CN116004393B - A mulberry Phellinus mulberry strain suitable for culturing mycelium from silkworm pupae and its cultivation method and application - Google Patents

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

A mulberry Phellinus mulberry strain suitable for culturing mycelium from silkworm pupae and its cultivation method and application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a mulberry Phellinus mulberry strain suitable for culturing mycelium from silkworm pupae and its application.

Background technique

Sanghuangporus sanghuang is a rare medicinal fungus that lives on the mulberry tree (Morus L). There are records of Phellinus linata in medical books of all dynasties since the Han Dynasty. In recent years, research at home and abroad has proven that Phellinus linteus is rich in various types of polysaccharides, flavonoids and their derivatives, sterols and other active substances, and has multiple medicinal effects such as improving immunity, anti-cancer, anti-inflammatory and antioxidant ^[1-3] , which has great industrial development and application value.

Despite this, people's understanding of Phellinus linteum is a gradual process: before 2012, there were no academic records or herbarium specimen records showing that there was Phellinus linteus fungus growing on mulberry trees in China ^[1] ; until 2012, Phellinus linata was discovered to be a previously undescribed new species ^[4] ; by 2016, Phellinus linteus and similar species were recognized as an independent new genus - Sanghuangporus; as of 2020, Phellinus linteus The number of species within the genus Poria has expanded to 14, including poplar (mulberry) yellow, martin (mulberry) yellow, sumac (mulberry) yellow, oak (mulberry) yellow, ^{etc. ,5-6]} , they are named after "mulberry" and are also called "phyllus linteus", not because they grow on mulberry trees, but just because they belong to the fungi of the genus Phellinus genus, and are not the same as real mulberry. (Mulberry mulberry) is a closely related species. The only one that can really be abbreviated as "Sanghuang" is the mulberry tree. The mulberry tree is specific to the tree species it grows on. It only grows on the trunk of the mulberry tree, and it is a medicinal fungus belonging to the genus Xanthomonas ^{[ 1,5]} .

Since the real Phellinus mulberry (Morus mulberry) is extremely rare, and the mulberry tree has extremely harsh growth conditions and is difficult to cultivate artificially, most of the so-called "mulberry huang" seen on the market are imitation mulberry (Morus mulberry). Closely related species), the so-called mulberry mulberry fruiting bodies widely cultivated in China, Japan and Korea are not mulberry mulberry mulberry huang, but mostly poplar (mulberry) huang ^[1,5] , and also include mulberry mulberry (mulberry) huang and miscellaneous mulberry huang. , and even Fibroporus hirsutum, etc.

At present, most of the methods for cultivating Phellinus linteus and imitation Phellinus linteus are using wood sections or bags to cultivate fruiting bodies. The disadvantages of cultivating fruiting bodies from wood segments or bags are as follows: a large amount of wood needs to be consumed, the cultivation cycle is long, the growth of fruiting bodies has strict environmental requirements, and the later stages of cultivation are exposed to high temperature and high humidity, which can easily cause contamination by miscellaneous bacteria. Therefore, the cost of cultivating fruiting bodies with wood segments or bags is high and the quality is difficult to control.

Large-scale cultivation of mycelium can replace fruiting body cultivation to a certain extent. The experimental results of several active ingredients and efficacy of Phellinus linteus mycelium are not inferior to those of fruiting bodies. Mycelium culture cycle is short, low cost, stable quality and good effect, which is a direction of Phellinus linteus industry ^[5] . Mycelium production is usually carried out by mycelial fermentation. However, fermentation culture has many shortcomings such as high process costs (requiring the purchase of fermentation tanks, cumbersome processes, etc.), easy mutation and degradation of mycelium, and most of them currently remain in the laboratory research stage.

Although most of the artificially cultivated Phellinus linteus currently sold on the market is imitation Phellinus linteus, the price is still expensive, which proves that there is an urgent need for breakthroughs and innovations in the following two aspects in the field of research and application of Phellinus linteus: 1. Obtaining authentic Phellinus linteus (Mulberry Phellinus) strain, this strain needs to be truly specific for parasitizing mulberry trees, and it must be classified as a fungus of the genus Xanthomonas; further, this strain needs to be resistant to mutation, aging, and stable quality , Suitable for artificial large-scale cultivation and other characteristics; 2. Breakthroughs and innovations in the cultivation technology of authentic mulberry trees and Phellinus mulberry. Only innovations in both Phellinus linteus strains and culture technology can truly promote the research and application of Phellinus linteus and help get rid of the current dilemma of my country's Phellinus linteus industry.

Bombyx mori L. is a metamorphic insect. After the silkworm larvae spins silk and forms a cocoon, it metamorphoses and develops into Silkworm chrysalis. Silkworm pupae are a valuable resource that can be used both as medicine and food ^[7] . There are records of its medicinal use in medical books such as "Compendium of Materia Medica" and "Essentials of Materia Medica" in ancient my country; in modern times, silkworm pupae are included as traditional Chinese medicines in "Dictionary of Chinese Medicine" and "Chronicles of Chinese Medicine" and are used as tonic drugs. Used to treat various conditions. Bombyx silkworm pupae are the only insect food included in the "List of Original and New Resource Foods for Common Food Raw Materials" approved by the Ministry of Health in 2004 ^[8] . Most of the silkworm areas in China have always had the habit of eating silkworm pupae.

Silkworm pupae are very rich in nutrients. Each 100g of dried silkworm pupae contains about 60g of crude protein, 30g of crude fat (pupa oil), 3 to 4g of chitin, 5g of sugar, as well as trace elements, vitamins, antimicrobial peptides, hormones, and lysozyme. and other biologically active substances. Bombyx mori pupa protein is a complete protein containing 18 amino acids ^[8-9] , which is very consistent with the ideal amino acid pattern recommended by FAO/WHO ^[10] . Silkworm pupae also contain a variety of trace elements (such as Zn, Mn, Se, etc.), as well as vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin D, niacin, etc.; silkworm pupae also contain a variety of hormone substances , such as prostatin, epinephrine, β-sitosterol, etc.; silkworm pupae also contain pupal phospholipids, antimicrobial peptides, lysozyme, adenine and other substances ^[11] .

Silkworm pupae have certain application prospects in the fields of food, daily chemicals, feed additives and fertilizers, traditional Chinese medicine and biomedicine. For example, silkworm pupae can be directly eaten as food, made into soy sauce, and used as fertilizers and additives for livestock and fishery feeds. Silkworm pupae are also used in the research of anti-hepatitis, hypertension, coronary heart disease, diabetes, and anti-cancer drugs.

Currently, there are three main applications for cultivating microorganisms using silkworm pupae. One is Beauveria bassiana, which is formed by infection of silkworm pupae or artificial inoculation with Beauveria bassiana ^[12] . As a traditional Chinese medicine in China, it has a sedative effect on the nerves and can treat epilepsy and other diseases ^[9] . The second type is Cordyceps militaris cultured from silkworm pupae, which is an ideal substitute for Cordyceps sinensis. The third method is to use silkworm pupae to cultivate Cordyceps cicadae, which is also a precious traditional Chinese medicinal material in China ^[13] .

Despite this, most of the current comprehensive utilization of silkworm pupae is still limited by various factors such as immature technical conditions and high costs. It is still at the research level and the usage is very limited; most of the remaining silkworm pupae are eaten directly or used as livestock Feed additives, but due to the special smell of silkworm pupae and technical reasons such as preservation, the actual dosage is also limited. Currently, dried silkworm pupae used as feed additives on the market generally sell for only 7,000 to 8,500 yuan per ton. Dry silkworm pupae sold through online e-commerce are generally used by fishing enthusiasts as bait, and the price does not exceed 10 yuan per catty. Sales are very limited. .

my country is the world's largest producer of sericulture and raw silk, with an annual output of more than 700,000 tons of fresh cocoons in recent years. After the silkworm larvae spins silk and forms a cocoon, about 20% of the weight of the fresh cocoon is silk (cocoon layer), and the other 80% is silkworm pupa. Silkworm pupae are the most important by-product of the silk reeling industry. Theoretically, China can produce more than 500,000 tons of fresh silkworm pupae and nearly 100,000 tons of dried silkworm pupae every year ^[14] . At present, most domestic silk reeling factories have a large amount of silkworm pupae accumulated every year, which cannot be effectively converted and utilized and have to be discarded at will, becoming "waste" of silkworm cocoon production and causing environmental pollution. There is an urgent need for breakthroughs and innovations in the comprehensive utilization technology of silkworm pupae.

So far, there are no reports on using silkworm pupae to cultivate authentic mulberry trees.

Since in the course of hundreds of millions of years of evolution, both silkworms and mulberry trees have adapted to mulberry trees, and silkworm pupae are a stage in the life development process of silkworms. There must be a certain co-evolutionary relationship between mulberry trees. Using silkworm pupae to cultivate mulberry trees and Phellinus mulberry has very important theoretical research and practical application value.

Contents of the invention

Technical problem: The technical problem to be solved by the present invention is to provide a mulberry Phellinus mulberry strain suitable for culturing mycelium using silkworm pupae as a culture medium.

The technical problem to be solved by the present invention is to provide the application of the above-mentioned mulberry Phellinus mulberry strain in preparing mulberry mulberry Phellinus mycelium.

Technical solution: A mulberry Phellinus strain No. 2, which is suitable for culturing mycelium from silkworm pupae, has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee (CGMCC for short) on September 17, 2021; address: Beijing No. 3, No. 1, Beichen West Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences; Postal Code: 100101), the collection number is CGMCC No. 23257.

The Sanghuangporus sanghuang strain of the present invention belongs to the family Hymenochaetaceae and the genus Sanghuangporus Sheng H.Wu, L.W. Zhou & Y.C.Dai in terms of classification. (Sanghuangporus sanghuang). The strain was isolated from the mulberry fruiting body of the mulberry tree on the trunk of a wild mulberry tree. After successive generations of screening and domestication, it was named Ye Sang Yuan No. 2.

The characteristics of the mulberry Phellinus mulberry strain Ye Sang Yuan No. 2 of the present invention are:

1. Biological characteristics

This strain is suitable for the culture medium made of silkworm pupae (60% silkworm pupae powder, 33.33% water, MgSO ₄ .7H ₂ O 0.67%, KH ₂ PO ₄ 0.67%, glucose 5.33%) under the conditions of static culture in the dark at 28°C. %; pH value 6.8~7.2) to cultivate mycelium and has the following characteristics:

(1) Fast growth: Put mycelium with a diameter of 0.5cm into a 9cm diameter petri dish (built-in 25g of silkworm pupa culture medium) and culture it in the dark at 28°C. The growth will start the next day, and the mycelium will cover the entire home. The average time on the surface of the silkworm pupa medium is 28 days (the mycelium has not yet turned yellow and mature at this time), and the average growth rate of the mycelium is 1.52mm/d; the average time from the time when the mycelium is inoculated into the silkworm pupa medium to when it turns yellow and mature and is harvested is 40d.

(2) Golden color: After the hyphae cover the entire culture medium and mature, they can secrete a large amount of yellow pigment, the color is golden, and the hyphae are well-proportioned.

(3) The strain is not easy to mutate: This strain has been cultivated in silkworm pupa medium for a long time (more than 4 years), and no degradation, mutation or other phenomena have been found. There is no need to rejuvenate and the mulberry mycelium can still be cultured normally.

(4) Firmness: After the mycelium turns yellow and matures, it has a high degree of firmness. The mycelium blocks are easy to separate and not easy to tear, making it suitable for separation and harvesting.

(5) High mycelium yield: Mycelium with a diameter of 0.5cm is inserted into a 9cm diameter petri dish (built-in 25g silkworm pupa culture medium) and cultured at 28°C in the dark for 40 days. The average number of bacteria isolated from each petri dish is The fresh weight of the mycelium is 2.06g (excluding a small amount of mycelium remaining in the culture medium and on the gauze), and the average weight of the mycelium obtained after drying is 0.43g.

(6) The mycelium is not easy to age: put the mycelium with a diameter of 0.5cm into a 9cm diameter petri dish (built-in 25g silkworm pupa culture medium) and culture it at 28°C in the dark. The average aging time of the grown mycelium is 49d.

(7) No peculiar smell: The separated mycelium block has no peculiar smell of silkworm pupa, but has the unique fragrance of mulberry tree.

(8) High polyphenol content: The average polyphenol content of the isolated mycelium reaches 39.51mg/g (dry powder).

2. Genetic characteristics

The mycelial DNA of the mulberry Phellinus mulberry strain No. 2 of the present invention is amplified by PCR with universal primers ITS1/ITS4 designed from the ribosomal gene rDNA internal transcribed spacer (ITS) sequence, and the obtained nucleic acid sequence size is 757bp. The obtained sequence was compared with the ITS sequences of Sanghuangporus, Phellinus and Inonotus, and No. 2 was compared with other mulberry trees of the genus Sanghuangporus. The ITS sequences have the highest similarity and are clustered into one category. Yesangyuan No. 2 is a mulberry Sanghuangporussanghuang strain that belongs to the family Hymenochaetaceae and the genus Sanghuangporus.

The application of a mulberry Phellinus mulberry strain suitable for culturing mycelium from silkworm pupae, the method includes the following steps:

1. Select silkworm pupae

The selected silkworm pupae must be healthy and disease-free (no rotten pupae, half-pupae, diseased pupae, stiff pupae, etc.). Preferably, the silkworm pupa is selected about 4 to 12 days after pupation. At this time, the silkworm pupa has formed, the skin color is yellow, the body is rich in protein, the compound eyes have not yet been colored, and the silkworm has not yet emerged into a silkworm moth. Grind the selected silkworm chrysalis into silkworm chrysalis powder, sieve it through a 12-mesh sieve and set aside.

2. Configure silkworm pupa culture medium

The silkworm pupa culture medium includes the above-mentioned silkworm pupa powder.

Further: including silkworm pupa powder, water, MgSO ₄ .7H ₂ O, KH ₂ PO ₄ and glucose.

Among them, the mass ratio of silkworm pupa powder, water, MgSO ₄ .7H ₂ O, KH ₂ PO ₄ and glucose is: 60:33.33: 0.67:0.67:5.33, and the pH value of the culture medium is 6.8 to 7.2.

Cut a round gauze with a diameter of 9cm, which is basically the same size as a 9cm petri dish. Use a hole punch to punch a 0.5cm diameter round hole in the middle of the gauze and set aside.

Weigh the above-mentioned silkworm pupa culture medium and place it in a 9cm diameter petri dish (weigh 25g for each culture dish). After the culture medium is flattened, place a piece of the above-mentioned circular gauze with holes on the surface of the culture medium and flatten the gauze. Place the gauze close to the surface of the silkworm chrysalis powder culture medium, sterilize it under high temperature and high pressure (121°C for 20 minutes), and cool it before use.

3. Mycelium culture

Use a sterilized hole punch to pick out a piece of No. 2 mycelium with a diameter of 0.5cm, and place it in the center hole of the gauze of the silkworm pupa culture medium (the inserted mycelium piece can directly contact the silkworm pupa at the hole position) culture medium), sealed with parafilm, and cultured in the dark at 28°C.

After about 40 days of cultivation, the mycelium of Yesangyuan No. 2 has matured, turned yellow, and is ready for harvest. When harvesting, open the sealing film, use sterilized tweezers to tear off the gauze with mycelium blocks, and then tear off the mycelium blocks attached to the gauze. The mycelium pieces are dried and ground into powder to serve as the finished product.

Beneficial effects: For the first time, the present invention uses the silkworm pupa medium to screen and domesticate the strains isolated from the fruiting bodies of the mulberry tree Moricolor, and obtains a mulberry tree strain named Ye Sang Yuan No. 2. This strain is suitable for cultivating mulberry mycelium in a medium made from silkworm pupae; the mycelium grown in the silkworm pupa medium has golden color, fast growth, high yield, firmness, resistance to mutation, resistance to aging, no odor, and polyphenols. With the advantages of high content, it is very suitable for the perennial factory production of mulberry mycelium using silkworm pupae as the substrate; the silkworm pupae culture medium is simple to configure, low cost, does not require wood consumption, does not damage the ecological environment, and does not require special equipment and complex cultivation environmental conditions. . Since silkworm pupae are wastes from sericulture production, this strain can be used in research on the treatment and comprehensive utilization of sericulture production wastes. Therefore, this strain has broad application prospects in production and research.

Description of the drawings

Figure 1 shows the mycelium blocks isolated from No. 2 No. 2 after culture in silkworm pupa medium.

Figure 2 shows the phylogenetic relationship between No. 2 No. 2 and other fungi of the genus Morinnoporus, Xyloporus and Fibroporus.

Figure 3 shows the mycelium powder produced from the mycelium produced in the silkworm pupa culture medium of No. 2 Ye Sang Yuan.

Detailed ways

The present invention will be described in further detail below with reference to examples, but the implementation of the present invention is not limited thereto.

Example 1. Collection of mulberry fruiting bodies and bacterial strain isolation and culture

(1) Based on the types and distribution of mulberry trees currently identified in my country (15 mulberry species, 4 varieties), examine the representative distribution areas of mulberry trees in my country. A total of 12 fruiting bodies born in different regions and specifically parasitic on mulberry trees were collected.

(2) Isolate the strains of the above 12 fruiting bodies and temporarily store them in ordinary PDA culture medium (200g of peeled potatoes, 20g of glucose, 20g of agar, natural pH value per liter of culture medium).

Example 2. Selection of silkworm pupae and configuration of silkworm pupae culture medium

(1) Selection of silkworm pupae

The selected silkworm pupae must first be healthy and disease-free, that is, there are no rotten pupae, half-pupae, diseased pupae, stiff pupae, etc.

Bombyx mori is a metamorphic insect. After the larvae completes cocooning on the cocoon, it begins to pupate. Silkworm pupae generally go through three development stages: the first stage is 1 to 3 days after the pupa body is formed, the second stage is about 4 to 12 days after pupation, and the third stage is 13 days after pupation, and the compound eyes have been colored. Before emerging into a silkworm moth. Among them, the pupa skin of the silkworm pupa in the above-mentioned first period has just formed, the pupa body is milky white or light yellow, the pupa body is soft and tender, and it is easy to break, so it is not suitable for drying and grinding to make silkworm pupa powder and making silkworm pupa culture medium; the third The compound eyes of silkworm pupae in three stages have been colored, and the pupal body has softened and turned brown. At this time, the body of the silkworm pupa has undergone rapid changes. The pupal skin is about to fall off and will emerge into silkworm moths. It is not suitable for drying and grinding into silkworm pupae. powder, and prepare silkworm pupa culture medium. Therefore, as a preferred option, the second period is selected, which is the silkworm pupa about 4 to 12 days after pupation. At this time, the skin color of the silkworm pupa is yellow, the body is rich in protein nutrition, the compound eyes are not yet colored, and it has not yet transformed into a silkworm moth. Suitable for drying and grinding to make silkworm chrysalis powder.

Grind the selected silkworm chrysalis into silkworm chrysalis powder, sieve it through a 12-mesh sieve, and set aside.

(2) Configuration of silkworm pupa culture medium

Prepare the silkworm pupa medium mixture: Each 100g of the mixture contains 90g of the above-mentioned silkworm pupa powder, add 1.0g of MgSO ₄ .7H ₂ O, 1.0g of KH ₂ PO ₄ and 8.0g of glucose, and mix thoroughly.

Add 50 ml of water to the above mixture and mix thoroughly to prepare the silkworm pupa culture medium. The contents of various components of the culture medium are: 60% silkworm pupa powder, 33.33% water, 0.67% MgSO ₄ .7H ₂ O, 0.67% KH ₂ PO ₄ , and 5.33% glucose; the pH value is 6.8 to 7.2.

Weigh the above-mentioned silkworm pupa culture medium, place it in a 9cm diameter petri dish (weigh 25g per petri dish), flatten it, sterilize it under high temperature and high pressure (121°C high pressure sterilization for 20 minutes), and cool it for later use.

Example 3. Preliminary culture of mulberry and Phellinus linteus strains

Select the above 12 mulberry Phellinus mulberry strains stored in PDA culture medium, pick out mycelium pieces with a diameter of 0.5cm with a sterilized hole punch, insert them into the center of the above silkworm pupa culture medium in a clean bench, and seal them with a parafilm. , placed in the dark at 28°C for static culture. Two days later, it was observed that the mycelium of the above-mentioned 12 strains started to grow in the silkworm pupa medium, and the mycelium grew in all directions on the surface of the silkworm pupa medium, indicating that the mulberry tree Phellinus mulberry can use the silkworm pupae as nutrients for its own growth.

Example 4. Long-term domestication of mulberry Phellinus linteus strain

After culturing the above 12 strains in the dark for 50 days at 28°C, use the same method to pick out mycelium pieces with a diameter of 0.5cm using a sterilized puncher, and then transfer them to the same silkworm pupae in a clean bench again. The base was sealed with parafilm and placed in the dark at 28°C for incubation. In this way, the transfer is performed every 50 days, and the transfer is repeated for 2 years.

Example 5. Strain screening

(1) Screen for unmutated strains

Two years later, the growth status of the above 12 mulberry S. mulberry lines in the silkworm pupa medium was investigated. Each strain has certain differences in its ability to produce yellow pigment and colony morphology under dark conditions, which can be distinguished by the naked eye. After multiple transfers, the hyphae of 4 strains grew slowly, produced very little yellow pigment, and grew sparsely. It was no longer possible to successfully cultivate mulberry mulberry fruiting bodies using bags with mulberry branches as the substrate. This shows that these four strains have undergone a certain degree of variation.

The other 8 strains produced more yellow pigments and had well-proportioned mycelial growth shapes. Mulberry fruiting bodies of Morus mulberry trees could be successfully cultured using bags with mulberry branches as the substrate, indicating that the strains did not mutate. The above eight strains include "Yesangyuan No. 1" (CGMCCNO.21079). Select the above 8 strains to continue culturing.

(2) Growth rate screening

Select the above 8 preliminary screened mulberry strains of Phellinus mulberry, use a sterilized hole punch to pick out mycelium with a diameter of 0.5cm, and insert it into the center of the same silkworm pupa culture medium again in the ultra-clean platform. Repeat for 3 more culture dishes, seal with parafilm, and culture at 28°C in the dark. The growth speed of each strain in the silkworm pupa medium is different, and the time for mycelium to cover the entire surface of the silkworm pupa medium ranges from 28 to 50 days. Eliminate the 3 strains with slow growth rate and retain the 5 strains whose mycelium covers the entire surface of the silkworm pupa medium for a shorter time (28d~35d). Continue to use the same silkworm pupa medium and let it stand in the dark at 28°C. nourish. The above five strains include "Yesangyuan No. 1" (CGMCC NO. 21079).

(3) Aging time screening

Select the above 5 strains, use a sterilized hole punch to pick out mycelium pieces with a diameter of 0.5cm, place them in the same silkworm pupa culture medium, seal them with a sealing film, and transfer each strain to more than 3 petri dishes. , continue to culture statically under dark conditions at 28°C, and investigate the aging time of each strain. After the mycelium matures, it gradually ages. The aged mycelium turns from yellow to brown, and the phenomenon of "spitting water" occurs (the surface of the mycelium secretes water in the shape of water beads), which is used as the aging standard. It was found that one strain began to age 5 days after mycelium covered the entire surface of the silkworm pupa medium, and this strain was eliminated. The time from mycelium covering the entire silkworm pupa medium surface to aging for the remaining four strains varied greatly, with averages of 9d, 12d, 16d, and 21d respectively. The above four strains include "Yesangyuan No. 1" (CGMCC NO. 21079). Four mulberry lines with relatively long aging time will be screened out for subsequent screening experiments.

(4) Mycelium firmness screening

Cut a round gauze with a diameter of 9cm, which is basically the same size as a 9cm petri dish. Use a hole punch to punch a circular hole with a diameter of 0.5cm in the middle of the gauze. Configure the silkworm pupa culture medium according to the method in Example 2. After the culture medium is flattened, place a circular gauze with the above holes on the surface of the culture medium. Flatten the gauze so that the gauze is close to the surface of the silkworm pupa powder culture medium. High temperature and high pressure Sterilize (high-pressure sterilization at 121°C for 20 minutes) and cool before use.

Select the above 4 strains, use a sterilized hole punch to pick out a piece of mycelium with a diameter of 0.5cm, and place it in the center of the gauze hole of the silkworm pupa culture medium (the inserted mycelium piece can touch the hole) Silkworm pupa medium), transfer each strain to more than 3 petri dishes, seal them with parafilm, and culture them statically in the dark at 28°C.

The mycelium blocks of the above four strains can grow normally after contacting the silkworm pupa powder medium, and the mycelium gradually stretches around until it covers the entire gauze. After the mycelium of each strain turns yellow and matures, open the sealing film of the petri dish, first use sterilized tweezers to tear off the gauze with mycelium, and then use tweezers to tear off the mycelium growing on the gauze.

Among the 4 strains of mulberry mulberry strains tested above, 3 strains (including "Yesangyuan No. 1") formed thin mycelium blocks, making it difficult to successfully separate structurally complete mycelium from gauze. The body is bulky, so the yield is low. The mycelium of only one strain of mulberry tree, Phellinus mulberry, is thicker and firm. It is not easy to break when separated from the gauze with tweezers. The mycelium separated from the gauze does not contaminate the silkworm pupa powder in the culture medium. ; Although a small amount of mycelium remains in the culture medium and gauze of this strain, most of the mycelial blocks can still be isolated in the end, and the structure of the mycelial blocks is relatively complete (Figure 1). This strain is regarded as the final Screened target strains.

Example 6. Investigation of biological properties of target strains

Through the above-mentioned domestication and step-by-step screening, a mulberry strain (target strain) was finally obtained. This strain is suitable for the culture medium made of silkworm pupae (60% silkworm pupae powder, 33.33% water, MgSO ₄ .7H ₂ O 0.67%, KH ₂ PO ₄ 0.67%, glucose 5.33%) under the conditions of static culture in the dark at 28°C. %; pH value 6.8~7.2) to cultivate mycelium and has the following characteristics:

(1) Fast growth: Put mycelium with a diameter of 0.5cm into a 9cm diameter petri dish (built-in 25g of silkworm pupa culture medium) and culture it in the dark at 28°C. The growth will start the next day, and the mycelium will cover the entire home. The average time on the surface of the silkworm pupa medium is 28 days (the mycelium has not yet turned yellow and mature at this time), and the average growth rate of the hyphae is 1.52mm/d; the hyphae can be harvested from the time they are inoculated into the silkworm pupa medium until they turn yellow and mature. The average is 40d.

(2) Golden color: After the hyphae cover the entire culture medium and mature, they can secrete a large amount of yellow pigment, the color is golden, and the hyphae are well-proportioned.

(3) The strain is not easy to mutate: This strain has been cultivated in silkworm pupa medium for a long time (more than 4 years), and no degradation or mutation has been found. It does not need to be rejuvenated, and the mulberry mycelium can still be cultured normally, and mulberry mycelium can also be cultivated. Branch bags are used to cultivate fruiting bodies.

(4) The mature mycelium has a high degree of firmness and is suitable for separation and collection: after the mycelium turns yellow and matures, it is relatively firm and can separate most of the mycelium blocks from the gauze. After the mycelium block is separated, although there is still a small amount of mycelium remaining in the culture medium and gauze, the structure of the finally separated mycelium block is intact and not easy to tear.

(5) High mycelium yield: Mycelium with a diameter of 0.5cm is inserted into a 9cm diameter petri dish (built-in 25g silkworm pupa culture medium) and cultured at 28°C in the dark for 40 days. The mycelium will turn yellow and mature, with an average of The fresh weight of the mycelium isolated from each petri dish is 2.06g (excluding a small amount of mycelium remaining in the culture medium and on the gauze), and the average weight of the mycelium obtained after drying is 0.43g;

(6) The mycelium is not easy to age: put the mycelium with a diameter of 0.5cm into a 9cm diameter petri dish (built-in 25g of silkworm pupa culture medium) and culture it at 28°C in the dark. The grown mycelium will cover the entire silkworm pupa culture. After the base surface (an average of 28 days), it takes an average of 21 days to gradually age (that is, from the time when the mycelium is connected to the culture medium to the aging of the mycelium, the average time is 49 days).

(7) No peculiar smell: The isolated round mycelium block has the unique fragrance of mulberry tree and Phellinus mulberry, without the peculiar smell of silkworm pupae.

(8) High polyphenol content: In view of the significant anti-inflammatory, anti-tumor, cell repair function and immune-enhancing activities of Phellinus linteus polyphenols, the target strain and "Yesangyuan No. 1" (CGMCC NO. 21079) were selected. According to the method described in step (4) of Example 5, use a sterilized hole punch to pick out mycelium pieces with a diameter of 0.5cm and inoculate them into a 9cm petri dish (built-in 25g silkworm pupa culture medium). , and set up three or more experimental replicates. After 40 days of static culture at 28°C in the dark, the mycelium grown from both strains has covered the entire surface of the silkworm pupa medium and turned golden yellow. At this time, the mycelium has matured but not aged; the mycelium is harvested body, dried and ground into powder. The polyphenol content in the mycelium was determined according to the method described in "Orthogonal Experiment to Optimize the Ultrasonic Extraction Process of Phellinus Phellinus Polyphenols" (Feng Ziwang et al., 2012). The results showed that the average polyphenol content of the target strain reached 39.51 mg/g (dry powder), which was higher than that of "Yesangyuan No. 1" (31.60 mg/g (dry powder)).

Example 7. ITS sequencing identification and naming of target strains

(1) ITS sequencing identification of target strains

Extract the mycelium DNA of the target strain and perform PCR with the universal primers ITSl (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') of the ribosomal gene rDNA internal transcribed spacer (ITS) sequence. After amplification, the amplification product was gel recovered and purified, and after sequencing, the size of the fragment was 757 bp.

Search the ITS sequences of Sanghuangporus, Phellinus and Inonotus from GenBank, and use MEGA7 software to construct a phylogenetic tree. The ITS sequence of No. 2 strain No. mulberry has the highest similarity with the ITS sequences of other mulberry trees of the genus Xanthopus mulberry (Genbank accession numbers are MN153568.1, JN642587.1, KT693244.1 and KT693275.1 respectively), and they are clustered together. category (Figure 2). It was confirmed that the target strain obtained was a Sanghuangporus sanghuang strain belonging to the family Hymenochaetaceae and the genus Sanghuangporus.

(2) Naming of target strains

Currently, there are 15 mulberry species and 4 varieties distributed in China ^[15] . Among them, there are only four cultivated types of mulberry trees (white mulberry and its varieties, Lu mulberry, Guangdong mulberry, and Ruisui mulberry), and the rest are wild types ^[16] . This strain is derived from a fruiting body parasitic on the trunk of a wild type of mulberry tree, and is named "Yesangyuan No. 2".

Example 8. Application of using silkworm pupae to cultivate mulberry mycelium of Phellinus moriensis.

In this example, the silkworm pupa culture medium was used to trial-produce Nosangyuan No. 2 mycelium, and the application of this strain in production was studied.

Cut a round gauze with a diameter of 9cm, which is basically the same size as a 9cm petri dish. Use a hole punch to punch a 0.5cm diameter round hole in the middle of the gauze and set aside.

Configure the silkworm pupa culture medium according to the method in Example 2, weigh the silkworm pupa culture medium, and place it on a 9cm diameter petri dish (weigh 25g for each culture dish). After the culture medium is flattened, a piece of culture medium is placed on the surface of the culture medium. Take the above-mentioned circular gauze with holes, flatten the gauze so that the gauze is close to the surface of the silkworm chrysalis powder culture medium, sterilize it under high temperature and high pressure (121°C high pressure sterilization for 20 minutes), and cool it for later use.

Use a sterilized hole punch to pick out the mycelium of Wild Mulberry No. 2 with a diameter of 0.5cm, and place it in the center of the gauze hole of the silkworm pupa culture medium (the inserted mycelium piece can contact the silkworm pupa culture medium at the hole position ), seal with parafilm, and incubate in the dark at 28°C.

After about 40 days of cultivation, the mycelium of Yesangyuan No. 2 has matured, turned yellow, and is ready for harvest. When harvesting, open the sealing film, use sterilized tweezers to tear off the gauze with mycelium pieces, and then tear off the mycelium pieces growing on the gauze.

Because the mycelium of Ye Sang Yuan No. 2 has the above-mentioned characteristics of fast growth, resistance to aging, and firmness, it is very suitable for direct large-scale production of mycelium using silkworm pupae as a culture medium. At present, waste silkworm pupae from reeling factories have been used to produce silkworm pupae culture. Base, small-scale production of mycelium, proving that the "Yesangyuan No. 2" strain is suitable for the long-term factory production of mulberry mycelium using silkworm pupae as the culture medium.

The medicinal efficacy of Phellinus linteus mycelium is not inferior to that of the fruiting bodies, which is a direction of Phellinus linteus industry ^[5] . Using silkworm pupa culture medium to produce medicinal fungus mulberry mulberry mycelium has the following advantages:

1. Compared with the traditional mycelium fermentation process to produce Phellinus linteus mycelium: The traditional fermentation method to produce mycelium often has shortcomings such as expensive fermentation equipment, high production costs, and cumbersome processes; at the same time, due to the genetics of the strains, etc. Factors: After multiple asexual reproductions, the Phellinus linteus strain is prone to aging, degradation, and mutation, making it difficult for actual production and application. In comparison, the configuration of the silkworm pupa culture medium is simple. "Yesangyuan No. 2" is cultured in the silkworm pupa culture medium. It only needs to be given a suitable temperature. There is no need for complex production process conditions and expensive equipment, and large-scale factoryization is required. The production cost is low; the strain is repeatedly transferred in the silkworm pupa culture medium many times without any degradation and mutation.

2. Compared with the traditional production of Phellinus phyllifolia fruiting bodies from bags or sections of wood: the production of Phellinus phyllifolia fruiting bodies from bags or sections of wood requires a large amount of wood, which does not meet the requirements of national environmental protection, and the production time is long, and can only be produced in one year. Once, it has high requirements on environmental conditions and strict requirements on temperature and humidity, which can easily cause various bacteria (such as Trichoderma, green mold, Neurospora, etc.), mildew and other contamination, as well as insect damage, rodent damage, etc. In comparison, the culture medium produced by silkworm pupae is simple in configuration. It can be used to cultivate mulberry mycelium without consuming any wood. The production method is sustainable and will not cause damage to the ecological environment. The production cost is low and the production cycle is short. It only takes about 40 days from the inoculation of the strain to the silkworm pupa culture medium to the harvesting after the mycelium matures, which can be used for large-scale indoor factory production all year round; the use of sealing film during the mycelium culture process can avoid the miscellaneous production of Phellinus linteus from bags or segments. Bacteria contamination and various harmful biological infringements, the yield is high. After drying the obtained mycelium and grinding it into powder, the finished product of mulberry mulberry mulberry yellow mycelium can be obtained (Figure 3).

Due to the wide range of sources of silkworm pupae, a large number of remaining silkworm pupae become waste every year in sericulture production. The sources are wide and the cost is low. Using Ye Sang Yuan No. 2 to produce mulberry mulberry mycelium can greatly increase the added value of sericulture production. At present, this strain has begun to be used in the treatment and comprehensive utilization of sericulture production waste.

Since the silkworm pupae are derived from the silkworm, the silkworm uses mulberry trees as its only source of food, and the wild fruiting bodies of Yesangyuan No. 2 are also parasitic on mulberry trees and use mulberry trees as the basis of nutrients. Therefore, the Yesangyuan No. 2 strain can be used as a source of food for silkworms and mulberry trees. The research materials on the co-evolution between mulberry, mulberry and mulberry huang have high research value.

The above descriptions are only embodiments of the present invention. It can be understood that simple modifications and replacements of the present invention should be included in the technical concept of the present invention without departing from the concept of the present invention.

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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.