CN112195104B - Culture medium for identifying phellinus igniarius of mulberry tree and preparation method and application thereof - Google Patents

Abstract

The invention discloses a culture medium for identifying phellinus igniarius, a preparation method and application thereof, and belongs to the technical field of biology. The culture medium for identifying phellinus igniarius of the invention comprises the following components: silkworm excrement powder KH₂PO₄、MgSO₄And water, the mass ratio of which is 41.25: 0.3: 0.15: 58.3, the PH value of the culture medium is 8.5-8.8, and then the culture medium is sterilized for 30min at 121 ℃ to prepare the phellinus igniarius identification culture medium. Only the real Phellinus igniarius hyphae can normally grow in the Phellinus igniarius identification culture medium, and other strains cannot normally grow in the Phellinus igniarius identification culture medium. The phellinus igniarius strain is cultured and stored in the identification culture medium for a long time, no degeneration, variation and other phenomena of the phellinus igniarius strain are found within 5 years, and the phellinus igniarius sporocarp can be successfully induced without rejuvenation. The culture medium is proved to be capable of identifying the real phellinus igniarius and is completely suitable for long-term storage of phellinus igniarius strains.

Description

Culture medium for identifying phellinus igniarius of mulberry tree and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of a culture medium for identifying phellinus igniarius.

Background

Phellinus linteus (Sanghuangporus sanghuang) is a very rare medicinal fungus parasitic on mulberry (Morus L.). Phellinus igniarius is one of the medicinal fungi with the best anticancer efficiency internationally recognized at present^[1]The element is the name of forest gold.

The use of Phellinus linteus is recorded in medical books of all generations since the Han dynasty in China. For example, Shennong Ben Cao Jing of the Han Dynasty, Ming Yi Bie Lu of the ceramic carry-in Jing of Wei jin, and Yao xing of the first Tang dynasty^[2]. Although the ancient people often have a relatively rough description of phellinus linteus, the name, texture, use method and the like of phellinus linteus are not the same, and the habitat of phellinus linteus is difficult to be examined, the phellinus linteus recorded in almost all ancient books is closely related to mulberry, and the mushrooms growing on other species of trees are not called as phellinus linteus by the ancient people.

In the last two decades, Phellinus Linteus has been increasingly researched by researchers in countries such as America, Japan and Korea and China due to its outstanding medicinal valueThe word "Phellinus linteus" is being abused gradually. Some scholars have classified the phellinus igniarius, so that only one kind of actual phellinus igniarius is determined, and the phellinus igniarius only grows on the trunk of the mulberry in the field^[3]We may refer to it as "phellinus linteus" for a while. The penmen undertakes the work of collecting and sorting mulberry resources, and personally surveys in the field for decades, so that the real wild phellinus igniarius is extremely rare, the shape of the fruiting body is extremely irregular, and the growth of the phellinus igniarius is extremely severe to the conditions of natural environment, mulberry varieties and the like; the wild yellow ansiolus-free mushroom growing on other tree species in similar shape is more, and the medicinal value of the phellinus igniarius is the highest, and the content of the medicinal components is far higher than that of the phellinus igniarius, the syringia amurensis and other miscellaneous yellows^[3]In consideration of commercial interest, most of the so-called "phellinus linteus" on the market are different in shape, have no property of the mulberry, but are sold in the shape of "phellinus linteus" or "phellinus linteus", are expensive, are difficult to identify by consumers, and gradually cause market confusion.

These so-called "phellinus linteus" can be basically classified into two categories: one is the fruiting body of Hymenochaetaceae (Hymenochaetaceae), such as: fumonisin yellow, honeysuckle yellow, oak yellow, poplar yellow and the like belonging to the genus of the genus fumonisin; another group are fruiting bodies of the family Polyporaceae (Polyporaceae), such as: rhus verniciflua Stokes belonging to Phellinus igniarius, Pinus densiflora belonging to Phellinus spp, and part of miscellaneous Phellinus igniarius.

In many documents, yellow ansiolus mushrooms growing on non-mulberry trees are not strictly defined by "phellinus linteus": in many research texts, the two types of yellow ansamitocybe are also mistaken for phellinus igniarius, and the picture of the true habitat of phellinus igniarius is rarely provided. The classification of Phellinus linteus is also once confused. Until 2014, some have found that phellinus igniarius is an independent new genus^[4]And, only Phellinus linteus grown on Morus alba, i.e. "Phellinus linteus" (Sanghuangpolus sanghuang) is authentic Phellinus linteus^[3]. The confusion of the market and the academic world on the knowledge of the phellinus igniarius at present severely restricts the development of the old and magical Chinese medicament phellinus igniarius in China, so that the phellinus igniarius is difficult to become a stable Chinese medicament formula.

In view of the outstanding medicinal value and the current situation of the phellinus igniarius, a simple and feasible method for identifying the phellinus igniarius is urgently needed to be established, the abuse of the phellinus igniarius can be gradually reduced by clearing what is the real phellinus igniarius, the phellinus igniarius is a very important medicinal fungus, the research and the application of the phellinus igniarius, which is a precious Chinese medicinal material, are really promoted, and the contribution is made to the promotion of the Chinese traditional medicine and pharmacy.

The mulberry is widely distributed and has strong viability, and living mulberry is not easy to be parasitized by edible fungi; mulberry is rich in latex, contains various alkaloids for inhibiting biological activity of glycosidase, and is a defensive substance toxic to most herbivorous insects^[5]. Silkworm (Bombyx mori L.) has been completely adapted to eating mulberry leaves after the evolution of billions of years, and the relation between silkworm and mulberry is typical of the co-evolution in nature, with the mulberry leaves as the only feed.

Silkworm excrement (silkworm excrement) is excrement generated after silkworm larvae eat mulberry leaves, is dark green and hexagonal, has natural faint scent, is a traditional Chinese medicine, and is commonly used as a baby headrest by people. Silkworm excrement has a dual effect on microorganisms: on one hand, compared with the conventional PDA culture medium for culturing edible fungus strains, the silkworm excrement has similar saccharides, fat, crude fiber, protein, vitamins and trace elements, and has higher nutritive value than alfalfa (dry powder), rice bran and wheat bran in a certain sense, so that the silkworm excrement can meet the normal growth requirement of the edible fungus strains^[6,7]. On the other hand, silkworm excrement also contains a plurality of antibacterial and antiviral bioactive substances. Phenols such as pectin, cecropin, lactone, 1-Deoxynojirimycin (DNJ), tannin, etc^[8-10]. Chlorophyll and its derivatives in faeces Bombycis have certain antimicrobial effect^[11,12]. Silkworm excrement contains unique components of chlorophyll and its derivatives, lutein, phytol (sterol), alkaloid, adrenocorticoid, uric acid (silkworm excrement), various nitrogen-free extracts, n-daucosterol, coumarin, etc.

Silkworm, which is a typical completely abnormal lepidopteran insect, develops from one to five years, particularly immediately before the five years of aging and mounting, internal organs drastically change, and the composition and content of substances in excrement (excrements) discharged are also greatly changed compared with those of excrements at 1 to 4 years of age.

Since the silkworms and the phellinus igniarius take the mulberry as a nutrient source in the evolution process of hundreds of millions of years and generate adaptability to the mulberry, the silkworm excrement is excrement of the silkworms, and the adaptability of the phellinus igniarius is detected by using the silkworm excrement, so that the simple and efficient phellinus igniarius identification culture medium is prepared, and has very important theoretical research and practical application values.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for identifying phellinus igniarius growing on mulberry, namely the phellinus igniarius and other phellinus igniarius (sporocarp which is easy to be confused with phellinus igniarius).

The technical scheme is as follows: in order to solve the technical problems, the invention adopts the following technical scheme:

a culture medium for identifying Phellinus Linteus comprises faeces Bombycis.

Preferably, the silkworm excrement is the silkworm excrement of the day before the 5 th instar of the silkworms are tufted, and the silkworm excrement is required to be healthy and pure (only a small amount of crushed mulberry leaves eaten by the silkworms are contained, and no silkworm breeding medicines such as anti-stiff powder and lime are contained).

Further, comprising: silkworm excrement powder KH₂PO₄、MgSO₄And water.

Wherein the faeces Bombycis powder and KH₂PO₄、MgSO₄The mass ratio of water is 41.25: 0.3: 0.15: 58.3 and the pH value is 8.5-8.8.

The preparation method of the culture medium for identifying the phellinus igniarius comprises the following steps:

(1) taking silkworm excrement of the day before the 5 th-age silkworm is cocooning;

(2) sieving fresh faeces Bombycis with 5 mesh sieve, and removing larger crushed folium Mori;

(3) drying at 60 deg.C for 10 hr, air-separating with electric fan, and manually selecting with tweezers to remove small folium Mori debris;

(4) placing on a 150-mesh sieve, washing with clear water once, and removing fine folium Mori debris attached to the surface of faeces Bombycis again to obtain pure 5-year-old silkworm faeces before cocooning;

(5) drying 5-instar silkworm excrement at 60 deg.C for 1 hr, and pulverizing with pulverizer to obtain pure 5-instar dry silkworm excrement powder;

(6) preparing a mulberry phellinus igniarius identification culture medium: weighing above dry faeces Bombycis powder, adding water and KH₂PO₄And MgSO₄Standing at room temperature for 40min to thoroughly absorb water, stirring, and adjusting pH with NaOH to obtain silkworm excrement powder and KH₂PO₄、MgSO₄The mass ratio of water is 41.25: 0.3: 0.15: 58.3, and the PH value of the culture medium is 8.5-8.8; obtaining the mulberry phellinus igniarius identification culture medium.

A method for identifying phellinus igniarius comprises the following steps:

(1) transferring the strain to be detected to a mulberry phellinus igniarius identification culture medium, and sealing with a sealing film;

(2) completely dark, standing and culturing at 28 deg.C for 10-12 days;

(3) under the culture conditions, only phellinus igniarius hyphae can normally grow in the culture medium, and other strains cannot normally grow in the phellinus igniarius identification culture medium.

The detailed steps for identifying phellinus igniarius in the invention are as follows:

(1) firstly, field investigation is carried out, and sporophores of the phellinus igniarius and various sporophores which are easy to be confused with the phellinus igniarius are collected (figure 1);

(2) the above various kinds of fruiting body strains were isolated and temporarily stored in PDA medium (FIG. 2A). The above strains can grow smoothly in PDA culture medium, and it is impossible to accurately distinguish which is true Phellinus igniarius only from the aspects of color, form, smell, etc.

(3) Taking the silkworm excrement of a day before the 5 th instar of a fresh silkworm is tufted, and requiring that the silkworm is healthy and disease-free and the silkworm excrement is pure (only containing a small amount of crushed mulberry leaves eaten by the silkworm, and no silkworm breeding medicines such as anti-stiff powder, lime and the like);

(4) primarily screening fresh silkworm excrement by using a sieve to remove larger crushed mulberry leaves;

(5) after drying, selecting again by electric fan wind, and manually selecting again by using tweezers to remove smaller mulberry leaf fragments;

(6) washing with clear water once, and further removing fine folium Mori debris attached to surface of faeces Bombycis to obtain pure 5-year-old silkworm faeces (except faeces Bombycis, without any other impurities).

(7) Drying again and then crushing by a crusher to obtain pure dry silkworm excrement powder before mounting at 5 ages;

(8) preparing a mulberry phellinus igniarius identification culture medium: weighing above dry faeces Bombycis powder, adding water and KH₂PO₄And MgSO₄Standing at room temperature for 40min to thoroughly absorb water, stirring, and adjusting pH with NaOH to obtain silkworm excrement powder and KH₂PO₄、MgSO₄The mass ratio of water is 41.25: 0.3: 0.15: 58.3, the PH value is 8.5-8.8;

(9) placing the above culture medium raw materials in a culture dish, spreading a layer, sterilizing at high temperature and high pressure, and placing in a super clean bench for cooling.

(10) Selecting various strains growing in the PDA culture medium, transferring into the identification culture medium, and standing and culturing in the dark at 28 deg.C for 10-12 days;

(11) under the above culture conditions, only Phellinus linteus mycelium can grow normally in the above culture medium, but the species parasitized by other strains to be tested are not Phellinus linteus, and can not grow normally in Phellinus linteus identification culture medium (FIG. 2B);

(12) storing Phellinus Linteus strain in the above identification culture medium for a long time, and culturing for 5 years without any degeneration and variation of Phellinus Linteus strain (FIG. 3); the pure mulberry twig is used for preparing a fruiting body fungus bag (without any additive substance), and the phellinus igniarius fruiting body of the mulberry can be smoothly cultured without rejuvenation (figure 4).

Has the advantages that: the invention is based on the coevolution relationship between the silkworm and the mulberry and between the phellinus igniarius and the mulberry for the first time, and the culture medium which is really suitable for the identification of the phellinus igniarius is manufactured by improving the 5-instar silkworm excrement. The invention has the following advantages: can intuitively and efficiently distinguish whether most yellow ansiola mushrooms on the market are real phellinus igniarius or not. The invention can overcome the difficulty that most people do not see the real wild phellinus igniarius and can not identify the phellinus igniarius from the appearance of the fruit body; the difficulty that various strains can grow smoothly on a PDA culture medium and cannot be distinguished accurately from the aspects of hypha color, form, smell and the like can be overcome; the culture medium is a green culture medium, the adopted identified raw material (silkworm excrement) is simple to obtain, natural, environment-friendly and non-toxic, and the culture medium is a reuse of silkworm breeding waste and has low cost; the identification method is simple, has strong operability and does not need to use more laboratory detection equipment. The culture medium formula for identifying phellinus igniarius provided by the invention can not only identify most of non-phellinus igniarius on the market, but also serve as an excellent culture medium for long-term culture and preservation of phellinus igniarius strains, and the phellinus igniarius strains are preserved for a long time without any phenomena of degradation, variation and the like, so that the phellinus igniarius sporocarp can be cultured smoothly without rejuvenation.

Drawings

FIG. 1 shows the wild collected Phellinus igniarius sporophore and various sporophores which are easily confused with Phellinus igniarius.

1. Cymoxanil yellow (gilin); 2. honeysuckle flower (tibet); 3. oak yellow (Zhejiang); 4. poplar yellow (Guizhou); 5. poplar yellow (Liaoning); 6. phellinus linteus (phellinus linteus) (Yunnan); 7. rhus verniciflua (Hunan); 8. pine yellow (tibet).

FIG. 2 shows the cultivation and identification of various strains. A: various species stored on PDA medium, B: the growth state of various strains in the phellinus igniarius identification culture medium. 1. Rhus verniciflua (Hunan); 2. pine yellow (tibetan); 3. pine yellow (Hunan); 4. martin yellow (Hubei); 5. cymoxanil yellow (gilin); 6. romantic yellow (black dragon river); 7. honeysuckle flower (tibet); 8. oak yellow (Zhejiang); 9. poplar yellow (Guizhou); 10. poplar yellow (Liaoning); 11. phellinus linteus (phellinus linteus) (Yunnan); 12. phellinus linteus (Phellinus linteus) (Tibetan).

FIG. 3 shows the growth of Phellinus linteus strain in Phellinus linteus identification medium for a long period (5 years) of culture (complete darkness).

FIG. 4 shows the fruiting body of Phellinus linteus induced by the culture medium stored in the culture medium for identifying Phellinus linteus.

Detailed Description

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

Example 1 isolation and culture of Phellinus Linteus and other bacterial species

(1) Yellow ansamitocybe (table 1) which is frequently mistaken for phellinus linteus in documents and networks; phellinus igniarius sporophore collected in field and various sporophore which is easy to be confused with Phellinus igniarius are shown in figure 1.

Table 1: phellinus igniarius and various sporophore which is easy to be confused with Phellinus igniarius

(2) Separating the above sporophore strains, and temporarily storing in PDA culture medium. The above strains can grow smoothly in PDA culture medium, and it is impossible to accurately distinguish which of them is true Phellinus linteus (FIG. 2A) only from the aspects of color, form, smell, etc.

Example 2 preparation of culture Medium for identifying Phellinus linteus

(1) Taking the silkworm excrement of a day before the 5 th instar of a fresh silkworm is tufted, and requiring that the silkworm is healthy and disease-free and the silkworm excrement is pure (only containing a small amount of crushed mulberry leaves eaten by the silkworm, and no silkworm breeding medicines such as anti-stiff powder, lime and the like);

(2) sieving fresh faeces Bombycis with 5 mesh sieve, and removing larger crushed folium Mori;

(3) drying at 60 deg.C for 10 hr, air-separating with electric fan, and manually selecting with tweezers to remove small folium Mori debris;

(4) placing on a 150-mesh sieve, washing with clear water once, and further removing fine folium Mori debris attached to surface of faeces Bombycis to obtain pure 5-year-old silkworm faeces (except faeces Bombycis, without any other impurities).

(5) Oven drying at 60 deg.C for 1 hr, and pulverizing with pulverizer (stainless steel traditional Chinese medicine pulverizer, Bjie BJ-800A) to obtain pure dry faeces Bombycis powder before mounting at 5 years old;

(6) preparing a mulberry phellinus igniarius identification culture medium: weighing above dry faeces Bombycis powder, adding water and KH₂PO₄And MgSO₄Standing at room temperature for 40min to thoroughly absorb water, stirring, and adjusting pH with NaOH to obtain silkworm excrement powder and KH₂PO₄、MgSO₄The mass ratio of water is 41.25: 0.3: 0.15: 58.3, PH 8.5-8.8;

(7) placing the above culture medium raw materials in a culture dish, spreading a layer (5-6 g for each 9cm culture dish bottom), sterilizing at 121 deg.C for 30min, and cooling on a super clean bench.

Example 3 identification of Phellinus linteus

(1) And (3) selecting various strains growing in the PDA culture medium, transferring the strains to the mulberry phellinus igniarius identification culture medium, and sealing with a sealing film.

(2) Completely dark, and standing and culturing at 28 deg.C for 10-12 days.

(3) Under the above culture conditions, only Phellinus linteus mycelium grew normally in the above culture medium, but neither of the species parasitized by other species tested was Phellinus linteus, and it could not grow normally in Phellinus linteus identification medium (FIG. 2B).

(4) Culturing Phellinus Linteus strain with the above identification medium for a long time, and culturing Phellinus Linteus strain without degeneration and mutation within 5 years (FIG. 3), and culturing Phellinus Linteus fruiting body without rejuvenation by making fruiting body bag with pure ramulus Mori (without any additive substance) (FIG. 4).

Reference documents:

1.Ikekawa T:Antitumor action of some Basidiomycetes,especially Phellinus linteus(Notes).Gann 1968,59(2):155.

2. bahaiying, Yangxing, Liqingjie, Tulipoul, Liyu, the herbal supplement of "sanghuang" was examined, and the fungus study 2017.

3. Wu-Shen-Hua, Huangguan, Chen Happy, Daiyehuan, Zhou-Li Wei and sang Huang classification and development prospects, and bacterium research 2016(4).

4. Daiyou, Tempoy, research on the kind of medicinal fungus Phellinus linteus, university of Beijing and forestry 2014(5) 1-6.

5.Konno K,Ono H,Nakamura M,Tateishi K,Hirayama C,Tamura Y,Hattori M,Koyama A,Kohno K:Mulberry latex rich in antidiabetic sugar-mimic alkaloids forces dieting on caterpillars.Proceedings of the National Academy of Sciences 2006.

6. Comprehensive utilization of faeces Bombycis in Osmanthus, Zhangyuan, feed research 2007(6), 000070-.

7. Preparing pectin, sodium zinc chlorophyllin and leaf protein from faeces Bombycis, Guangxi university; 2014.

8. liuling, namely preparing sodium copper chlorophyllin and extracting effective components in the sodium copper chlorophyllin by using silkworm excrement, which is university of Guangxi; 2007.

9.Asano N,Nash R,Molyneux R,Fleet G:Sugar-mimic glycosidase inhibitors:natural occurrence,biological activity and prospects for therapeutic application.2000,11(8):1645-1680.

10. chenweidong, Xiaogeng, Liaoxingtai, sericulture resource substance research and development progress Chinese herbal medicine 1999(9) 52-55.

11. Sunbbo, Li Yong, Wuhongli, Wuhui, silkworm excrement and the research progress of the extraction process, China silkworm 2009,30(4):18-21.

12. The research on chemical components of the silkworm excrement, namely, the strong tin, the Li xing Cui, the Wang Lei, the Chen Run, the Chinese traditional medicine journal 2008(21) 2493 and 2496.

Claims (6)

1. A culture medium for identifying phellinus igniarius of a mulberry tree, which is characterized by comprising the following components: silkworm excrement powder KH₂PO₄、MgSO₄And water.

2. The medium for identifying Phellinus linteus according to claim 1, wherein the faeces powder is faeces Bombycis powder prepared from faeces Bombycis of the previous day of mounting at 5-year-old silkworm.

3. According to the rightThe medium for identifying Phellinus Linteus of claim 1, wherein the culture medium comprises faeces Bombycis powder and KH₂PO₄、MgSO₄The mass ratio of water is 41.25: 0.3: 0.15: 58.3 and the pH value is 8.5-8.8.

4. The preparation method of the culture medium for identifying the phellinus igniarius is characterized by comprising the following steps of:

(1) taking silkworm excrement of the day before the 5 th-age silkworm is cocooning;

(2) primarily screening, and removing mulberry leaves;

(3) drying at 60 deg.C for 10 hr, selecting faeces Bombycis, and removing impurities;

(4) placing in a 150-mesh sieve, washing with clear water once, and removing fine folium Mori debris attached to surface of faeces Bombycis again to obtain pure 5-year-old silkworm faeces before mounting;

(5) drying 5-instar silkworm excrement at 60 deg.C for 1 hr, and pulverizing with pulverizer to obtain pure 5-instar dry silkworm excrement powder;

(6) preparing a mulberry phellinus igniarius identification culture medium: weighing above dry faeces Bombycis powder, adding water and KH₂PO₄And MgSO₄Standing at room temperature for 40min to thoroughly absorb water, stirring, and adjusting pH with NaOH to obtain silkworm excrement powder and KH₂PO₄、MgSO₄The mass ratio of water is 41.25: 0.3: 0.15: 58.3, and the PH value of the culture medium is 8.5-8.8; obtaining the mulberry phellinus igniarius identification culture medium.

5. A method for identifying phellinus igniarius is characterized by comprising the following steps:

(1) transferring the strain to be detected to the mulberry phellinus igniarius identification culture medium prepared according to claim 4, and sealing the culture medium with a sealing film;

(2) culturing under the condition of complete darkness;

(3) under the culture conditions, only phellinus igniarius hyphae can normally grow in the culture medium, and other strains cannot normally grow in the phellinus igniarius identification culture medium.

6. The method for identifying Phellinus linteus according to claim 5, wherein in step (2), the culture conditions are static culture at 28 ℃ for 10-12 days.

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