12 days.
The black fungus strain is obtained by screening, domesticating and cultivating Bai-color wild black fungus, and the screening and domesticating method is different from that of the embodiment 1 in that:
in the steps (1) to (5), the tissue separation and purification method of the fruiting body is as follows: sterilizing the surface of the black fungus by adopting an ethanol solution with the volume percentage of 70% for 2min, cutting tissue blocks with the size of 2cm multiplied by 2cm, continuously sterilizing by adopting the ethanol solution with the volume percentage of 70% for 1min, washing by adopting sterile water for 2 times, naturally airing, inoculating into a PAD culture medium, and culturing at the constant temperature of 27 ℃ until hyphae grow to fill the test tube; selecting mycelium at the front section of the test tube, transferring the mycelium to a test tube slant culture medium for culture, and repeatedly selecting and transplanting for 3 times according to the method to obtain a purified strain; the test tube slant culture medium comprises the following components: 200g of potato, 20g of glucose, 20g of agar and 1000ml of water;
in the steps (1) to (5), the black fungus cultivation and domestication method adopts bag type clinker cultivation, and the cultivation steps are as follows:
(1) preparing a fungus bag: selecting a polyethylene angle-folded bag long bag of 15cm multiplied by 55cm multiplied by 0.004cm, bagging the cultivation material, placing the bag in a sterilization pot for sterilization, raising the temperature to 100 ℃ within 4 hours after the cultivation material is put into the pot, and sterilizing the bag for 15 hours at constant temperature, wherein the fire can not be stopped in the period; naturally cooling the temperature in the pot to 60 ℃ after sterilization, taking out the material rod when the temperature is hot, moving the material rod into a cooling chamber or an inoculation chamber, and inoculating according to the aseptic operation requirement when the temperature of the material rod is reduced to below 28 ℃; when inoculating, 3 holes are drilled on the bag body for inoculation, the diameter of each hole is 1.5cm, and the depth of each hole is 2 cm; the cultivation material comprises the following components in parts by mass: 43 parts of mixed wood chips, 12 parts of wheat bran, 0.8 part of gypsum, 0.8 part of lime, 0.5 part of cane sugar, 13 parts of cottonseed hulls and 38 parts of mulberry branch chips;
(2) hypha culture: after inoculation, placing the fungus sticks in a dark, dry and well ventilated environment for hypha culture, wherein the environmental temperature of a culture site is 26 ℃, the air humidity is below 70%, the hypha culture time is 2 months, and pile turning, pile scattering and ventilation are carried out according to the environmental temperature and the hypha growth condition;
(3) puncturing holes: when hyphae grow over the fungus bags and a small amount of black fungus base is formed, pricking holes are formed when the hyphae are physiologically mature; the bag-removing-free pricking is adopted to produce the ears, the number of the pricking holes is 150 per bag, the aperture is 0.4cm, and the hole depth is 0.5 cm. Stacking the punctured fungus sticks in a shape like a Chinese character 'jing', opening doors and windows at the same time, and culturing the fungi for 8 days;
(4) arranging the fields: when the black fungus bases are formed at most of the openings of the fungus sticks, the fungus sticks are arranged in a greenhouse under the sunny or cloudy weather condition that the temperature is stable below 22 ℃; furrowing is well done before the discharge, the east-west furrowing trend is carried out, the furrowing width is 1.1m, the length is not limited, the furrowing ditch width is 40cm, and lime is scattered on furrowing surfaces; the short bag mushroom sticks can be directly vertically placed on the furrowing surface, and the long bag mushroom sticks need to be erected on the furrowing surface to form a frame, so that the mushroom sticks lean against the frame obliquely; when the fungus sticks are placed, the distance between the two fungus sticks is 10cm, so that ventilation and light receiving are facilitated;
(5) and (3) ear emergence management: after 3 days of discharge, starting to manage the water content; spraying water with water mist spraying belt for 4 times every day; after the ear slices grow to be more than 1cm, the water spraying frequency can be reduced, and the water spraying amount per time is increased; when the temperature is higher than 25 ℃, water is sprayed in the morning and evening, so that water spraying at high temperature is avoided; stopping water spraying 2 days before harvesting, and stopping water spraying 8 days after harvesting; when raining for a plurality of continuous days, a thin film is adopted for rain sheltering;
(6) harvesting: when the ear is unfolded and the ear root is contracted and thinned, the agaric is mature and can be harvested; stopping water for 2 days or getting clear after rain, collecting the ear after the ear is closed;
(7) and (3) pest control: the prevention is mainly used for preventing the pollution of other bacteria such as neurospora, trichoderma and the like from the production and preparation of the bacteria sticks, and a sterilization closing, an inoculation closing and a hypha culture closing are well performed; preventing the nematode from being infected by the spray water or soil.
The black fungus planted in the method is detected as follows:
(1) genetic difference analysis
The ISSR analysis of genetic difference of the black fungus strains adopts 16 strains and 13 ISSR primers, the serial numbers of the 16 black fungus strains are specifically shown in the following table 1, and the sequences of the 13 ISSR primers are shown in the following table 2.
TABLE 1
TABLE 2
| Numbering | Primer name | Primer sequences | Temperature of renaturation |
| 1 | P3 | GA GA GA GA GA GA GA GA T | 53.1 |
| 2 | P4 | GA GA GA GA GA GA GA GAC | 56.0 |
| 3 | P5 | AG AG AG AG AG AG AG AGG | 56.0 |
| 4 | P6 | AG AG AG AG AG AG AG AGC | 56.0 |
| 5 | P9 | GSGTGTGTGTGTGTGTGT | 52.0 |
| 6 | P10 | AG AG AG AG AG AG AG AGT | 54.0 |
| 7 | P16 | TC TC TC TC TC TC TC TC C | 56.0 |
| 8 | P21 | AG AG AG AG AG AG AG AGYA | 54.0 |
| 9 | P22 | AG AG AG AG AG AG AG AGYC | 56.0 |
| 10 | P25 | GA GA GA GA GA GA GA GAYC | 56.0 |
| 11 | P26 | GA GA GA GA GA GA GA GAYT | 54.0 |
| 12 | P27 | GA GA GA GA GA GA GA GAYG | 56.0 |
| 13 | P32 | GA GA GA GA GA GA GA GAA | 54.0 |
As can be seen from the graphs in FIGS. 3-15, the 13 primers of the application can amplify stable bands for 16 black fungus strains, and the ISSR analysis of the genetic difference of the strains shows that the difference between the banding patterns and the number of Guiyun No. 3 and other strains is large. The ISSR clustering analysis results are shown in the ISSR dendrogram of FIG. 2, and the results show that: the genetic similarity coefficient of Guiyun No. 3 and other strains is below 0.68, and the strains have far genetic relationship, which shows that the Guiyun No. 3 strain has larger genetic difference with other black fungus strains and is a new black fungus strain.
(2) Antagonism experiment
Antagonistic experiments of the black fungus Guiyun No. 3 strain and the new family No. 4, 916 and Baiyun No. 6 strains are respectively shown in the figures 16, 17 and 18. As can be seen from the graphs of 16-18, the Guiyun No. 3 strain has obvious hypha antagonistic reaction with the Guangxi main cultured black fungus strains of the new families No. 4, No. 916 and No. 6, and the strains are incompatible, which indicates that the Guiyun No. 3 strain is obviously different from the currently popularized and cultured black fungus strain and is a new black fungus strain.
(3) Morphological characteristic analysis of the black fungus strain
As can be seen from the comparison of the guiyun No. 3 subelement on the left in fig. 19 with the new family No. 4 subelement on the right in fig. 19, the 916 fruiting body on the left in fig. 20, the black 3 fruiting body on the right in fig. 20, and the guiyun No. 3 fungus stick on the left in fig. 21 with the new family No. 4 fungus stick on the right in fig. 21: the morphological characteristics of Guiyun No. 3 black fungus are different from those of other black fungus varieties in different degrees, and the Guiyun No. 3 black fungus is single-sheet, has no vein or few vein, is soft and elastic, has smooth and smooth mouthfeel, and is popular with consumers. The fresh product is yellow brown, bright and transparent; the dried product has black brown abdominal surface, gray black back surface, excellent appearance and wide market prospect.
(4) Measurement of foam expansion Rate
1. Measurement method
The soaking rate of the black fungus selected from Ty071, 916, YE1 and YE2 is detected with the black fungus Guiyun No. 3 of the invention, and the cultivation methods of the black fungus selected from Ty071, 916, YE1 and YE2 and the black fungus Guiyun No. 3 of the invention are the same as the example 1. The determination method comprises the following steps: weighing 10g of air-dried black fungus respectively, soaking in clear water for 4-6 hours, taking out, draining water (absorbing surface water by using absorbent paper), weighing wet weight W1, spreading the black fungus overnight (about 12 hours), then placing in an oven at 60 ℃ for drying, weighing dry weight W2, and calculating the foam expansion ratio of W1 to W2. The measurements were repeated twice, the first measurement results are shown in Table 3 below, and the first measurement results are shown in Table 4 below.
TABLE 3
TABLE 4
As can be seen from tables 3 and 4, the foaming rate of Guiyun No. 3 of the invention is more than 20.0, which is far higher than that of other black fungus strains.
(5) The results of comparing the iron and polysaccharide contents of Guiyun No. 3 and other Auricularia strains according to the present invention are shown in Table 5 below.
TABLE 5
| Bacterial strains | Guiyun No. 3 | New science No. 4 | |
916 | YE1 | Black three |
| Polysaccharide content (g/100g) | 4.15 | 2.90 | 2.60 | 3.42 | 3.50 | 3.19 |
| Iron content (mg/Kg) | 166 | 85 | 53.3 | 36.3 | 71.1 | 37.8 |
As can be seen from Table 5, the Guiyun No. 3 sporophore of the present invention contains 166mg/Kg of iron, which is much higher than that of the currently popularized and cultivated black fungus, and the Guiyun No. 3 sporophore polysaccharide content of the present invention is also higher than that of other comparative strains.
The instant brewing food prepared from the black fungus of the application comprises the following components: the food and the preparation method are as follows:
preparing edible fungus powder:
example 4
An edible fungus powder is prepared by the following method: the edible fungus powder is obtained by cleaning the black fungus planted in the embodiment 1, steaming at high temperature and high pressure for 30min, drying at constant temperature of 60 ℃ until the water content of the black fungus is 12%, and performing ultra-fine powder crushed.
Example 5
An edible fungus powder is prepared by the following method: the edible fungus powder is obtained by cleaning the black fungus planted in the embodiment 1, steaming at high temperature and high pressure for 20min, drying at constant temperature of 60 ℃ until the water content of the black fungus is 15%, and performing ultra-fine powder crushed.
Example 6
An edible fungus powder is prepared by the following method: the edible fungus powder is obtained by cleaning the black fungus planted in the embodiment 1, then steaming at high temperature and high pressure for 25min, drying at constant temperature of 60 ℃ until the water content of the black fungus is 13%, and carrying out ultra-fine powder crushed.
(II) preparing instant brewing food
Example 7
An instant brewing food comprises the following components in percentage by mass: 5% of edible fungus powder, 5% of walnut powder, 5% of corn powder, 60% of rice powder, 20% of black rice powder and 5% of sugar in example 4.
Example 8
An instant brewing food comprises the following components in percentage by mass: 80% of edible fungus powder, 15% of rice powder and 5% of sugar in example 4.
(III) preparing instant noodles
Example 9
An instant brewing instant noodle comprises the following components by mass percent: 20% of edible fungus powder and 80% of flour in example 4.
Example 10
An instant brewing instant noodle comprises the following components by mass percent: 50% of edible fungus powder and 50% of flour in example 4.
The edible fungus powder prepared in the embodiments 4, 4 and 6 is mixed according to the solid-liquid ratio of 1 g: 5ml of the mixture is mixed with water and stirred into paste, and then the paste is left to stand and respectively expands by 200 percent, 250 percent and 227 percent.
The instant brewing foods in the embodiment 7 and the embodiment 8 are brewed and eaten, the brewing effect is obvious when the instant brewing foods are brewed by boiled water at 100 ℃, and pasty foods such as rice paste and the like obtained by brewing have high adhesiveness, high stability, high iron content and good health care effect.
The instant noodles in the embodiment 9 and the embodiment 10 are brewed and eaten, more instant noodles are obtained compared with the instant noodles brewed in the common aspect, the brewing rate of the embodiment 9 is improved by 14.5 percent compared with the common aspect, the brewing rate of the embodiment 10 is improved by 10.2 percent compared with the common aspect, and the instant noodles have the effects of compressing wheaten food and being convenient to carry.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (2)
1. A Auricularia auricula (Auricularia auricular-judae) strain is characterized in that the strain is Auricularia auricula (Auricularia auricular-judae), and the preservation number is CGMCC No. 15367; the black fungus strain is obtained by separating, domesticating and culturing a hundred-color wild black fungus; the fruiting body soaking rate of the black fungus cultured by the black fungus strain is not less than 20.0, the iron content is not less than 166mg/Kg, and the polysaccharide content is not less than 4.15g/100 g; the fruiting body of Auricularia is in single piece shape, has no vein or little vein, and has soft and elastic ear, yellow brown color before drying, bright color and transparency, and dark brown abdomen and gray black back after drying.
2. The application of the black fungus strain as claimed in claim 1, wherein the black fungus fruiting body obtained by cultivating the black fungus strain is prepared into instant black fungus powder, and the instant black fungus powder is prepared by the following method: the instant black fungus powder is obtained by cleaning black fungus, then steaming at high temperature and high pressure for 20-30 min, drying at constant temperature of 60 ℃ until the water content of the black fungus is 12-15%, and carrying out superfine grinding.
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| CN112568064B (en) * | 2020-11-23 | 2022-04-05 | 河北省科学院生物研究所 | Black fungus strain JH40 and application thereof |
| CN113174338B (en) * | 2021-05-28 | 2022-12-20 | 广西壮族自治区农业科学院 | Black fungus tissue isolation culture medium and application thereof |
| CN115380764B (en) * | 2021-10-13 | 2024-05-14 | 江西农业大学 | Xylopsis fragrans and domestication cultivation method of Xylopsis fragrans |
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