CN116004393B - Phellinus linteus strain suitable for culturing mycelium with silkworm pupa, and culturing method and application thereof - Google Patents
Phellinus linteus strain suitable for culturing mycelium with silkworm pupa, and culturing method and application thereof Download PDFInfo
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Abstract
A Phellinus linteus strain suitable for culturing mycelium with silkworm pupa and its culturing method and application belong to the biotechnology field. The Phellinus linteus strain of the present invention has been preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.23257 in the 9 th month 17 day 2021. The strain is suitable for culturing silkworm pupa, and culture medium (silkworm pupa powder 60%, water 33.33%, and MgSO) prepared from silkworm pupa 4 .7H 2 O 0.67％、KH 2 PO 4 0.67%, glucose 5.33%; the culture medium has the advantages of fast growth, high yield, firmness, easy separation of mycelium blocks from the culture medium, difficult variation, difficult aging, no peculiar smell, high polyphenol content and the like, is suitable for the perennial industrial production of the Phellinus linteus mycelium by using a silkworm chrysalis culture medium, has no mixed bacteria pollution, and does not need specific equipment and complex culture environment conditions; the silkworm pupa culture medium has simple preparation, low cost, no need of consuming wood and no damage to ecological environment.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Phellinus linteus strain suitable for culturing mycelium by silkworm pupae and application thereof.
Background
Phellinus linteus (Sanghuangporus sanghuang) is a rare medicinal fungus parasitized on mulberry (Morus L). Phellinus linteus is described in the past medical books from Han dynasty. In recent years, domestic and foreign researches prove that the Phellinus linteus is rich in active substances such as various polysaccharides, flavone and derivatives thereof, sterols and the like, and has multiple medicinal effects of improving immunity, resisting cancer, resisting inflammation, resisting oxidization and the like [1-3] Polar toolIndustrial development and application value.
Nevertheless, people's knowledge of Phellinus linteus is a progressive process: before 2012, no academic records and specimen records of the specimen library show that Phellinus linteus fungus growing on Morus alba is present in China [1] The method comprises the steps of carrying out a first treatment on the surface of the Until 2012, sang Huangcai was found to be a new species not previously described [4] The method comprises the steps of carrying out a first treatment on the surface of the By 2016, phellinus linteus and similar species were identified as independent new genus, genus Sang Huangkong (Sanghuang porus); as of 2020, the genus of Sang Huangkong was propagated to 14 species, including Phellinus linteus (Phellinus linteus) except for Morus Sang Huangwai, phellinus linteus (Phellinus linteus) Rebauhinia, phellinus linteus (Phellinus linteus) and Quercus acutissima (Phellinus linteus) [1,5-6] They are also known under the name "Phellinus" and are not grown on Morus alba, but merely because they belong to the genus Sang Huangkong fungi and true Phellinus linteus (Phellinus linteus) is a closely related species. The method can be really abbreviated as 'Phellinus linteus', but only has the same type of Phellinus linteus; phellinus linteus and grown tree species have specificity, grow only on the trunk of mulberry, and are medicinal fungi belonging to Sang Huangkong genus [1,5] 。
Because true Phellinus linteus (Phellinus linteus) is extremely rare, and the addition of Phellinus linteus is extremely harsh to the growing environment, artificial cultivation is difficult, so-called "Phellinus linteus" found in the market is mostly imitated Phellinus linteus (the kindred species of Phellinus linteus), so-called Phellinus linteus fruiting bodies widely cultivated in China, japan and Korean are not Phellinus linteus, and are cultivated in poplar (Phellinus linteus) Huang Juduo [1,5] And also includes syringa amurensis (Phellinus linteus) and Phellinus linteus, and even Phellinus linteus.
At present, most of methods for culturing Phellinus linteus Huang Jifang adopt cut log or bag material for culturing fruiting body. The fruiting body cultivated by log or bag has the following disadvantages: the method has the advantages of consuming a large amount of wood, along with long culture period, harsh environmental requirements for fruiting body growth, and extremely easy contamination of mixed bacteria due to exposure to high temperature and high humidity in the later period of culture. Therefore, the fruiting body is cultivated by the log or the bag material, which has high cost and difficult quality control.
The mycelium is used for mass culture, and the mycelium can replace fruiting body culture to a certain extent. Test results of several effective components and efficacy of Phellinus linteus myceliumIs not fed to fruiting body, has short mycelium culture period, low cost, stable quality and good effect, and is one direction of Phellinus linteus industry [5] . Mycelium production is usually carried out in the manner of mycelium fermentation. However, the fermentation culture has the defects of high process cost (a fermentation tank needs to be purchased, the process is complicated, and the like), easy variation and degradation of hypha, and the like, and most of the processes are in laboratory research stage at present.
Although the artificially cultivated Phellinus linteus sold in the market at present is mostly imitated Phellinus linteus, the price is still high, and the research and application fields of Phellinus linteus are proved to have breakthrough and innovation in the following two aspects: 1. obtaining a authentic Phellinus linteus strain which is truly parasitic to the mulberry with specificity and is a fungus belonging to Sang Huangkong genus in classification; furthermore, the strain has the characteristics of difficult variation, difficult aging, stable quality, suitability for artificial mass culture and the like; 2. breaks through and innovates in the aspect of the culture technology of authentic mulberry Phellinus linteus. Only innovations in both the Phellinus linteus strain and the cultivation technology can truly assist in researching and applying Phellinus linteus, and help to get rid of the current dilemma of Phellinus linteus industry in China.
Silkworm (Bombyx mori l.) belongs to an allergic insect, and after spinning and cocooning, silkworm larvae develop to silkworm pupae (Silkworm chrysalis). The silkworm chrysalis is a valuable resource for both medicine and food [7] . Medical records are recorded in medical books of Ben Cao gang mu (compendium of materia Medica) and Ben Cao Pin Hui Jing (essence of materia Medica) in ancient China; in modern times, silkworm pupa are recorded by Chinese medicines such as "Chinese medicine Dathesaurus" and "Chinese medicine Zhi" as tonic medicines for treating various diseases. The silkworm chrysalis is the only insect food in the 'raw and new resource food list of common food raw materials' approved by the Ministry of health in 2004 [8] Most silkworm areas in China always have habits of eating domestic silkworm chrysalis.
The silkworm chrysalis has rich nutrient components, and each 100g of dried silkworm chrysalis contains 60g of crude protein, 30g of crude fat (chrysalis oil), 3-4 g of chitin, 5g of sugar, trace elements, vitamins, antibacterial peptides, hormones, lysozyme and other bioactive substances. The silkworm chrysalis protein is a complete protein and contains 18 amino acids [8-9] Very close to the ideal amino acid pattern suggested by FAO/WHO [10] . The silkworm pupa also contains various microelements (such as Zn, mn, se, etc.), and vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin D, nicotinic acid, etc.; the silkworm chrysalis contains various hormone substances, such as prostatophor, epinephrine, beta-sitosterol and the like; the silkworm pupa also contains pupa phospholipid, antibacterial peptide, lysozyme, adenine, etc [11] 。
The silkworm chrysalis has a certain application prospect in the fields of food, daily chemicals, feed additives, fertilizers, traditional Chinese medicines, biological medicines and the like. Such as: the silkworm chrysalis can be directly used as food for eating, soy sauce production, fertilizer and additive for feeds of livestock, fishery and the like; the silkworm chrysalis is also applied to the research of anti-hepatitis, hypertension, coronary heart disease, diabetes and anticancer drugs.
At present, three applications of culturing microorganisms by using silkworm pupae are mainly available. A beauveria bassiana pupa is formed by silkworm pupa infection or artificial inoculation of beauveria bassiana (Beauveria bassiana) [12] As a traditional Chinese medicine in China, the Chinese medicine has tranquilization effect on nerves and can treat epilepsy, carbuncle and other symptoms [9] . The second is Cordyceps militaris cultivated by silkworm pupa, which is an ideal substitute for Cordyceps militaris. Thirdly, the cordyceps sobolifera (Cordyceps cicadae) cultivated by silkworm pupa is also a rare traditional Chinese medicine in China [13] 。
Nevertheless, most comprehensive utilization of domestic silkworm chrysalis is still limited by various factors such as immature technical conditions, high cost and the like, and still stays at a research level, so that the use amount is very limited; the rest silkworm chrysalis is mostly eaten directly or used as a feed additive for domestic animals, but the actual dosage is limited due to the special smell of the silkworm chrysalis, fresh keeping and other technical reasons. The dry silkworm chrysalis used as a feed additive in the market at present is generally sold at 7000-8500 yuan/ton, and the dry silkworm chrysalis sold by a network electronic commerce is generally used as bait by fishing enthusiasts, the selling price is not more than 10 yuan/jin, and the sales volume is very limited.
China is the first country of world mulberry and raw silk production, and fresh cocoons produced in years are more than 70 ten thousand tons. Silkworm (silkworm)After the larva spits and cocoons, about 20% of the weight of the fresh cocoons is silk (cocoon layer), and the other 80% is silkworm chrysalis. Silkworm chrysalis is the most main byproduct in silk reeling industry, and theoretically, more than 50 ten thousand tons of fresh silkworm chrysalis and more than 10 ten thousand tons of dried silkworm chrysalis can be produced annually in China [14] . At present, most silk reeling factories in China accumulate a large number of silkworm chrysalis every year, cannot be effectively converted and utilized, and can not be discarded at will, so that the silkworm chrysalis is a waste for silkworm cocoon production, and environmental pollution is caused. The comprehensive utilization technology of the silkworm chrysalis at home is urgently required to be broken through and innovated.
To date, there is no report on the cultivation of authentic Phellinus linteus by using silkworm chrysalis.
Since the silkworms and Sang Huangjun use the mulberry as a nutrient source in the evolution process of hundreds of millions of years, adaptability is generated to the mulberry, silkworm pupae are a stage in the life development process of the silkworms, a certain co-evolution relationship is necessarily existed among the silkworms, the Phellinus linteus and the mulberry, and the silkworm pupae are utilized for culturing the Phellinus linteus, so that the silkworm has very important theoretical research practical application value.
Disclosure of Invention
Technical problems: the invention aims to provide a Phellinus linteus strain suitable for culturing mycelium by taking silkworm chrysalis as a culture medium.
The invention also solves the technical problem of providing the application of the Phellinus linteus strain in preparing Phellinus linteus mycelium.
The technical scheme is as follows: a Phellinus linteus strain wild Sang Yuan suitable for culturing mycelium with silkworm pupae is preserved in China general microbiological culture Collection center (CGMCC; address: north Xiyi Hirschu No. 1, 3 in the Kogyo area of Beijing city; postal code: 100101) at the year 09 month 17 of 2021, and the preservation number is CGMCC No.23257.
Phellinus linteus (Sanghuangporus sanghuang) strain of the invention belongs to Phellinus linteus (Sanghuangporus sanghuang) belonging to Hymenochaetaceae (Hymenochaetaceae) and Sang Huangkong genus (Sanghuangporus Sheng H.Wu, L.W. Zhou & Y.C.Dai) in classification. The strain is obtained by separating Phellinus linteus fruiting body on the trunk of a wild type mulberry, and is named as wild Sang Yuan number 2 after generation-by-generation screening and domestication.
The Phellinus linteus strain wild Sang Yuan No.2 of the invention is characterized in that:
1. biological characteristics
The strain is suitable for culture medium (silkworm pupa powder 60%, water 33.33%, mgSO) prepared from silkworm pupa under dark static culture condition at 28deg.C 4 .7H 2 O 0.67％、KH 2 PO 4 0.67%, glucose 5.33%; PH value of 6.8-7.2) and has the following characteristics:
(1) The growth speed is high: inoculating mycelium with diameter of 0.5cm into a culture dish with diameter of 9cm (25 g silkworm pupa culture medium is built in), standing at 28deg.C, and culturing in dark, wherein the growth is started in the next day, the average time for mycelium to cover the whole silkworm pupa culture medium surface is 28d (the mycelium is not yet yellow and mature), and the average growth speed of mycelium is 1.52mm/d; hyphae were harvested from inoculation into the silkworm pupa medium until yellowing and maturation, with a time average of 40d.
(2) Golden color: after the mycelium is fully distributed in the whole culture medium and developed and matured, a large amount of yellow pigment can be secreted, the color is golden, and the mycelium is uniform in morphology.
(3) The strain is not easy to mutate: the strain is cultured by using a silkworm chrysalis culture medium for a long time (more than 4 years), no degradation, mutation and other phenomena are found, and the Phellinus linteus mycelium can be normally cultured without rejuvenation.
(4) And (3) firmness: after the mycelium turns yellow and matures, the firmness is high, the mycelium blocks are easy to separate and not easy to tear, and the mycelium blocks are suitable for separation and collection.
(5) The mycelium yield is high: after mycelia with the diameter of 0.5cm are inoculated into a culture dish with the diameter of 9cm (25 g of silkworm pupa culture medium is placed in the culture dish) and subjected to dark stationary culture at 28 ℃ for 40d, the fresh weight of the mycelia separated from each culture dish is 2.06g (the mycelia do not contain a small amount of mycelia remained in the culture medium and on gauze), and the average weight of the mycelia obtained after drying is 0.43g.
(6) Mycelium is not easy to age: mycelium with the diameter of 0.5cm is inoculated into a culture dish with the diameter of 9cm (25 g of silkworm pupa culture medium is placed in the culture dish) and subjected to dark stationary culture at the temperature of 28 ℃, and the aging time of the grown mycelium is 49d on average.
(7) No peculiar smell: the mycelium blocks obtained by separation have no foreign odor of domestic silkworm chrysalis and have faint scent of mulberry and mulberry Huang Teyou.
(8) High polyphenol content: the mycelium polyphenol content obtained by separation is up to 39.51mg/g (dry powder) on average.
2. Genetic characterization
The Phellinus linteus strain wild Sang Yuan No.2 mycelium DNA of the invention is subjected to PCR amplification by a universal primer ITSl/ITS4 designed by a sequence of a transcription spacer region (internal transcribed spacer, ITS) in ribosomal gene rDNA, and the obtained nucleic acid sequence has the size of 757bp. The obtained sequence was aligned with the ITS sequences of Sang Huangkong genus (Sanghuang porus), phellinus (Phellinus) and Phellinus (Inonotus), and the wild Sang Yuan No.2 had the highest similarity with the ITS sequences of other Phellinus of Sang Huangkong genus, and was classified as such. Wild Sang Yuan is a Phellinus linteus (Sanghuangporus sanghuang) strain belonging to the genus Hymenochaetaceae, sang Huangkong in classification.
The application of the Phellinus linteus strain suitable for culturing mycelium with silkworm pupae comprises the following steps:
1. selecting silkworm pupae
The selected silkworm pupa is required to be healthy and free of diseases (free of rotted pupa, half pupa, dead pupa, stiff pupa and the like). Preferably, silkworm pupa is selected about 4-12 days after pupation, at this time, the silkworm pupa is formed, the skin color is yellow, the in vivo protein is rich in nutrition, compound eyes are not colored, and silkworm moth is not formed yet. Grinding selected silkworm chrysalis into silkworm chrysalis powder, and sieving with 12 mesh sieve.
2. Preparing silkworm pupa culture medium
The silkworm pupa culture medium comprises the silkworm pupa powder.
Further: comprises silkworm chrysalis powder, water and MgSO 4 .7H 2 O、KH 2 PO 4 Glucose.
Wherein, silkworm pupa powder, water and MgSO 4 .7H 2 O、KH 2 PO 4 The mass ratio of glucose is as follows: 60:33.33:0.67:0.67:533, the pH value of the culture medium is 6.8-7.2.
A round gauze with the diameter of 9cm is cut, and the size of the round gauze is basically consistent with that of a 9cm culture dish. A round hole with the diameter of 0.5cm is punched in the middle of the gauze for standby.
Weighing the silkworm pupa culture medium, placing in 9cm diameter culture dishes (25 g for each culture dish), spreading the culture medium, placing a piece of perforated round gauze on the surface of the culture medium, flattening the gauze to enable the gauze to be closely attached to the surface of silkworm pupa powder culture medium, sterilizing at high temperature and high pressure (sterilizing at 121 ℃ for 20 min), and cooling for later use.
3. Mycelium culture
Selecting wild Sang Yuan # 2 mycelium blocks with diameter of 0.5cm with sterilized puncher, placing at the central hole position of gauze of the silkworm pupa culture medium (the inserted mycelium blocks can directly contact the silkworm pupa culture medium at the hole position), sealing with sealing film, and standing at 28deg.C under dark condition.
Culturing for about 40 days, and collecting mycelium of wild Sang Yuan No.2 after the mycelium is mature and yellow. When collecting, opening the sealing film, tearing off the gauze with mycelium blocks by using sterilized forceps, and tearing off the mycelium blocks attached to the gauze. And (5) drying the mycelium blocks, and grinding the mycelium blocks into powder to obtain a finished product.
The beneficial effects are that: the invention firstly separates the Phellinus linteus fruiting body to obtain the strain, and uses a silkworm chrysalis culture medium to screen and domesticate step by step to obtain the Phellinus linteus strain named as wild Sang Yuan No. 2. The strain is suitable for culturing Phellinus linteus mycelium with culture medium prepared from silkworm pupa; the mycelium grows in the silkworm chrysalis culture medium, has the advantages of golden yellow color, fast growth, high yield, firmness, difficult variation, difficult aging, no peculiar smell, high polyphenol content and the like, and is very suitable for industrialized production of the Phellinus linteus mycelium all the year round by taking silkworm chrysalis as a matrix; the silkworm pupa culture medium has the advantages of simple configuration, low cost, no need of consuming wood, no damage to ecological environment, no need of special equipment and complex culture environment conditions. As the silkworm chrysalis belongs to the waste of the mulberry production, the strain can be applied to the treatment and comprehensive utilization research of the mulberry production waste. Therefore, the strain has wide application prospect in production and research.
Drawings
FIG. 1 is a mycelium pellet isolated after culture of wild Sang Yuan No.2 in a silkworm chrysalis medium.
FIG. 2 is a phylogenetic relationship between wild Sang Yuan No.2 and other fungi of the genus Sang Huangkong, phellinus and Folium.
FIG. 3 is a mycelium powder produced by mycelium produced in a silkworm chrysalis medium under the condition of wild Sang Yuan No. 2.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
EXAMPLE 1 Phellinus linteus fruiting body collection and strain isolation culture
(1) According to the types (15 varieties, 4 varieties) and distribution of the mulberries which are found out in China at present, the representative distribution area of the mulberries in China is examined. The fruiting bodies of Phellinus linteus which are specially parasitic in different areas are collected for 12 times.
(2) The above 12 fruiting body strains were isolated and temporarily stored in a common PDA medium (200 g of peeled potato, 20g of glucose, 20g of agar, and natural pH per liter of medium).
Example 2 selection of silkworm pupae and arrangement of silkworm pupae Medium
(1) Selection of silkworm chrysalis
The selected silkworm pupae firstly require health and are free of diseases, namely: no rotten pupa, half pupa, dead pupa, stiff pupa, etc.
Silkworm belongs to metamorphosis insects, and pupation starts after the cocooning of larvae is finished. Silkworm pupae undergo roughly three developmental stages: the first period is 1-3 days after pupa formation, the second period is about 4-12 days after pupation, and the third period is 13 days after pupation, the compound eyes are colored until the compound eyes emerge as silkworm moth. Wherein, the silkworm pupa skin in the first period is just formed, the pupa body is milky white or light yellow, the pupa body is soft and tender and is extremely easy to break, the silkworm pupa skin is not suitable for being ground into silkworm pupa powder after being dried, and a silkworm pupa culture medium is prepared; the silkworm pupa compound eye in the third period is colored, the pupa body becomes soft and turns brown, at the moment, the silkworm pupa body undergoes abrupt change, the pupa skin is about to fall off and is about to emerge as silkworm moth, and the silkworm pupa compound eye is not suitable for being dried and ground into silkworm pupa powder, and a silkworm pupa culture medium is prepared. Therefore, preferably, the second period is selected, namely, the silkworm pupa is about 4 to 12 days after pupation, at the moment, the skin of the silkworm pupa is yellow, the protein in the body is rich in nutrition, compound eyes are not colored, the silkworm pupa is not converted into silkworm moth, and the silkworm pupa is suitable for being dried and ground into silkworm pupa powder.
Grinding selected pupa Bombycis into pupa Bombycis powder, and sieving with 12 mesh sieve.
(2) Preparation of silkworm pupa culture medium
Preparing a silkworm pupa culture medium mixture: every 100g of the mixture contains 90g of silkworm chrysalis powder, and MgSO is added 4 .7H 2 O 1.0g、KH 2 PO 4 1.0g and 8.0g of glucose are fully and uniformly mixed.
Adding 50ml of water into the mixture, and fully and uniformly stirring to prepare the silkworm chrysalis culture medium. The culture medium comprises the following components in percentage by weight: 60% of silkworm chrysalis meal, 33.33% of water and MgSO (MgSO) 4 .7H 2 O 0.67％、 KH 2 PO 4 0.67%, glucose 5.33%; the PH value is 6.8-7.2.
Weighing the silkworm pupa culture medium, placing in 9cm diameter culture dishes (25 g for each culture dish), spreading, sterilizing at high temperature and high pressure (121 deg.C for 20 min), and cooling.
EXAMPLE 3 preliminary culture of Phellinus linteus strain
Selecting the 12 Phellinus linteus strains stored in PDA culture medium, picking mycelium blocks with diameter of 0.5cm with sterilized puncher, inoculating into the culture medium center of silkworm pupa in a super clean bench, sealing with sealing film, and standing at 28deg.C in dark for culturing. After two days, the mycelia of the 12 strains are observed to start to grow in the silkworm pupa culture medium, and the mycelia grow around the surface of the silkworm pupa culture medium, which indicates that the mulberry Sang Huangneng can utilize silkworm pupa as a self-growing nutrient substance.
EXAMPLE 4 Long-term domestication of Phellinus linteus strains
After the 12 strains are subjected to dark stationary culture for 50d at 28 ℃, mycelium blocks with the diameter of 0.5cm are picked up by a puncher after sterilization in the same method, and are transferred to the same silkworm pupa culture medium again in a super clean bench, sealed by a sealing film, and still subjected to dark stationary culture at 28 ℃. The transfer is repeated every 50d for 2 years.
Example 5 strain screening
(1) Screening of non-variant strains
After 2 years, the growth state of the above 12 mulberry leaves Huang Zhu in the silkworm chrysalis medium was examined. Each strain has a certain difference in the capability of generating yellow pigment under dark condition and colony morphology, and can be distinguished by naked eyes. After multiple switching, the hyphae grow slowly, the yellow pigment produced is little, the hyphae grow sparsely, and the bag material with the mulberry twigs as the matrix can not be used for smoothly culturing the fruiting bodies of the mulberry Phellinus, so that the 4 strains have a certain degree of variation.
In addition, the yellow pigment produced by 8 strains is more, the mycelium growth morphology is symmetrical, and the bag material with mulberry branches as a matrix can be used for smoothly culturing the fruiting body of the mulberry Phellinus, which indicates that the strain does not have variation. The 8 strains contain a wild Sang Yuan number (CGMCC No. 21079). The above 8 strains were selected for further cultivation.
(2) Growth rate screening
Selecting the above 8 preliminarily screened mulberry and mulberry Huang Zhu lines, picking mycelium with diameter of 0.5cm with a sterilized puncher, inoculating the same silkworm pupa culture medium in the center of the super clean bench again, repeatedly transferring more than 3 culture dishes for each line, sealing with sealing film, and culturing in dark at 28deg.C. The growth speed of each strain in the silkworm chrysalis culture medium is different, and the time for mycelium to cover the whole surface of the silkworm chrysalis culture medium is 28-50 d. 3 strains with slower growth speed are eliminated, 5 strains (28 d-35 d) with shorter time of mycelium being fully distributed on the surface of the whole silkworm pupa culture medium are reserved, and the same silkworm pupa culture medium is continuously used for dark static culture at 28 ℃. The 5 strains contain a wild Sang Yuan number (CGMCC No. 21079).
(3) Aging time screening
Selecting the 5 strains, picking mycelium blocks with the diameter of 0.5cm by using a sterilized puncher, placing the mycelium blocks in the same silkworm pupa culture medium, sealing the silkworm pupa culture medium by using a sealing film, repeatedly transferring more than 3 culture dishes for each strain, continuously performing stationary culture under the dark condition at the temperature of 28 ℃, and investigating the aging time of each strain. The mycelium gradually ages after maturing, and the aged mycelium turns from yellow to brown, and the phenomenon of water spitting appears (water is secreted from the surface of the mycelium to be in the shape of water drops), which is used as an aging standard. It was found that 1 strain began to age after mycelium covered the entire silkworm pupa medium surface for 5d, and that strain was eliminated. The time from the mycelium covering the whole silkworm pupa culture medium surface of the other 4 strains to aging is greatly different, and the average time is 9d, 12d, 16d and 21d respectively. The 4 strains contain a wild Sang Yuan number (CGMCC No. 21079). 4 mulberry and mulberry Huang Zhu lines with relatively long aging time were selected and used for the subsequent screening experiments.
(4) Mycelium firmness screening
A round gauze with the diameter of 9cm is cut, and the size of the round gauze is basically consistent with that of a 9cm culture dish. A round hole with the diameter of 0.5cm is punched in the middle of the gauze by a puncher. A silkworm pupa culture medium was prepared according to the method of example 2, the culture medium was flattened, a piece of the perforated round gauze was placed on the surface of the culture medium, the gauze was flattened, the gauze was closely adhered to the surface of the silkworm pupa powder culture medium, sterilized at high temperature under high pressure (121 ℃ C. For 20 min), and cooled for use.
Selecting the 4 strains, picking mycelium blocks with diameter of 0.5cm by using a sterilized puncher, placing the mycelium blocks in the center of a gauze hole of the silkworm pupa culture medium (the inserted mycelium blocks can be contacted with the silkworm pupa culture medium at the hole position), transferring more than 3 culture dishes for each strain, sealing by a sealing film, and standing for culture at 28 ℃.
The mycelium blocks of the 4 strains can grow normally after contacting with the silkworm chrysalis meal culture medium, and the mycelium gradually extends to the periphery until the mycelium blocks cover the whole gauze. After the mycelium of each strain turns yellow and matures, the sealing film of the culture dish is opened, the gauze with the mycelium is torn off by using the sterilized tweezers, and then the mycelium growing on the gauze is torn off by using the tweezers.
Among the above-mentioned 4 strains of Phellinus linteus strain, 3 strains (including "wild Sang Yuan No. 1") formed mycelium blocks thinner, and it was difficult to smoothly separate mycelium blocks having a complete structure from gauze, so that the yield was low. The mycelium of Phellinus linteus of 1 strain is thicker, the mycelium block is firm, the mycelium block is not easy to break when being separated from gauze by forceps, and the mycelium separated from the gauze is not stained with silkworm pupa powder in a culture medium; although a small amount of mycelium remains in the culture medium and gauze, most mycelium blocks can be finally separated, and the mycelium blocks have a complete structure (figure 1), so that the strain is used as a target strain finally screened.
EXAMPLE 6 investigation of biological Properties of target Strain
And (3) through the domestication and step-by-step screening, obtaining a Phellinus linteus strain (target strain) finally. The strain is suitable for culture medium (silkworm pupa powder 60%, water 33.33%, mgSO) prepared from silkworm pupa under dark static culture condition at 28deg.C 4 .7H 2 O 0.67％、KH 2 PO 4 0.67%, glucose 5.33%; PH value of 6.8-7.2) and has the following characteristics:
(1) The growth speed is high: inoculating mycelium with diameter of 0.5cm into a culture dish with diameter of 9cm (25 g silkworm pupa culture medium is built in), standing at 28deg.C, and culturing in dark, wherein the growth is started in the next day, the average time for mycelium to cover the whole silkworm pupa culture medium surface is 28d (the mycelium is not yet yellow and mature), and the average growth speed of mycelium is 1.52mm/d; the mycelium can be collected after being inoculated to a silkworm chrysalis culture medium until the mycelium turns yellow and matures, and the average time is 40d.
(2) Golden color: after the mycelium is fully distributed in the whole culture medium and developed and matured, a large amount of yellow pigment can be secreted, the color is golden, and the mycelium is uniform in morphology.
(3) The strain is not easy to mutate: the strain is cultured by using a silkworm chrysalis culture medium for a long time (more than 4 years), no degradation, mutation and other phenomena are found, the Phellinus linteus mycelium can be still cultured normally without rejuvenation, and fruiting bodies can be also cultured by using mulberry twig bags.
(4) The mature mycelium has high firmness and is suitable for separation and collection: after the mycelium turns yellow and matures, the mycelium is firm, and most mycelium blocks can be separated from the gauze. After the mycelium blocks are separated, a small amount of mycelium residues still exist in the culture medium and gauze, but the mycelium blocks finally separated are complete in structure and are not easy to tear.
(5) The mycelium yield is high: inoculating mycelium with diameter of 0.5cm into 9cm culture dish (25 g silkworm pupa culture medium is placed therein) with diameter of 28 ℃ for dark static culture for 40d, yellowing and maturing mycelium, separating mycelium from each culture dish to obtain mycelium fresh weight of 2.06g (without a small amount of mycelium remained in the culture medium and on gauze), and drying to obtain mycelium with average weight of 0.43g;
(6) Mycelium is not easy to age: mycelium with the diameter of 0.5cm is inoculated into a culture dish with the diameter of 9cm (25 g of silkworm pupa culture medium is placed in the culture dish) for dark stationary culture at the temperature of 28 ℃, and after the grown mycelium covers the whole surface of the silkworm pupa culture medium (average 28 d), the mycelium is gradually aged after the average 21d (namely, the mycelium is inoculated into the culture medium and the time from the mycelium aging is 49 d).
(7) No peculiar smell: the separated round mycelium blocks have the faint scent of mulberry and mulberry Huang Teyou and have no foreign odor of silkworm chrysalis.
(8) High polyphenol content: in view of the remarkable anti-inflammatory, anti-tumor, cell repair and immunopotentiating activities of Sang Huangduo phenolic compounds, two Phellinus linteus strains of target strain and "wild Sang Yuan No. (CGMCC No. 21079) were selected, mycelium blocks with a diameter of 0.5cm were picked up by a sterilized puncher according to the method described in step (4) of example 5, inoculated into 9cm dishes (25 g silkworm pupa medium was placed therein), and three or more experimental replicates were set. After 40d of dark static culture at 28 ℃, the mycelia grown from both strains are covered on the whole silkworm pupa culture medium surface and turn to golden yellow, and at the moment, the mycelia are mature but not aged; collecting mycelium, oven drying, and grinding into powder. The polyphenol content in the mycelia was determined according to the method described in "orthometric test, preferably Sang Huangduo phenol ultrasonic extraction Process" (Feng Ziwang et al 2012). The results showed that the average polyphenol content of the target strain was 39.51mg/g (dry powder) higher than "wild Sang Yuan No. 1" (31.60 mg/g (dry powder)).
Example 7 ITS sequencing identification and naming of target Strain
(1) ITS sequencing identification of target strains
Extracting mycelium DNA of a target strain, carrying out PCR amplification by using universal primers ITSl (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') of transcription spacer (internal transcribed spacer, ITS) sequences in ribosomal gene rDNA, recovering and purifying amplified products by gel, and sequencing, wherein the size of the fragment is 757bp.
ITS sequences of Sang Huangkong genus (Sanghuang porus), phellinus genus (Phellinus) and Folium (Inonotus) were searched for from GenBank, and a phylogenetic tree was constructed using MEGA7 software. Wild Sang Yuan strain ITS sequence is most similar to ITS sequences of other Phellinus linteus of Sang Huangkong genus (Genbank accession numbers are MN153568.1, JN642587.1, KT693244.1 and KT693275.1, respectively), and is classified (FIG. 2). The target strain obtained was confirmed to be a Phellinus linteus (Sanghuangporus sanghuang) strain belonging to the genus Hymenochaetaceae (Hymenochaetaceae), sang Huangkong in classification.
(2) Naming of target strains
At present, the mulberry distributed in China has 15 mulberry seeds and 4 varieties [15] . Wherein only 4 of the cultivated mulberry trees (white mulberry and its variety, lu mulberry, guangdong mulberry, rui mulberry) are cultivated, and the rest are wild type [16] . The strain originates from a fruiting body of a trunk of a wild type mulberry, and is named "wild Sang Yuan No. 2".
EXAMPLE 8 application of silkworm pupa to cultivation of Phellinus linteus mycelium
In the embodiment, the silkworm pupa culture medium is used for producing wild Sang Yuan No.2 mycelium in a test mode, and the application of the strain in production is studied.
A round gauze with the diameter of 9cm is cut, and the size of the round gauze is basically consistent with that of a 9cm culture dish. A round hole with the diameter of 0.5cm is punched in the middle of the gauze for standby.
A silkworm pupa culture medium was prepared according to the method of example 2, the silkworm pupa culture medium was weighed, placed in 9cm diameter dishes (25 g for each dish), flattened, placed on the surface of the culture medium with one piece of the above perforated round gauze, flattened to make the gauze closely adhere to the silkworm pupa powder culture medium surface, autoclaved at high temperature (121 ℃ for 20 min), and cooled for later use.
Selecting wild Sang Yuan # 2 mycelium with diameter of 0.5cm with sterilized puncher, placing in the center of gauze hole of silkworm pupa culture medium (the inserted mycelium block can contact with silkworm pupa culture medium at hole position), sealing with sealing film, and standing at 28deg.C in dark condition for culturing.
Culturing for about 40 days, and collecting mycelium of wild Sang Yuan No.2 after the mycelium is mature and yellow. When collecting, opening the sealing film, tearing off the gauze with mycelium blocks by using sterilized forceps, and tearing off mycelium blocks growing on the gauze.
The wild Sang Yuan No.2 mycelium has the characteristics of quick growth, difficult aging, firmness and the like, is very suitable for directly producing the mycelium on a large scale by taking silkworm chrysalis as a culture medium, and is prepared into a silkworm chrysalis culture medium by taking waste silkworm chrysalis from a silk reeling factory at present, and mycelium is produced on a small scale, so that the wild Sang Yuan No.2 strain is proved to be suitable for industrial production of Phellinus linteus mycelium throughout the year by taking silkworm chrysalis as the culture medium.
The medicinal efficacy of Phellinus linteus mycelium is not delivered to fruiting body, and is one direction of Phellinus linteus industry [5] . The method for producing the medicinal fungus Phellinus linteus mycelium by using the silkworm pupa culture medium has the following advantages:
1. compared with the traditional mycelium fermentation process for producing Phellinus linteus mycelium: the traditional method for producing mycelium by fermentation often has the defects of expensive fermentation equipment, high production cost, complex procedures and the like; meanwhile, due to factors such as hereditary of strains, the Phellinus linteus strains are subjected to multiple asexual propagation, and are easy to age, degenerate and mutate, and are difficult to truly and practically produce and apply. In comparison, the silkworm pupa culture medium is simple in configuration, the wild Sang Yuan No.2 is cultured in the silkworm pupa culture medium, and only proper temperature is needed, so that complex production process conditions and high-price equipment are not needed, and the cost of large-scale industrial production is low; the strain is repeatedly transferred to the silkworm chrysalis culture medium for many times, and no degeneration and variation phenomenon exists.
2. Compared with the traditional production of Phellinus linteus fruiting body by using bags or log: the bag material or the log is used for producing the Phellinus linteus fruiting body, a large amount of wood is consumed, the national environmental protection requirement is not met, the production time is long, the Phellinus linteus fruiting body can be produced only once a year, the environmental condition requirement is high, the temperature and humidity requirement is severe, and various miscellaneous bacteria (such as trichoderma, viride, neurospora, and the like), mildew and the like are extremely easy to pollute, and insect pests, rats and the like are extremely easy to cause. In comparison, the culture medium for preparing silkworm pupa is simple in configuration, the Phellinus linteus mycelium is cultured by the culture medium, no wood is consumed, the production mode is sustainable, and the ecological environment is not damaged; the production cost is low; the production period is short, and the collection is only about 40 days after the strain is inoculated to a silkworm chrysalis culture medium until the mycelium is mature, so that the indoor industrial mass production can be realized throughout the year; the sealing film is used in the mycelium culture process, so that the pollution of mixed bacteria and the invasion of various harmful organisms in the production of Phellinus linteus by using bag materials or log can be avoided, the yield is high, and the obtained mycelium is dried and ground into powder to obtain the Phellinus linteus mycelium finished product (figure 3).
As the source of the silkworm chrysalis is wide, a large amount of residual silkworm chrysalis becomes waste in the production of silkworm chrysalis every year, the source is wide, the cost is low, the wild Sang Yuan No.2 is utilized to produce the Phellinus linteus mycelium, and the added value of the silkworm production can be greatly improved. At present, the strain is applied to the treatment and comprehensive utilization of mulberry production waste.
The silkworm chrysalis is derived from silkworm, the silkworm takes mulberry as a sole source of food, wild fruiting body of wild Sang Yuan No.2 is also parasitic to mulberry, and the mulberry is taken as a basis of nutrient substances, so that the wild Sang Yuan No.2 strain can be used as a research material for co-evolution among silkworm, mulberry and Phellinus linteus, and has high research value.
The foregoing description is merely exemplary of the invention, and I expect it will be understood that simple modifications and substitutions of the invention are intended to be included within the spirit of the invention without departing from the spirit of the invention.
Reference is made to:
[1] wu Shenghua and Dai Yucheng the species analysis of Phellinus linteus of medicinal fungi [ J ]. The journal of fungus, 2020, v.39; no.203 (05): 6-19
[2] Chen Tijiang, wu Jinzhong, ji Jianying, et al, analysis of fruiting body components of Morus Huang Zaipei and microscopic morphological observation [ J ]. Bacterial research, 2005,3 (1): 30-34
[3]MENGDI,ZHANG,YU,et al.Inonotus sanghuang Polyphenols Attenuate Inflammatory Response Via Modulating the Crosstalk Between Macrophages and Adipocytes[J].Frontiers in Immunology,2019,10:1-12
[4] Wu Shenghua A precious medicinal fungus "Phellinus linteus" species is named [ J ]. Edible and medicinal fungus 2012 (03): 177-179
[5] Wu Shenghua, huang Guanzhong, chen Yuping, et al, classification and development prospect of Phellinus linteus [ J ]. Bacterial research 2016 (4): 187-200
[6]WU S H,CHANG C C,WEI C L,et al.Sanghuangporus toxicodendri sp.nov.(Hymenochaetales,Basidiomycota)from China[J].MycoKeys,2019,57:101-111
[7] Chen Qingai silkworm chrysalis function and its application [ J ] silkworm report 2010,41 (2): 42-43
[8] Mu Lixia, liao Sentai, showcase, etc. silkworm chrysalis protein comprehensive utilization research progress [ J ]. Guangdong agricultural science, 2011 (23): 106-109
[9] Xi Yunhong and Liang Ju pharmaceutical effects of silkworm and its by-products [ J ]. Sichuan silkworm industry, 2009 (02): 52-53
[10] Zhang Yan, chen Yegao, hailina, etc. silkworm chrysalis amino acid composition and its nutritive value [ J ]. Yunnan chemical industry, 2002,29 (006): 22-23
[11] Song Yanqing, deng Shuhai, sui Zhi, etc. medicinal components of silkworm chrysalis and extraction process thereof, research profile [ J ]. J.Chinese journal of biochemical medicine, 2006 (05): 306-309
[12] Wang Jinhua research and application of active substances of Bombyx Batryticatus and pupa Beauveri [ J ]. Shizhen national medicine, 2003,14 (008): 492-494
[13] Tang Shunming, zeng Guangyuan, etc. A method for artificially culturing Cordyceps sinensis with silkworm pupae as host.2017:
[14] zhu Xiangrui A lecture for diversified utilization of mulberry resources (8) [ J ]. A report of mulberry, 2018,49 (04): 53-54
[15] Xia Mingjiong the characteristic properties and distribution of Morus plants of Morus genus [ J ]. Chinese silkworm industry, 2015,36 (004): 8-14
[16] Xia Mingjiong classification and study of Morus alba [ J ]. Chinese silkworm industry, 2015,036 (003): 7-13.
Claims (4)
1. Phellinus linteus strain suitable for culturing mycelium with silkworm pupaSanghuangporus sanghuang) Strains, the classification of which is named: phellinus linteusSanghuangporus sanghuang) The wild Sang Yuan No.2 strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 09 and 17 of 2021, and has a preservation address of CGMCC No.23257, namely, no. 3 of North Xiyun No. 1, the Korean region of Beijing, and a preservation number of the strain.
2. The method for culturing a Phellinus linteus strain suitable for culturing mycelium with silkworm pupae according to claim 1, comprising the steps of:
(1) Selecting silkworm chrysalis: selecting healthy and disease-free silkworm chrysalis for about 4-12 days, grinding the silkworm chrysalis into silkworm chrysalis powder, and sieving the silkworm chrysalis powder by a 12-mesh sieve for later use;
(2) Preparing a silkworm pupa culture medium: taking the silkworm chrysalis powder, water and MgSO prepared in the step (1) 4 .7H 2 O、KH 2 PO 4 Glucose was 60% by mass: 33.33:0.67:0.67:5.33, wherein the PH value of the culture medium is 6.8-7.2, and the culture medium is sterilized for 20min at 121 ℃ to obtain the silkworm pupa culture medium;
(3) Mycelium culture: the mulberry and Phellinus linteus are treatedSanghuangporus sanghuang) Inoculating mycelium blocks of wild Sang Yuan No.2 onto the silkworm pupa culture medium prepared in the step (2), sealing with a sealing film, standing at 28deg.C in dark for culturing, and collecting mycelium blocks of wild Sang Yuan No.2 with mature growth and yellow color.
3. The use of the Phellinus linteus strain suitable for culturing mycelium with silkworm pupa according to claim 1 for culturing mycelium with silkworm pupa.
4. Mulberry and mulberryHuang Peiyang A, characterized in that it comprises silkworm pupa powder and MgSO 4 .7H 2 O、KH 2 PO 4 The mass ratio of glucose to water is 60:33.33:0.67:0.67:5.33, the pH of the culture medium is 6.8-7.2.
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