CN116004393A - Phellinus linteus strain suitable for culturing mycelium with silkworm pupa, and culturing method and application thereof - Google Patents

Abstract

The invention discloses a Phellinus mulberry strain suitable for cultivating mycelium with silkworm chrysalis, a cultivation method and application thereof, and belongs to the field of biotechnology. The mulberry Phellinus strain described in the present invention has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee on September 17, 2021, and the preservation number is CGMCC No.23257. The strain is suitable for cultivation in silkworm chrysalis, and the mycelium is grown in a culture medium made of silkworm chrysalis (silkworm chrysalis powder 60%, water 33.33%, MgSO ₄ .7H ₂ O 0.67%, KH ₂ PO ₄ 0.67%, glucose 5.33%; pH value 6.8 Growth in ~7.2) has the advantages of fast growth, high yield, firmness and easy separation of mycelium blocks from the medium, not easy to mutate, not easy to age, no peculiar smell, high polyphenol content, etc., suitable for perennial industrial production with silkworm chrysalis medium The Phellinus mycelium of the mulberry tree has no contamination by bacteria, and does not require specific equipment and complicated cultivation environment conditions; the silkworm pupa culture medium is simple to prepare, low in cost, does not need to consume wood, and does not damage the ecological environment.

Description

A mulberry Phellinus strain suitable for cultivating mycelium with silkworm chrysalis and its cultivation method and application

technical field

The invention belongs to the field of biological technology, and in particular relates to a mulberry Phellinus strain suitable for cultivating mycelium with silkworm pupae and application thereof.

Background technique

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

Nevertheless, people's understanding of Phellinus is a gradual process: before 2012, there were no academic records and herbarium specimen records showing that there were Phellinus fungi growing on mulberry trees in China ^[1] ; until 2012, Phellinus was discovered as a previously undescribed new species ^[4] ; until 2016, Phellinus and related species were recognized as an independent new genus-Sanghuangporus; as of 2020, Phellinus The number of species within the genus of Poria has been expanded to 14, including poplar (mulberry) yellow, mulberry (mulberry) yellow, sumac (mulberry) yellow, oak (mulberry) yellow, etc. ^{[1 ,5-6]} , they are named after "Sang", also known as "Panghuang", not because they grow on mulberry trees, but only because they belong to the fungus of the genus Phellinus, and the true Phellinus (Mulberry Phellinus) are closely related species. The only thing that can be referred to as "Sang Huang" for short is the mulberry tree Phellinus sangha; the mulberry tree Phellinus is specific to the tree species it grows on, and it only grows on the trunk of the mulberry tree, and it is a medicinal fungus belonging to the genus Phellinus ^{[ 1,5]} .

Because the real Phellinus (Mulberry Phellinus) is extremely rare, and the mulberry tree Phellinus is extremely harsh on the growth environment, it is difficult to cultivate artificially. closely related species), the so-called Sanghuang fruiting bodies widely cultivated in China, Japan, and Korea are not Sanghuang mulberry, but mainly poplar (Sang) Huang ^[1,5] , and also include Baodingxiang (Sang) Huang and Zashuhuang , and even Poreophora coarseum.

At present, most of the methods for cultivating Phellinus and imitating Phellinus adopt segmented wood or bag materials to cultivate fruiting bodies. There are the following disadvantages in the cultivation of fruiting bodies with segmented wood or bag materials: a large amount of wood needs to be consumed, the cultivation period is long, the growth of fruiting bodies is demanding on the environment, and the later stage of cultivation is exposed to high temperature and high humidity, which can easily cause bacterial contamination. Therefore, the cost of cultivating the fruiting body with segment wood or bag material is high and the quality is difficult to control.

Massive cultivation of mycelium can replace fruiting body cultivation to a certain extent. The test results of several active components and efficacy of Phellinus mycelium are not inferior to fruiting bodies. The mycelium culture period is short, the cost is low, the quality is stable, and the effect is good, which is a direction of the Phellinus industry ^[5] . Mycelium production is usually carried out by mycelium fermentation. However, fermentation culture has many defects such as high process cost (need to purchase fermentation tanks, cumbersome procedures, etc.), easy variation and degradation of mycelia, and most of them remain in the laboratory research stage at present.

Although most of the artificially cultivated Phellinus sold in the market are imitation Phellinus, the price is still expensive, which proves that in the field of research and application of Phellinus, it is urgent to make breakthroughs and innovations in the following two aspects: 1. Obtain authentic Phellinus (Mulberry Phellinus) strain, the strain needs to be truly specific and parasitic on the mulberry tree, and is a fungus belonging to the genus Phellinus Phellinus in classification; further, the strain needs to be resistant to variation, aging, and stable quality 1. It is suitable for artificial large-scale cultivation; 2. It has made breakthroughs and innovations in the cultivation technology of the authentic mulberry tree Phellinus japonica. Only innovations in both Phellinus strains and culture techniques can really help the research and application of Phellinus, and help to get rid of the current predicament of Phellinus industry in my country.

The silkworm (Bombyx mori L.) is a metamorphic insect, and the silkworm larva develops into a silkworm chrysalis after spinning and cocooning. Silkworm chrysalis is a valuable resource for both medicine and food ^[7] . In ancient my country, medical books such as "Compendium of Materia Medica" and "Essentials of Materia Medica" have its medicinal records; in modern times, silkworm chrysalis is included in "Chinese Medicine Dictionary" and "Chinese Medicine Chronicle" as a tonic drug. Used to treat various conditions. Silkworm chrysalis is the only insect food included in the "Original and New Resource Food List of Common Food Raw Materials" approved by the Ministry of Health in 2004 ^[8] , and most silkworm areas in China have always had the habit of eating silkworm chrysalis.

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

Silkworm chrysalis has a certain application prospect in the fields of food, daily chemical, feed additive and fertilizer, traditional Chinese medicine and biomedicine. For example: silkworm chrysalis can be directly eaten as food, made soy sauce, fertilizer and feed additives for livestock and fishery; silkworm chrysalis can also be used in the research of anti-hepatitis, hypertension, coronary heart disease, diabetes, and anticancer drugs.

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

Nevertheless, most of the current comprehensive utilization of silkworm chrysalis is still limited by various factors such as immature technical conditions and high cost, and it is still at the research level, and the usage is very limited; most of the remaining silkworm chrysalis are directly eaten or used as livestock However, due to the special smell of silkworm chrysalis and technical reasons such as preservation, the actual dosage is also limited. At present, the price of dried silkworm chrysalis used as feed additives on the market is only 7,000-8,500 yuan/ton. Dried silkworm chrysalis sold on the Internet are generally used as bait by fishing enthusiasts, and the price is no more than 10 yuan/catties. The sales volume is 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 silkworm larvae spin and cocoon, about 20% of the fresh cocoon weight is silk (cocoon layer), and the other 80% is silkworm chrysalis. Silkworm chrysalis is the most important by-product of the silk reeling industry. In theory, China can produce more than 500,000 tons of fresh silkworm chrysalis and nearly 100,000 tons of dry silkworm chrysalis per year ^[14] . At present, most domestic silk reeling factories accumulate a large number of silkworm chrysalis every year, which cannot be effectively transformed and utilized and have to be discarded at will, becoming "waste" of silkworm cocoon production, causing environmental pollution. There is an urgent need for breakthroughs and innovations in the comprehensive utilization technology of silkworm chrysalis.

So far, there is no report on the cultivation of authentic mulberry Phellinus using silkworm chrysalis.

Since in the evolution process of hundreds of millions of years, both silkworm and Phellinus mulberry have adapted to mulberry tree by using mulberry tree as their source of nutrients, while silkworm chrysalis is a stage in the life development process of silkworm, silkworm, mulberry tree Phellinus, There must be a certain co-evolutionary relationship between mulberry trees. Using silkworm chrysalis to cultivate Phellinus mulberry has very important theoretical research and practical application value.

Contents of the invention

Technical problem: the technical problem to be solved in the present invention is to provide a strain of Phellinus mulberry strain suitable for cultivating mycelium with silkworm pupae as a medium.

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

Technical solution: A mulberry Phellinus strain Yesangyuan No. 2, which is suitable for cultivating mycelia from silkworm pupae, has been preserved in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short; address: Beijing) on September 17, 2021 No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, City, Institute of Microbiology, Chinese Academy of Sciences; Zip code: 100101), the preservation number is CGMCC No.23257.

The mulberry tree Phellinus sanghuang (Sanghuangporus sanghuang) bacterial strain described in the present invention belongs to the Sanghuangporus Sheng H.Wu, L.W. Zhou&Y.C.Dai (Sanghuangporus Sheng H.Wu, L.W. Zhou&Y.C.Dai) on classification. (Sanghuangporus sanghuang). The strain was isolated from the fruiting bodies of Phellinus mulberry mulberry on the trunk of a wild type mulberry tree, and was named Ye Sangyuan No. 2 after successive generations of screening and domestication.

The present invention's mulberry Phellinus phylloxera strain No. 2 is characterized in that:

1. Biological characteristics

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

(1) Fast growth: put the mycelium with a diameter of 0.5cm into a 9cm-diameter petri dish (built-in 25g silkworm chrysalis medium) and culture it in the dark at 28°C. The growth will start the next day, and the mycelium will cover the whole house. The average time on the surface of the silkworm chrysalis culture medium is 28 days (the mycelium has not yet turned yellow and mature), and the average growth rate of mycelia is 1.52mm/d; 40d.

(2) Golden color: Mycelia cover the entire culture medium and mature, and can secrete a large amount of yellow pigment with golden color and well-proportioned mycelium shape.

(3) The bacterial strain is not easy to mutate: the bacterial strain has been cultivated with the silkworm chrysalis medium for a long time (more than 4 years), and no degeneration, variation, etc. have been found, and the mulberry Phellinus mycelium can still be normally cultivated without rejuvenation.

(4) Firmness: After the mycelium becomes yellow and mature, the degree of firmness is high, and the mycelium block is easy to separate and not easy to tear, so it is suitable for separation and harvesting.

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

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

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

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

2. Genetic features

No. 2 mycelia DNA of Mulberry Phellinus strain Ye Sangyuan of the present invention, through the universal primer ITS1/ITS4 of ribosomal gene rDNA internal transcription spacer (internal transcribed spacer, ITS) sequence design, carry out PCR amplification, the nucleic acid sequence size that obtains is 757bp. Alignment of the obtained sequence with the ITS sequences of Sanghuangporus, Phellinus and Inonotus, Ye Sangyuan 2 and other mulberry trees of the genus Phellinus The ITS sequence similarity is the highest, clustered into one category. Ye Sang Yuan No. 2 is a strain of Sanghuangporus sanghuang belonging to the family Hymenochaetaceae and the genus Sanghuangporus.

An application of the mulberry Phellinus phylloxera strain suitable for cultivating mycelium with silkworm chrysalis, the method comprises the following steps:

1. Select silkworm chrysalis

The selected silkworm pupae are required to be healthy and disease-free (no rotten pupae, half-pupated pupae, dead pupae, dead pupae, etc.). As preferably, choose the silkworm chrysalis of about 4～12 days after pupation, the silkworm chrysalis of this moment has formed, and skin color is yellow, and protein nutrition is abundant in the body, and compound eye is not yet colored, and has not emerged into silkworm moth yet. Grind the selected silkworm chrysalis into silkworm chrysalis powder, sieve through a 12-mesh sieve and set aside.

2. Configure silkworm chrysalis culture medium

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

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

Among them, the mass ratio of silkworm chrysalis 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 6.8-7.2.

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

Take by weighing above-mentioned silkworm chrysalis culture medium, place the petri dish of 9cm diameter (each petri dish takes by weighing 25g), after substratum is flattened, the circular gauze after above-mentioned punching is placed on the substratum surface, gauze is flattened, Make the gauze close to the surface of the silkworm chrysalis powder culture medium, sterilize under high temperature and high pressure (121° C. for 20 minutes), cool down and set aside.

3. Mycelium culture

Pick the Ye Sang Yuan No. 2 mycelium block with a diameter of 0.5 cm with a sterilized hole puncher, and place it in the central hole position of the gauze of the above-mentioned silkworm pupa culture medium (the inserted mycelium block can directly contact the silkworm pupae at the hole position. Culture medium), sealed with parafilm, and cultured statically at 28°C in the dark.

After about 40 days of cultivation, the mycelia of Ye Sang Yuan No. 2 matured and turned yellow in color, ready to be harvested. When harvesting, the sealing film is opened, the gauze with the mycelium block is torn off with sterilized tweezers, and then the mycelium block attached to the gauze is torn off. The mycelium block is dried and ground into powder to be used as a finished product.

Beneficial effects: the present invention isolates the strain obtained from the fruiting body of Phellinus mulberry for the first time, screens and acclimates step by step with silkworm chrysalis culture medium, and obtains a strain of Phellinus mulberry, which is named Ye Sangyuan No. 2. The strain is suitable for cultivating mulberry Phellinus mycelia in the medium made by silkworm chrysalis; the mycelium growing in the medium of silkworm chrysalis has golden color, fast growth, high yield, firmness, not easy to mutate, not easy to age, no peculiar smell, polyphenols With the advantages of high content, it is very suitable for the perennial industrial production of mulberry Phellinus mycelium using silkworm chrysalis as the substrate; the silkworm chrysalis culture medium is simple to configure, low in cost, does not need to consume wood, does not destroy the ecological environment, does not require specific equipment and complicated cultivation environment conditions . Since the silkworm chrysalis belongs to the waste of sericulture production, the strain can be applied to the research on the treatment and comprehensive utilization of sericulture production waste. Therefore, the strain has broad application prospects in production and research.

Description of drawings

Fig. 1 is the mycelium block isolated after culturing No. 2 Ye Sang Yuan in silkworm chrysalis culture medium.

Fig. 2 shows the phylogenetic relationship between Yesangyuan 2 and other fungi of the genera Phellinus, Xyloporus and Fibropora.

Fig. 3 is the mycelium powder that Ye Sang Yuan No. 2 is produced from the mycelium produced in the silkworm chrysalis culture medium.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Embodiment 1, mulberry Phellinus phellinus fruiting body collection and bacterial classification separation and cultivation

(1) According to the currently identified mulberry species (15 mulberry species, 4 varieties) and distribution in my country, investigate the representative distribution area of mulberry trees in my country. A total of 12 fruiting bodies that were born in different regions and specifically parasitized on Phellinus mulberry were collected.

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

Embodiment 2, the selection of silkworm chrysalis and the configuration of silkworm chrysalis culture medium

(1) Selection of silkworm chrysalis

The selected silkworm chrysalis should first be healthy and disease-free, that is, no rotten chrysalis, semi-pupated chrysalis, sick dead chrysalis, dead chrysalis, etc.

Bombyx mori is a metamorphic insect, and the larvae begin to pupate after cocooning on the cocoon. The silkworm chrysalis generally goes through three developmental stages: the first stage is 1-3 days after pupal body formation, the second stage is about 4-12 days after pupation, and the third stage is 13 days after pupation, when the compound eyes are colored Until it emerges as a silkworm moth. Wherein, the above-mentioned silkworm chrysalis pupa skin of the first period has just formed, and the pupa body is milky white or light yellow, and the pupa body is soft and tender, and is easy to break, so it is not suitable for drying and grinding into silkworm pupa powder, and making silkworm pupa culture medium; The compound eyes of the silkworm chrysalis in the three periods have been colored, and the chrysalis has become soft and turned brown. At this time, the body of the chrysalis undergoes drastic changes, and the pupa skin is about to fall off, and it is about to emerge as a silkworm moth, and it is not suitable for drying and grinding into silkworm chrysalis. Powder, and make silkworm chrysalis culture medium. Therefore, as a preference, select the second period, which is about 4 to 12 days after pupation, the silkworm chrysalis. At this time, the silkworm chrysalis skin color is yellow, the protein in the body is rich in nutrition, the compound eyes have not yet colored, and have not yet transformed into silkworm moths. It is suitable for drying and grinding into silkworm chrysalis powder.

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

(2) Configuration of Bombyx mori chrysalis culture medium

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

Add 50ml of water to the above mixture, stir well, and then make silkworm chrysalis culture medium. The contents of various components in the medium are: silkworm chrysalis powder 60%, water 33.33%, MgSO ₄ .7H ₂ O 0.67%, KH ₂ PO ₄ 0.67%, glucose 5.33%; pH value 6.8-7.2.

The silkworm pupa culture medium was weighed, placed in a 9 cm diameter petri dish (each petri dish weighed 25 g), flattened, sterilized by high temperature and high pressure (121° C. for 20 min), and cooled for later use.

Embodiment 3, the preliminary cultivation of Phellinus Phellinus strain

Select the above-mentioned 12 Phellinus mulberry strains stored in the PDA medium, pick out a mycelium block with a diameter of 0.5cm with a sterilized hole puncher, insert the above-mentioned Bombyx mori chrysalis medium center in the ultra-clean bench, and seal it with a parafilm , placed in the dark at 28°C for static culture. Two days later, it was observed that the mycelia of the above-mentioned 12 strains all started to grow in the silkworm chrysalis medium, and the mycelium grew around on the surface of the silkworm chrysalis medium, indicating that Phellinus mulberry can use the silkworm chrysalis as a nutrient for its own growth.

Embodiment 4, long-term domestication of Phellinus mulberry strain

After the above-mentioned 12 bacterial strains were cultured in the dark at 28°C for 50 days, in the same way, a 0.5cm-diameter piece of mycelia was picked out with a sterilized hole puncher, and then transferred to the same silkworm pupa for culture in the ultra-clean bench. base, sealed with parafilm, and kept at 28°C in the dark for static culture. In this way, it is switched every 50d, and the switching is repeated for 2 years.

Embodiment 5, bacterial strain screening

(1) Screening of unmutated strains

After 2 years, the growth state of the above-mentioned 12 Phellinus mulberry strains in the silkworm chrysalis culture medium was investigated. There are certain differences in the ability of each strain to produce yellow pigment and the shape of colonies under dark conditions, which can be distinguished by naked eyes. There are 4 strains after multiple transfers, the growth of mycelium slows down, the yellow pigment produced is very little, and the mycelium grows sparsely. It has been impossible to successfully cultivate the fruiting body of Phellinus mulberry tree with the bag material of mulberry branch as the substrate. It shows that these 4 strains have produced a certain degree of variation.

The other 8 strains produced more yellow pigments, and the mycelia grew in a uniform shape. The fruiting bodies of Phellinus mulberry were successfully cultivated with the bag material of mulberry branches, which indicated that the strains did not mutate. The above 8 strains include "Yesangyuan No. 1" (CGMCC NO.21079). The above 8 strains were selected for further cultivation.

(2) Growth rate screening

Select the above 8 initially screened mulberry Phellinus strains, pick out mycelia with a diameter of 0.5cm with a sterilized hole puncher, and insert the same silkworm pupa culture medium center again in the ultra-clean bench, each strain Transfer to more than 3 culture dishes repeatedly, seal with parafilm, and culture in the dark at 28°C. The growth speed of each strain in the silkworm pupae culture medium was different, and the time for the hyphae to cover the entire surface of the silkworm pupae culture medium ranged from 28d to 50d. Eliminate the 3 strains with slower growth rate, keep 5 strains whose mycelia covered the entire surface of the silkworm chrysalis medium for a short time (28d～35d), continue to use the same silkworm chrysalis medium to 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 puncher to pick out mycelium blocks with a diameter of 0.5cm, place them in the same silkworm pupa culture medium, seal with a parafilm, and transfer each strain to more than 3 culture dishes repeatedly , continue static culture at 28°C in the dark, 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" (water is secreted from the surface of the mycelium in the shape of water droplets) appears, which is used as the aging standard. It was found that one strain began to age 5 days after the mycelium covered the entire surface of the silkworm chrysalis medium, and the strain was eliminated. The time from the mycelium of the remaining 4 strains covering the entire surface of the silkworm chrysalis culture medium to aging varies greatly, with an average of 9d, 12d, 16d, and 21d, respectively. The above four strains include "Yesangyuan No. 1" (CGMCC NO.21079). Four Phellinus Phellinus strains with relatively long aging time were screened out for subsequent screening experiments.

(4) Mycelium Firmness Screening

Cut out a circular gauze with a diameter of 9 cm, which is basically the same size as a 9 cm petri dish. A circular hole with a diameter of 0.5 cm was punched in the middle of the gauze with a punch. Configure the silkworm chrysalis culture medium according to the method in Example 2. After the culture medium is flattened, a circular gauze after the above-mentioned perforation is placed on the surface of the culture medium, and the gauze is flattened so that the gauze is close to the surface of the silkworm chrysalis powder culture medium. Sterilize (autoclave at 121°C for 20 minutes), cool and set aside.

Select above-mentioned 4 strains, pick the mycelia piece of diameter 0.5cm with the puncher after the sterilization, place the gauze hole center of above-mentioned silkworm chrysalis culture medium (the mycelium piece that inserts can touch the home in the hole position. Silkworm chrysalis culture medium), each strain was transferred to more than 3 culture dishes, sealed with parafilm, and cultured statically at 28°C in the dark.

The mycelium blocks of the above four strains can grow normally after contacting the silkworm chrysalis powder medium, and the mycelium gradually extends around until covering the entire gauze. After the mycelium of each bacterial strain turns yellow and mature, the petri dish sealing film is opened, the gauze with mycelium is torn off with sterilized tweezers, and the mycelium grown on the gauze is torn off with tweezers.

Among the 4 strains of Phellinus mulberry strains tested above, 3 strains (including "Yesangyuan No. 1") formed thin mycelium blocks, and it was difficult to successfully separate the mycelium with complete structure from the gauze. body, so the yield is low. The mycelium of only one strain of Phellinus mulberry is thicker, and the mycelium block is 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 chrysalis powder in the medium ; although a small amount of mycelium remains in the culture medium and gauze, most of the mycelium blocks can still be isolated at last, and the structure of the mycelium blocks is relatively complete (Fig. 1). Screened target strains.

Embodiment 6, the investigation of the biological character of target bacterial strain

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

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

(2) Golden color: Mycelia cover the entire culture medium and mature, and can secrete a large amount of yellow pigment with golden color and well-proportioned mycelium shape.

(3) The strain is not easy to mutate: the strain has been cultivated with silkworm pupa culture medium for a long time (more than 4 years), and no degeneration, variation, etc. have been found, and there is no need for rejuvenation. The branch bag material cultivates the fruiting body.

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

(5) High yield of mycelium: After inserting mycelium with a diameter of 0.5 cm into a 9 cm petri dish (built-in 25 g of silkworm pupae culture medium) at 28°C for 40 days in the dark, the mycelium turns yellow and matures, with an average The fresh weight of the mycelium obtained by separating the petri dish reaches 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 reaches 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 chrysalis medium) and culture it in the dark at 28°C, and the grown mycelium will cover the entire silkworm chrysalis for cultivation After the basal surface (average 28d), it takes an average of 21d to gradually age (that is, the average time from the time when the mycelium is inserted into the culture medium to the aging of the mycelium is 49d).

(7) No peculiar smell: the separated round mycelium block has the unique fragrance of Phellinus mulberry and no peculiar smell of silkworm chrysalis.

(8) High polyphenol content: In view of the significant anti-inflammation, anti-tumor, cell repair function and immune-enhancing activities of Phellinus polyphenols, the target strain and "Yesangyuan No. 1" (CGMCC NO. 21079) were selected to A mulberry Phellinus strain, according to the method described in embodiment 5 steps (4), picks the mycelium block of diameter 0.5cm with the puncher after the sterilization, inoculates to 9cm culture dish (built-in 25g silkworm chrysalis substratum) , and set more than three experimental repetitions. After standing in the dark at 28°C for 40 days, the mycelia grown from the two strains have covered the entire surface of the silkworm chrysalis culture medium and turned golden yellow. At this time, the mycelium has matured but not aged; the mycelia were harvested body, dried and ground into powder. According to the method described in "Optimized Phellinus Polyphenols Ultrasonic Extraction Technology by Orthogonal Experiment" (Feng Ziwang et al., 2012), the content of polyphenols in the mycelium was determined. The results showed that the average polyphenol content of the target strain was 39.51 mg/g (dry powder), higher than that of "Yesangyuan No. 1" (31.60 mg/g (dry powder)).

Example 7, ITS sequencing identification and naming of the target strain

(1) ITS sequencing identification of the target strain

Extract the mycelium DNA of the target strain, and perform PCR with the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') of the ribosomal gene rDNA internal transcription spacer (internal transcribed spacer, ITS) sequence Amplification, gel recovery and purification of the amplified product, after sequencing, the size of the fragment is 757bp.

The ITS sequences of Sanghuangporus, Phellinus and Inonotus were searched from GenBank, and the phylogenetic tree was constructed using MEGA7 software. The ITS sequence of Ye Sangyuan No. 2 strain has the highest similarity with the ITS sequences of other Morus phellinus of Phellinus genus (Genbank accession numbers are MN153568.1, JN642587.1, KT693244.1 and KT693275.1), and they are clustered into one class (Figure 2). It was confirmed that the obtained target strain was a strain of Sanghuangporus sanghuang belonging to the family Hymenochaetaceae and the genus Sanghuangporus in classification.

(2) Naming the target strain

There are currently 15 mulberry species and 4 varieties of mulberry distributed in China ^[15] . Among them, there are only 4 cultivated types of mulberry trees (white mulberry and its variants, Lusang, Guangdong mulberry, Ruisui mulberry), and the rest are wild types ^[16] . The strain originated from a fruiting body parasitic on the trunk of a wild type mulberry tree, and the strain was named "Yesangyuan No. 2".

Embodiment 8, the application of cultivating mulberry Phellinus mycelia with silkworm chrysalis

In this example, the No. 2 mycelium of Ye Sang Yuan was produced on the medium of silkworm pupae, and the application of the strain in production was studied.

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

Configure the silkworm chrysalis medium according to the method in Example 2, take the silkworm chrysalis medium, place a 9cm diameter petri dish (each petri dish takes 25g), after the culture medium is flattened, place a piece on the surface of the culture medium The circular gauze after the above-mentioned perforation is flattened so that the gauze is close to the surface of the silkworm chrysalis powder culture medium, sterilized under high temperature and high pressure (121° C. and high pressure sterilization for 20 min), and cooled for later use.

Pick the wild sangyuan No. 2 mycelium with a diameter of 0.5cm with a sterilized hole puncher, and place it in the center of the gauze hole of the above-mentioned silkworm chrysalis medium (the inserted mycelium block can touch the silkworm chrysalis medium at the hole position ), sealed with parafilm, and cultured statically at 28°C in the dark.

After about 40 days of cultivation, the mycelia of Ye Sang Yuan No. 2 matured and turned yellow in color, ready to be harvested. When harvesting, the sealing film is opened, the gauze with the mycelium block is torn off with sterilized tweezers, and the mycelium block grown on the gauze is torn off.

Because the mycelium of Yesangyuan No. 2 has the above-mentioned characteristics of fast growth, not easy to age, and firmness, it is very suitable for direct large-scale production of mycelium using silkworm chrysalis as a medium. At present, the silkworm chrysalis discarded in the silk reeling factory has been used to produce silkworm chrysalis culture Based on the small-scale production of mycelium, it proved that the "Yesangyuan No. 2" strain is suitable for the perennial industrial production of mulberry Phellinus mycelium using silkworm pupae as the medium.

The medicinal efficacy of Phellinus mycelium is not lost to the fruiting body, which is a direction of Phellinus industry ^[5] . The production of the medicinal fungus Phellinus Phellinus mycelia with silkworm chrysalis medium has the following advantages:

1. Compared with the production of Phellinus mycelium by traditional mycelium fermentation process: the traditional method of fermentation mycelia production often has disadvantages such as expensive fermentation equipment, high production costs, and cumbersome procedures; Factors, Phellinus strains are prone to aging, degeneration, and variation after repeated asexual reproduction, and it is difficult to actually produce and apply them. In comparison, the silkworm chrysalis medium is simple to configure. "Yesangyuan No. 2" is cultivated in the silkworm chrysalis medium, and only needs to be given a suitable temperature. There is no need for complicated production process conditions and expensive equipment, and large-scale factory production The production cost is low; the bacterial strain is transferred repeatedly in the culture medium of silkworm chrysalis without any degeneration and variation phenomenon.

2. Compared with the production of Phellinus fruiting bodies by bag materials or wood sections: the production of Phellinus fruiting bodies by bag materials or wood sections needs to consume a lot 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 pollution, as well as pests and rodents. In comparison, the culture medium for the production of silkworm chrysalis is simple in configuration to cultivate the mycelia of Phellinus mulberry tree without consuming any wood. The production method is sustainable and will not cause damage to the ecological environment; the production cost is low; the production cycle is short, It is only about 40 days from the inoculation of the strain to the culture medium of the silkworm pupae to the harvesting of the mycelium after maturity, which can be used for large-scale indoor factory production all year round; the use of sealing film in the process of mycelium cultivation can avoid the miscellaneous production of Phellinus japonica from bag materials or wood segments Bacterial pollution and various harmful organisms attack, the yield is high, the obtained mycelium is dried, and after being ground into powder, the finished product of mulberry Phellinus mycelium can be obtained (Figure 3).

Due to the wide range of silkworm pupae sources, a large number of surplus silkworm pupae become wastes in sericulture production every year. The source is wide and the cost is low. Using Ye Sangyuan No. 2 to produce mulberry Phellinus mycelium can greatly increase the added value of sericulture production. At present, the strain has been applied to the treatment and comprehensive utilization of sericulture production waste.

Because silkworm chrysalis is derived from silkworm, silkworm uses mulberry tree as the only source of food, and the wild fruiting body of Ye Sang Yuan No. 2 also parasitizes on mulberry tree, and uses mulberry tree as the basis of nutrients, therefore, Ye Sang Yuan No. 2 bacterial strain can be used as silkworm, mulberry tree The research material of co-evolution between Phellinus mulberry and Phellinus mulberry has very high research value.

The above description is only an embodiment of the present invention, and it can be understood that, on the premise of not departing from the concept of the present invention, simple modifications and replacements of the present invention should be included in the technical concept of the present invention.

references:

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[4] Wu Shenghua. Rectification of the precious medicinal fungus "Panghuang" species [J]. Edible and Medicinal Fungi, 2012(03):177-179

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Claims (6)

1. Phellinus linteus strain suitable for culturing mycelium with silkworm pupa, the classification of the strain is named as: phellinus linteus (Sanghuangporus sanghuang) wild Sang Yuan No.2 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the day of 09 and 17 of 2021, and the preservation address is North Chen Xiyu No. 1 and 3 of the area of Beijing, chaoyang, and the preservation number is CGMCC No.23257.

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: inoculating Phellinus linteus (Sanghuangporus sanghuang) and wild Sang Yuan # 2 mycelium blocks on the silkworm pupa culture medium prepared in the step (2), sealing with a sealing film, standing at 28deg.C in dark condition for culturing, and collecting the wild Sang Yuan # 2 mycelium after development and yellowing.

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. The application of silkworm chrysalis in Phellinus linteus culture medium is characterized in that silkworm chrysalis is ground into powder and added into Phellinus linteus culture medium.

5. A mulberry and mulberry Huang Peiyang base, which is characterized by comprising the following substances: silkworm chrysalis powder and MgSO ₄ .7H ₂ O、KH ₂ PO ₄ Glucose, water.

6. A Phellinus linteus culture medium as set forth in claim 5, wherein silkworm pupa powder, 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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