Phellinus igniarius strain rich in polysaccharide and general flavone as well as cultivation method and application of phellinus igniarius strain

Abstract

The invention provides a phellinus igniarius strain rich in polysaccharide and total flavone as well as a cultivation method and application thereof, relates to the technical field of biology, and aims to solve the problems that a phellinus igniarius variety with high yield and high polysaccharide and total flavone content is lacked in the prior art, and an existing cultivation method is difficult to meet the effects of high yield and income increase. The phellinus igniarius strain disclosed by the invention is phellinus igniarius, is named as Harmulberry No.1, and is preserved in China General Microbiological Culture Collection Center on October 31, 2024, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.41588. In the cultivation process of the phellinus igniarius sporocarp, a cultivar culture medium is prepared from, by mass, 78% of oak sawdust, 15% of trametes robiniophila cultivation waste, 5% of corn flour, 1% of gypsum powder and 1% of cane sugar. The phellinus igniarius variety disclosed by the invention has potential development and application values and can be used for preparing health-care foods or medicines.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a strain of Phellinus igniarius rich in polysaccharide and total flavonoids, and a cultivation method and application thereof. Background Art

[0002] Phellinus igniarius belongs to the phylum Basidiomycetes, class Agaricaceae, order Psoriatica, family Psoriatica, genus Phellinus, also known as mulberry yellow mushroom, mulberry yellow mushroom, etc. Phellinus igniarius has a long history of medicinal use and has been used in the field of traditional Chinese medicine for more than 2,000 years. It has a wide range of physiological activities and medicinal value and is known as "forest gold". At present, there are traditional Chinese medicine products such as mulberry yellow pills and mulberry yellow oral liquid on the market, which shows that it has high health and medicinal value.

[0003] With the rapid development of the mulberry mushroom industry in recent years, the requirements for the quality of mulberry mushroom strains are becoming higher and higher. The existing technology lacks high-quality mulberry mushroom varieties with high yield, high polysaccharide and total flavonoids content; the survival rate of wild domesticated mulberry mushroom is low, and the collection process is prone to pollution, which brings inconvenience to the wild domestication of mulberry mushroom; and the existing mulberry mushroom cultivation materials are mostly based on mulberry sawdust, and use relatively expensive auxiliary materials such as wheat bran and corn flour, which increases production costs. Summary of the invention

[0004] The problem to be solved by the present invention is: The existing technology lacks mulberry linterus varieties with high yield and high polysaccharide and total flavonoid content, and the existing cultivation methods are difficult to achieve the effects of high yield and increased income.

[0005] The present invention adopts the following technical solutions to solve the above technical problems: The present invention provides a strain of Phellinus linteus, which is Phellinus linteus ( Sanghuangporus sp. ), named Hassan 1, was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41588.

[0006] The present invention also provides a Phellinus linterus fruiting body for cultivating the Phellinus linterus strain.

[0007] The present invention also provides a method for cultivating the fruiting body of Phellinus igniarius, comprising the following steps: (1) inoculating the mother strain of Phellinus igniarius according to claim 1 into a liquid culture medium to obtain a liquid strain; (2) sterilizing the cultivation medium, inoculating the liquid spawn into the cultivation medium for spawning, opening the fungus bag when the mycelium is fully grown, and then performing fruiting management, and finally harvesting the fruiting bodies of Phellinus igniarius to obtain finished products; The cultivation culture medium comprises, by mass ratio, 78% of oak sawdust, 15% of Sophora japonica cultivation waste, 5% of corn flour, 1% of gypsum powder and 1% of sucrose.

[0008] Furthermore, in step (2), the mushroom bag is opened in a circular manner around the mushroom bag.

[0009] Furthermore, in step (2), the cultivated seeds are placed in a rice seedling raising shed for mushroom production management, the temperature for mushroom production management is 24-28° C., the relative humidity of the air is 85%-95%, and sufficient scattered light and oxygen are provided.

[0010] The present invention also provides an application of the Phellinus igniarius fruiting body in the preparation of health-care food or medicine.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains the Phellinus igniarius variety Hasan No. 1 through wild domestication. The mycelium of the strain grows robustly, has good growth potential, and has a high fruiting body yield. The polysaccharide and total flavonoids contents of the fruiting bodies reach 6.72% and 86.85% respectively, while the polysaccharide content of the existing Phellinus igniarius varieties is generally only 0.5%-5%, and the total flavonoids content is generally only 5%-60%. The Phellinus igniarius variety of the present invention has a strong ability in synthesizing polysaccharides and total flavonoids, has potential development and application value, and can be used for preparing health foods or medicines.

[0012] The present invention not only can utilize wastes, but also can greatly save production costs by adding waste residues of Sophora japonica fungus to the culture medium during the cultivation and management process, and is helpful to further increase the polysaccharide content of Phellinus igniarius; at the same time, the adopted annular opening method of the mushroom bag, compared with the traditional crescent-shaped opening method, expands the mushroom production area, and is helpful to effectively utilize the nutrients in the cultivation material; and the secondary utilization of the rice seedling raising shed during the cultivation process of the present invention increases the varieties that can be planted in the rice seedling raising shed, and also greatly increases the planting income. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a picture of the fruiting body of Hassan No. 1 in an embodiment of the present invention; Figure 2 Schematic diagram of the structure of the sterile box in an embodiment of the present invention; Figure 3 Schematic diagram of the structure of a simple mouth opener in an embodiment of the present invention. DETAILED DESCRIPTION

[0014] In order to enable those skilled in the art to better understand the scheme of the present invention, exemplary implementations or embodiments of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described implementations or embodiments are only implementations or embodiments of a part of the present invention, not all of them. Based on the implementations or embodiments of the present invention, all other implementations or embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.

[0015] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0016] Embodiment 1:

[0017] 1. Strain Collection The fungus was collected from Laoyeling, Shangzhi County. Ten wild Phellinus igniarius strains were collected. Through separation and domestication, a strain with strong mycelium growth, good growth and high content of active ingredients was obtained, named Hasan No. 1. The specific method is as follows: After collecting wild Phellinus linteus fruiting bodies, simply disinfect the surface, brush off surface impurities with a clean brush, wipe the solid outer surface with 75% alcohol 5 times for disinfection, use a scalpel to cut the fruiting bodies longitudinally, put them in a simple sterile box, and transport them back to the laboratory for subsequent wild fruiting body extraction. Figure 2 As shown, the sterile box is a square closed container with fixed piles inside, and the Phellinus linteus fruiting bodies are fixed in the sterile box by piercing the fruiting bodies with the fixed piles. This method can make the cross section of the Phellinus linteus fruiting bodies continue to grow during transportation, reduce contamination, and improve the survival rate.

[0018] The invention improves the wild resource collection tools, cuts the fruiting bodies in the wild, and allows the cross-sections of the Phellinus igniarius fruiting bodies to continue to grow during transportation, thereby achieving mycelium rejuvenation, reducing the wild resource pollution rate and improving the wild domestication survival rate.

[0019] 2. Strain Isolation 2.1. Culture medium preparation Culture medium formula: 200 g potato (peeled), 20 g glucose, 2 g peptone, 2 g yeast powder, 20 g agar and 1000 ml water, natural pH.

[0020] Weigh 200g of peeled potatoes, cut into thin slices, wash, add 1000ml of water and boil, boil for another 20min after the water boils, then filter with gauze, take the filtrate, if the filtrate is less than 1000ml, add 1000ml to make up, then add 20g of agar and cook until melted, then add 20g of glucose, 2g of peptone and 2g of yeast powder to melt, divide into test tubes for sterilization while hot, the test tubes should be filled with about one-fifth to one-quarter of the height, seal the test tubes with cotton plugs, and autoclave at 121℃ for 20min. After sterilization, place on a slant for later use.

[0021] 2.2. Tissue Isolation Take out the rejuvenated wild Phellinus linteus fruiting bodies, rinse them several times with sterile water in a sterile room, and use sterile paper to fully absorb the surface moisture, and then soak them in 0.1% mercuric chloride for 5 minutes. After taking them out, rinse them several times with sterile water (you can also directly apply 75% alcohol on the surface of the fruiting bodies) to disinfect the surface. Then, quickly pick up the wild Phellinus linteus fruiting bodies about 0.5cm above the flame of the alcohol lamp with an inoculation needle. 2 The fruiting body tissue blocks were inoculated on the test tube slant culture medium, and 5 test tubes were separated for each fruiting body.

[0022] 2.3. Cultivation The inoculated test tubes were placed in a constant temperature incubator at 25°C and numbered SH-1 to SH-10.

[0023] 3. Mycelial Growth Test 3.1. Culture medium preparation Plate culture medium formula: 200g potato, 20g glucose, 20g agar and 1000ml water.

[0024] Weigh 200g of peeled potatoes, cut into thin slices, wash, add 1000ml of water and boil, boil for another 20min after the water boils, then filter with gauze, take the filtrate, if the filtrate is less than 1000ml, add 1000ml, then add 20g of agar and cook until melted, then add 20g of glucose and melt, autoclave at 121℃ for 20min, after sterilization, pour into a sterile plate with a diameter of 9cm in the clean bench for use.

[0025] 3.2. Inoculation and culture Take 0.5cm on the slope of the test tube 2 The mother culture blocks were inoculated on the plate culture medium (d=9cm) and cultured at a constant temperature of 25℃. When the mycelium covered the plate, a 0.7cm diameter puncher was used to take out the uncontaminated blocks of the same age and size and transferred them to the center of the plate culture medium (d=9cm). The mycelium was cultured at a constant temperature of 25℃. After the mycelium germinated, the growth, uniformity and color of the mycelium were observed. The results are shown in Table 1.

[0026] Table 1 Strain number Growth Uniformity color SH-1 ++++ tidy yellow SH-2 ++ tidy Light yellow SH-3 ++ tidy tan SH-4 +++ tidy yellow SH-5 +++ tidy yellow SH-6 + irregular tan SH-7 ++ tidy Light yellow SH-8 + irregular tan SH-9 + irregular Light yellow SH-10 ++ tidy yellow Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0027] The Phellinus igniarius strains with robust mycelium growth and good growth were selected and numbered as SH-1, SH-4 and SH-5.

[0028] Embodiment 2: 1. Culture medium optimization 1.1 Liquid culture preparation Liquid culture medium formula: 100g potato, 50g cottonseed hull, 15g glucose, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.3ml glycerol and 1000ml water; pH is 5-6.

[0029] Weigh 100g of peeled potatoes, cut into thin slices, wash, add 1000ml of water and boil, boil for 5min after the water boils, add 50g of cottonseed hulls, boil for another 15min, then filter with gauze, take the filtrate, if the filtrate is less than 1000ml, add 1000ml, then add 15g of glucose, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate and 0.3ml of glycerol to melt, divide into Erlenmeyer flasks while hot, the filling amount is about two-fifths of the volume of the Erlenmeyer flask, and autoclave at 121℃ for 20min to obtain liquid culture medium.

[0030] Use a hole punch to take 5-8 pieces of 0.5cm 2 The mother culture block is inoculated into a liquid culture medium, and cultured at 20-25° C. and 140-160 r / min with shaking to obtain a liquid culture medium.

[0031] 1.2 Cultivar preparation Cultivation medium A: 78% mulberry sawdust, 15% wheat bran, 5% corn flour, 1% gypsum powder and 1% sucrose.

[0032] Select fresh, mold-free culture medium, put wheat bran, corn flour and gypsum powder together in proportion and mix them evenly, then add mulberry sawdust in proportion, put sucrose into water to melt, stir the mixed dry material and sucrose water evenly, prepare the cultivation medium (water content 60wt%), put it into 17x35cm sized bacteria bags, put 1.5kg of bacteria medium in each bag, sterilize it at normal pressure for 12h, and let it stand until the temperature drops to 25℃.

[0033] Cultivation medium B: 78% oak sawdust, 15% Sophora japonica fuciformis cultivation material, 5% corn flour, 1% gypsum powder and 1% sucrose.

[0034] Choose fresh, mold-free culture medium, put wheat bran, corn flour and gypsum powder together in proportion and mix them evenly, then add oak sawdust in proportion, put sucrose into water to melt, stir the mixed dry material and sucrose water evenly, prepare the cultivation medium (water content 60wt%), put it into 17x35cm bags, put 1.5kg of medium into each bag, sterilize it at normal pressure for 12h, and let it stand until the temperature drops to 25℃.

[0035] Among them, the Sophora japonica cultivation material in the cultivation medium B formula is the waste residue of Sophora japonica AS-7, Sophora japonica AS-7 (deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 24, 2022, with the deposit number CGMCC No.40140.).

[0036] Under sterile conditions, liquid strains were inoculated into culture medium A and B, and then moved into the greenhouse for uniform culture. The culture conditions were: temperature 22-28°C, relative air humidity 50-60%, good ventilation, and light avoidance. The results of bag filling time, mycelial growth and uniformity of the three strains are shown in Table 2.

[0037] Table 2

[0038] Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0039] The results in Table 2 show that when the waste residue of Sophora japonica AS-7 is added to the culture medium, the time for Phellinus igniarius to fill the bag is shorter, and the mycelium grows denser and stronger. In addition, the use of oak sawdust instead of mulberry sawdust can reduce the cultivation cost.

[0040] 2. Optimization of opening method Opening method 1: When the mycelium is fully grown, use a blade (sterilize it in boiling water for 30 minutes before use) to cut a crescent-shaped incision on one side of the bag full of mycelium, and place it upright in the mushroom greenhouse.

[0041] Opening method 2: When the mycelium is fully grown, use a simple opener to make a circular incision around the bag and place it upright in the greenhouse. Figure 3 As shown, the simple opener is equipped with two parallel blades with a distance of 3-5 cm, which cut a circle at the upper third of the mushroom bag, and finally achieve ring-shaped mushroom production.

[0042] The bags of Phellinus igniarius with different opening methods were managed for fruiting, with a temperature of 24-28°C, a relative humidity of 85%-95%, and sufficient scattered light and oxygen. When the cap began to become leathery and yellow-brown mist-like spores were ejected from the back, they were harvested. The results are shown in Table 3.

[0043] Table 3 SH-1 output (g / bag) SH-4 output (g / bag) SH-5 output (g / bag) Opening method 1 29.7 23.8 25.4 Opening method 2 74.3 51.4 63.1 The results in Table 3 show that after the Phellinus ignia bags were ring-cut for export, the fruiting body yield increased significantly. Ring-cutting for export can make full use of the nutrients in the cultivation material in the Phellinus ignia bags and increase the yield.

[0044] Embodiment 3: 1. Cultivar Preparation SH-1, SH-4, SH-5 and existing varieties SH-BJ (Beijing Jixunyuan Edible Fungi Research Institute) and SH-DB (Northeast Edible Fungi Research Institute) were used as test materials. Cultivated seeds were prepared according to the cultivar preparation method in Example 2. The cultivar culture medium was cultivar culture medium B, and the opening method was opening method 2.

[0045] 2. Rice seedling cultivation experiment Cultivated varieties of SH-1, SH-4, SH-5 and existing varieties SH-BJ and SH-DB were prepared and placed in an idle greenhouse after rice seedling cultivation for mushroom production. During the cultivation process in the rice seedling greenhouse, the temperature was 24-28°C, the relative humidity of the air was 85%-95%, and sufficient scattered light and oxygen were provided. The humidification method used drip irrigation belts to moisten the ground and increase the air humidity by transpiration, so as to reduce the pollution caused by direct humidification of open water, save labor and humidification facilities, and reduce costs.

[0046] When the cap begins to become leathery and the back ejects yellow-brown mist-like spores, it is harvested. The yield, fruiting body color, and contamination rate of SH-1, SH-4, SH-5 and existing varieties SH-BJ and SH-DB were tested, and the results are shown in Table 4.

[0047] Table 4 variety Pollution rate (%) Mushroom uniformity Fruiting body color Output (g / bag) SH-1 2 +++ yellow 71.5 SH-4 9 ++ yellow 48.1 SH-5 4 +++ yellow 64.7 SH-BJ 16 ++ yellow 43.9 SH-DB 23 ++ yellow 46.8 Among them, in the uniformity of mushroom production, "+" means uneven mushroom production, "++" means relatively uniform mushroom production, and "+++" means very uniform mushroom production. Table 4 shows that compared with the mainstream varieties SH-BJ and SH-DB in the prior art, the mulberry ignia variety SH-1 has uniform mushroom production, low contamination rate and significantly improved yield when planted in rice seedling greenhouses.

[0048] 3. Active ingredient content determination test The secondary metabolite content of SH-1, SH-4, SH-5 and the existing varieties SH-BJ and SH-DB was determined. The polysaccharide, total flavonoids, total phenols and triterpenoid contents of the samples were determined according to the method of Wang Weike et al. (Wang Weike, Lu Na, Yan Jing, et al. Effects of growth years on the nutrition, active ingredients and antioxidant activity of Baumbergia lintei fruiting bodies cultivated in logs [J]. Journal of Mycology, 2021, 40(03): 668-680.). The glucose standard curve equation was γ1=0.01χ1-0.0138 (R 2 =0.9984); the equation of the rutin standard curve was γ2=0.003χ2-0.015 (R 2 =0.9997); The equation of the gallic acid standard curve was γ3=0.0193χ3+0.0145 (R 2 =0.9990); the equation of the oleanolic acid standard curve was γ4=0.0073χ4-0.01 (R 2 =0.9970). The test results are shown in Table 5.

[0049] Table 5 Test items SH-1 SH-4 SH-5 SH-BJ SH-DB Polysaccharide (%) 6.72 4.09 3.26 3.85 3.60 Total flavonoids (%) 86.85 29.76 34.17 24.09 22.47 Total phenols (%) 17.71 9.92 16.31 10.43 8.59 Triterpenes (%) 2.14 1.55 1.37 1.92 1.26 As shown in Table 5, the polysaccharide content and total flavonoids content in the mulberry ignia variety SH-1 are significantly higher than those in SH-4, SH-5 and the existing varieties SH-BJ and SH-DB, and are more prominent than the existing technology. At the same time, the total phenol and triterpene content are also higher than other varieties.

[0050] 4. Identification and preservation Morphological identification The fruiting body of the SH-1 variety of Phellinus igniarius is sessile, smooth on the surface, and covered with short yellow hairs; the color is bright yellow during the growth period, and becomes darker and yellow-brown during maturity ( Figure 1 ).

[0051] Molecular Biology Identification ITS sequencing was performed on the SH-1 strain. Through amplification and DNA sequencing, the nucleotide sequence of the SH-1 strain is shown in Sequence Table 1. Molecular biological identification showed that the SH-1 strain belonged to the phylum Basidiomycetes, class Agaricaceae, order Puccinellales, family Puccinellaceae, and genus Phellinus. Based on comprehensive morphological identification, the SH-1 strain belonged to Phellinus igniarius. Sanghuangporus sp. .

[0052] On October 31, 2024, the Phellinus igniarius strain SH-1 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was classified and named Phellinus igniarius, named Hassan No. 1, and the deposit number is CGMCC No.41588.

[0053] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A strain of Phellinus linteus, characterized in that The strain is Phellinus linteus ( Sanghuangporus sp. ), named Hassan 1, was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41588.

2. A method for cultivating the Phellinus linterus fruiting body of the Phellinus linterus strain according to claim 1.

3. A method for cultivating the fruiting body of Phellinus igniarius according to claim 2, characterized in that: The following steps are included: (1) inoculating the mother strain of Phellinus igniarius described in claim 1 into a liquid culture medium to obtain a liquid strain; (2) sterilizing the cultivation medium, inoculating the liquid spawn into the cultivation medium for spawning, opening the fungus bag when the mycelium is fully grown, and then performing fruiting management, and finally harvesting the fruiting bodies of Phellinus igniarius to obtain finished products; The cultivation culture medium comprises, by mass ratio, 78% of oak sawdust, 15% of Sophora japonica cultivation waste, 5% of corn flour, 1% of gypsum powder and 1% of sucrose.

4. The method for cultivating Phellinus igniarius fruiting bodies according to claim 3, characterized in that: In step (2), the mushroom bag is opened in a circular manner around the mushroom bag.

5. The method for cultivating Phellinus igniarius fruiting bodies according to claim 4, characterized in that: In step (2), the cultivated seeds are placed in a rice seedling raising shed for mushroom production. The temperature for mushroom production is 24-28° C., the relative humidity of the air is 85%-95%, and sufficient scattered light and oxygen are provided.

6. Use of the Phellinus igniarius fruiting body according to claim 2 in the preparation of health food or medicine.

Images