CN112931059A - Phellinus igniarius strain and cultivation method thereof - Google Patents

Abstract

The invention provides a phellinus igniarius strain and a cultivation method thereof, belonging to the technical field of biology. The Phellinus linteus strain is specifically Phellinus linteus QJF-2 with the preservation number of CGMCC No. 19653. The phellinus igniarius QJF-2 strain provided by the invention has high medicinal value, is authentic phellinus igniarius, and has obviously higher medicinal effect than common poplar phellinus igniarius.

Description

Phellinus igniarius strain and cultivation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a phellinus igniarius strain and a cultivation method thereof.

Background

Phellinus linteus is a rare medicinal fungus growing on mulberry, is known as forest gold, has been used for over 2000 years in China, is recorded in detail in the medical function of Phellinus linteus in the past herbal works, and has the main functions of treating dysentery, night sweat, metrorrhagia, bloody stranguria, abdominal pain, rectocele, bloody discharge, leukorrhagia, amenorrhea, diarrhea, prolonging life and the like. Modern researches show that the ganoderma lucidum polysaccharide extract has obvious effects of resisting tumors and oxidation, enhancing immunity, preventing and treating rheumatoid arthritis, reducing blood fat, inhibiting uric acid and the like, has high medicinal value, is proved to be superior to medicinal fungi such as ganoderma lucidum and the like particularly in the aspect of resisting tumors, becomes a hot spot for research and development of domestic and foreign medicinal preparations and health care products, and has good economic benefit and social benefit.

Health consciousness of modern people urges the generation of phellinus igniarius fever, the price is also raised in a step-by-step mode, wild phellinus igniarius resources are very limited, people are prompted to explore an artificial cultivation mode of phellinus igniarius, but real phellinus igniarius (Sanghuanghuan) is difficult to domesticate artificially, even if domestication is successful, the yield and quality of sporocarp are also very unstable, and large-scale artificial cultivation in the real sense is difficult to realize. At present, most of the phellinus linteus cultured successfully on the market is the phellinus linteus and phellinus linteus with the shape and appearance very similar to those of phellinus linteus, and the phellinus linteus are also commonly called phellinus linteus, but the property of the phellinus linteus is greatly different from that of the real phellinus linteus. Therefore, the artificial domestication of the real phellinus igniarius is carried out, and a high-yield and stable-yield cultivation technical system is established, so that the method has great significance for the healthy and stable development of the phellinus igniarius industry.

Disclosure of Invention

The invention provides a genuine phellinus igniarius strain (QJF-2) with good drug effect, and a bag material cultivation and cut-log cultivation method thereof, and also provides a solid cultivation method of phellinus igniarius mycelium and a liquid cultivation method of phellinus igniarius mycelium.

The invention provides a phellinus igniarius strain of mulberry,

the Phellinus linteus strain is specifically Phellinus linteus QJF-2 with the preservation number of CGMCC No. 19653.

The invention also provides a cultivation method of the phellinus igniarius strain, which comprises the following steps:

spraying microbial agent on the cut of the fungus bag with the Phellinus igniarius mycelium, and bagging to induce the Phellinus igniarius;

preferably, the microbial agent comprises a selenium-enriched microbial leaf fertilizer.

Further, the cultivation method comprises bag cultivation or wild-like cultivation.

Further, the bag cultivation method comprises the following steps:

(a1) uniformly stirring the compost, putting the compost into a fungus bag, sterilizing at high pressure, and cooling;

(a2) adding the cultivated species into the culture material obtained in the step (a1), and culturing at constant temperature in a dark place until hyphae grow over the fungus bags;

(a3) when the outdoor temperature reaches above 25 ℃, transferring the fungus bags obtained in the step (a2) into a cultivation shed for color conversion, cutting a cut on the fungus bags after the color conversion is finished, spraying a microbial agent to the cut, bagging, vertically placing on a fungus bed in the cultivation shed, and keeping the air humidity in the cultivation shed at 80% -85%;

(a4) after the cut part turns yellow, the bag is removed, the water is sprayed, the air humidity is controlled to be 85% -90%, and ventilation is carried out until harvesting.

Further, the wild-imitating cultivation comprises the following steps:

(b1) cutting the wood sections into short wood sections with the length of 17-20 cm, chopping into thin sections, splicing into wood section bundles, putting into clear water for soaking, taking out, putting into a fungus bag, filling culture materials in gaps and on the surfaces of the wood section bundles, tying fungus bag openings, and sterilizing at normal pressure to obtain fungus sticks;

(b2) adding the cultivated species into the fungus sticks obtained in the step (b1), and culturing at constant temperature in a dark place until hyphae overgrow the fungus sticks;

(b3) when the outdoor temperature reaches above 25 ℃, transferring the fungus sticks obtained in the step (b2) into a cultivation shed for color conversion, cutting a cut on the fungus sticks after the color conversion is finished, spraying a microbial agent to the cut, bagging, vertically placing on a fungus bed in the cultivation shed, and keeping the humidity of air in the shed to be 85-90%;

(b4) after the cut part turns yellow, the bag is removed, the water is sprayed, the air humidity is controlled to be 85% -90%, and ventilation is carried out until harvesting.

Further, the step (b3) further includes overwintering management, specifically: after winter, the sunshade net at the top of the cultivation shed is directly covered on the surface of the fungus stick, and the sunshade net is uncovered to the top of the cultivation shed in the next 4 months for fruiting management.

The invention also provides a solid-state culture method of the phellinus igniarius strain, which comprises the following steps:

(c1) inoculating the Phellinus linteus mycelium onto sterilized carrot or potato slices, and culturing at 28 deg.C;

(c2) and (c1) crushing the carrot slices or potato slices full of phellinus igniarius mycelia obtained in the step (c1), drying, and crushing again to obtain phellinus igniarius powder.

The invention also provides a liquid fermentation method of the phellinus igniarius strain, which comprises the following steps:

(d1) preparing a liquid fermentation culture medium, and sterilizing; wherein the liquid fermentation medium comprises carrot extract;

(d2) inoculating phellinus igniarius mycelium into the liquid fermentation culture medium obtained in the step (d1), standing at room temperature, and performing shaking culture to obtain phellinus igniarius mycelium pellets;

(d3) and (d2) filtering the phellinus igniarius mycelium pellets obtained in the step (d2), drying and crushing to obtain phellinus igniarius mycelium powder rich in carotene.

The invention also provides the phellinus igniarius powder prepared by the solid culture method.

The invention also provides phellinus igniarius hypha powder rich in carotene and prepared by the liquid fermentation method.

The invention has the following advantages:

(1) the QJF-2 strain provided by the invention has high medicinal value, is authentic phellinus igniarius, and has obviously higher medicinal effect (such as anti-tumor cell effect) than common phellinus igniarius.

(2) The bag material cultivation method and the wild-simulated cultivation method of the QJF-2 strain provided by the invention have the advantages of fast yellowing, fast spawn running, stable fruiting body development, small fluctuation of yield and quality, short production period, low management cost and the like.

(3) The phellinus igniarius mycelium powder obtained by the solid culture and liquid culture method provided by the invention contains rich carotene, terpenes, crude polysaccharide and other bioactive substances, the content of the phellinus igniarius mycelium powder is far higher than that of phellinus igniarius sporocarp, and the developed product can be directly eaten and has good taste.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

One embodiment of the invention provides a phellinus igniarius strain, which is specifically a phellinus igniarius strain

Phellinus igniarius QJF-2 with preservation number of CGMCC No. 19653.

Wild phellinus linteus is collected from mulberry forest in the lake Qiandao of Chunan county, Hangzhou, Zhejiang, and is subjected to tissue isolation culture, mycelium is collected, ITS identification and sequencing result show that the wild phellinus linteus has the highest ITS sequence homology with Sanghuangtrung.

The Phellinus linteus strain is identified as authentic Phellinus linteus (Sanghuangpolus sanghuang) by combining experimental data such as sporophore morphology, mycelium morphology, microscopic characteristics and ITS identification.

The phellinus igniarius strain is domesticated and cultivated for a plurality of times, the domesticated sporocarp is divided into components, the strain is preserved in the common microorganism center of China general microbiological culture Collection management Committee (CGMCC) in 6-month and 4-day 2020, the address is No. 3 of the Ministry of microorganisms of the national academy of sciences, the Zealand No.1 of Beijing, the Ministry of China, the postal code is 100101, the preservation number is CGMCC No.19653, the name is QJF-2, and the classification name is as follows: phellinus linteus (Sanghuangporus sanghuang).

The 736bp ITS sequence of the phellinus igniarius QJF-2 is shown in a sequence table SEQ ID No. 1.

The phellinus igniarius QJF-2 can be stably cultivated in bags and cut-log, and the effect of fruiting bodies on resisting tumor cells is obviously superior to that of phellinus igniarius of poplar.

Specifically, an embodiment of the present invention provides a method for cultivating the above-mentioned phellinus linteus strain,

the method comprises the following steps: spraying microbial agent on the cut of the fungus bag with the Phellinus igniarius mycelium, and bagging to induce the Phellinus igniarius.

In one embodiment of the invention, the microbial agent comprises a selenium-enriched microbial leaf fertilizer.

Specifically, the selenium-rich microbial foliar fertilizer can be a commercially available selenium-rich microbial foliar fertilizer under the brand name of agriculture, Hara, and concretely comprises biological fermentation products, chitosan, composite probiotics, chelated trace elements, small molecular peptides, special disease-resistant growth promoting factors, selenium and the like.

In an embodiment of the invention, the cultivation method comprises bag cultivation or wild-like cultivation.

Specifically, the bag cultivation method comprises the following steps:

(a1) uniformly stirring the compost, putting the compost into a fungus bag, sterilizing at high pressure, and cooling;

(a2) adding the cultivated species into the culture material obtained in the step (a1), and culturing at constant temperature in a dark place until hyphae grow over the fungus bags;

(a3) when the outdoor temperature reaches above 25 ℃, transferring the fungus bags obtained in the step (a2) into a cultivation shed, cutting a cut on the fungus bags, spraying a microbial agent at the cut, bagging, vertically placing on a fungus bed in the cultivation shed, and keeping the air humidity in the cultivation shed to be 80-85%;

(a4) after the cut part turns yellow, the bag is removed, the water is sprayed, the air humidity is controlled to be 85% -90%, and ventilation is carried out until harvesting.

Specifically, in the step (a1), the culture material comprises the following components in percentage by weight: 75-80% of miscellaneous wood chips, 13-18% of wheat bran, 0-3% of soybean meal, 0-2.5% of sugar, 1.5% of quicklime and 65% of water.

Further, in the step (a1), the miscellaneous wood chips are mulberry wood chips, oak (quercus mongolica) wood chips or oak wood chips.

Further, in the step (a2), the constant temperature was 28 ℃.

Further, in the step (a3), the shape of the cutting opening is crescent.

Further, in the step (a2), the cultivated species cover the material surface 1/2 of the cultivation material.

Further, in the step (a3), the sandy soil is sieved in advance.

Specifically, the wild-imitating cultivation comprises the following steps:

(b1) cutting the wood sections into short wood sections with the length of 17-20 cm, chopping into thin sections, splicing into wood section bundles, putting into clear water for soaking, taking out, putting into a fungus bag, filling culture materials in gaps and on the surfaces of the wood section bundles, tying fungus bag openings, and sterilizing at normal pressure to obtain fungus sticks;

(b2) adding the cultivated species into the fungus sticks obtained in the step (b1), and culturing at constant temperature in a dark place until hyphae overgrow the fungus sticks;

(b3) when the outdoor temperature reaches more than 25 ℃, transferring the fungus sticks obtained in the step (b2) into a cultivation shed, cutting a cut on the fungus sticks, spraying a microbial agent at the cut, bagging, vertically placing on a fungus bed in the cultivation shed, and keeping the humidity of air in the shed to be 85-90%;

(b4) spraying water after the cut turns yellow, controlling the air humidity at 85% -90%, and ventilating until harvesting.

Specifically, in the step (b1), the culture medium is a mixture of wood chips and wheat bran.

Further, in the step (b1), the constant temperature was 28 ℃.

Further, in the step (b1), the soaking time is 15-20 min.

Further, in the step (b2), the cultivated species covers above the material level 1/2.

In an embodiment of the present invention, in the method for cultivating phellinus linteus strain, the step (b3) further includes overwintering management. The overwintering management specifically comprises the following steps: after winter, the sunshade net at the top of the cultivation shed is directly covered on the surface of the fungus bag, and the sunshade net is uncovered to the top of the cultivation shed in the next 4 months for conventional fruiting management. The phellinus igniarius can be harvested only in three years, and the water spraying is stopped one week before harvesting.

In the prior art, although the phellinus igniarius sporocarp is difficult to obtain in a large quantity, mycelium of the phellinus igniarius sporocarp can be obtained in a solid or liquid fermentation mode, and researches show that the content of certain nutrient components of the phellinus igniarius mycelium is basically consistent with that of the sporocarp (Yujie, leaf, etc., 2018), so the mycelium obtained by solid or liquid fermentation has good application value and development prospect, and the situation that the phellinus igniarius sporocarp is deficient can be relieved.

At present, conventional materials such as mulberry twig sawdust, bran, cottonseed hulls and the like are mainly used as main raw materials in a solid and liquid fermentation culture medium of phellinus igniarius, and mycelia obtained by utilizing the conventional formulas have poor nutritional effects and poor mouthfeel, and are not suitable for direct eating.

An embodiment of the present invention provides a solid-state culture method of the phellinus linteus strain, including the following steps:

(c1) inoculating the Phellinus linteus mycelium onto sterilized carrot or potato slices, and culturing at 28 deg.C for 30 days;

(c2) and (c1) crushing the carrot slices or potato slices full of phellinus igniarius mycelia obtained in the step (c1), drying, and crushing again to obtain phellinus igniarius powder. The Phellinus Linteus powder is rich in carotene or nutrients (including carbohydrate, protein, vitamins, etc.) contained in potato.

The embodiment of the invention also provides the phellinus igniarius powder prepared by any one of the methods.

An embodiment of the present invention further provides a liquid fermentation method of the phellinus linteus strain, including the following steps:

(d1) preparing a liquid fermentation culture medium, and sterilizing; wherein the liquid fermentation medium comprises carrot extract;

(d2) inoculating phellinus igniarius mycelium into the liquid fermentation culture medium obtained in the step (d1), standing at room temperature, and performing shaking culture to obtain phellinus igniarius mycelium pellets;

(d3) filtering Phellinus linteus mycelium pellet, oven drying, and pulverizing to obtain Phellinus linteus mycelium powder rich in carotene.

Specifically, the liquid fermentation medium comprises 200g of carrot extract, 30g of soybean meal extract, 20g of glucose, 1.0g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 110 mg of vitamin B and 1L of water.

Specifically, in the step (d2), the mixture was left standing at room temperature for 1 day.

Further, in the step (d2), the temperature of the shaking culture was 28 ℃.

Further, in the step (d2), the rotation speed of the shaking culture is 150 rpm.

Further, in the step (d2), the period of shaking culture was 15 days.

An embodiment of the invention also provides phellinus igniarius hypha powder rich in carotene and prepared by any one of the methods.

The present invention will be described in detail with reference to examples.

Example 1Morphological characteristics and classification and identification of phellinus igniarius strain QJF-2

(1) Morphological characteristics of Phellinus linteus strain QJF-2: the second fruiting body layer and the third fruiting body layer are superposed, are in a horseshoe shape or a fan shape, are woody and hard, have dark yellow to brown fungus cap back surfaces and yellow abdomen surfaces, and are compact in texture; culturing on PDA culture medium for 14 days, wherein the diameter of colony is 90mm, the texture is villiform, yellow, and the reverse side is brown; observing the hypha with light brown color, multiple branches and diaphragm by a microscope, wherein the diameter of the hypha is 2.5-4.5 mu m. The morphological characteristics indicate that the mulberry phellinus igniarius is authentic.

(2) ITS identification of Phellinus linteus strain QJF-2

Extracting DNA of phellinus igniarius strain QJF-2 by a conventional method, and performing PCR amplification by using ITS1 and ITS4 as primers, wherein the primer sequences of ITS1 and ITS4 are as follows:

ITS1:5'--3'-TCCGTAGGTGAACCTGCGG

ITS4:5'--3'-TCCTCCGCTTATTGATATGC

the PCR amplification system is as follows: premix 12.5. mu.L, ITS1 and ITS4 each 1. mu.L, DNA template 2. mu.L, using ddH₂O was supplemented to 25. mu.L.

Amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 3min, annealing at 55 ℃ for 50S, and extension at 72 ℃ for 45S for 33 cycles; fully extend for 7min at 72 ℃.

Detecting the PCR product by 1.2% agarose gel electrophoresis, wherein the target fragment is about 700bp, sending the PCR product to Shanghai Biotech company for sequencing, and the sequence information is shown in 736bp ITS sequence of Phellinus igniarius QJF-2 in sequence table SEQ ID No. 1.

The sequences were Blast aligned to show the highest ITS sequence homology to Sanghuangpolus sanghuang.

According to the comprehensive analysis of the experimental data such as the sporophyte morphology, the mycelium morphology, the microscopic characteristics, the ITS identification and the like of the strain, the phellinus igniarius strain is proved to be authentic phellinus igniarius, and the latin name of the phellinus igniarius strain is sanghuang huang.

Example 2Cultivation experiment of Phellinus linteus QJF-2

(1) Preparing stock seeds: 81% of sawdust (half of coarse sawdust and half of fine sawdust), 15% of wheat bran, 2% of bean cake, 0.5% of lime, white sugar and 1% of gypsum are mixed according to the weight percentage, water is added, the mixture is fully and uniformly stirred, the water content is controlled to be 62% -65%, the mixture is bagged, the specification of a cultivation bag is a polypropylene bag with the thickness of 17cm multiplied by 33cm multiplied by 0.05cm, a rubber band is tied, the mixture is sterilized by high pressure, and the mixture is kept for 150min at the temperature of 125 ℃. And (3) after the fungus bags are sterilized, taking out the fungus bags, spreading the fungus bags for cooling, inoculating the phellinus igniarius QJF-2 test tube seeds under the aseptic condition, and culturing the phellinus igniarius at the temperature of 28 ℃ for 40 days in the dark to obtain the phellinus igniarius QJF-2 stock seeds.

(2) Preparation of cultivars: the composition and preparation method of the culture material of the cultivated species are shown in step (1), the stock seeds obtained in step (1) are inoculated on the culture material of the cultivated species according to the proportion of 5 percent by weight, and the cultivated species are cultivated for 40 days under the condition of 28 ℃ and dark place, so that the cultivated species of phellinus igniarius QJF-2 are obtained.

(3) Bag cultivation method of phellinus igniarius QJF-2

Mixing 78% of mixed wood chips, 18% of wheat bran, 2% of bean pulp, 1% of sugar and 1% of quicklime according to the weight percentage, adding water, fully and uniformly stirring, controlling the water content to be 62-65%, filling the mixture into a polypropylene fungus bag with the thickness of 17cm multiplied by 35cm multiplied by 0.05cm, and keeping the mixture at the temperature of 121-126 ℃ for 3 hours;

placing the sterilized fungus bags into a cooling chamber to be cooled to room temperature, inoculating the cultivated species in the step (2) into a culture material according to the proportion of 5 percent by weight under the aseptic condition, covering the cultivated species with a material surface of more than 1/2, immediately sealing the inoculated cultivated species by using a bacterium filtering sealing film or a jacket bag, and culturing the cultivated species in the dark at the temperature of 28 ℃ and under good ventilation conditions for 50 days until hyphae grow over the fungus bags;

before the fungus bags enter a cultivation shed, a pit is dug according to the depth of 3 cm-4 cm and the distance of plant row spacing of 10cm multiplied by 10cm or 12cm multiplied by 12cm, the pit is sprayed with the green mold, the fungus bags with the surfaces covered with the faint yellow mycelia are moved into the cultivation shed, a sterilization blade is used for cutting a cut (cutting a fungus skin and a crescent shape) on the fungus bags, the fungus bags are inverted in the pit and buried with moist sandy soil, the cultivation shed is divided into A, B areas, a microbial agent is sprayed to the cut of the phellinus igniarius fungus bags in the area B, the bags are sleeved, and then the fungus bags are vertically placed on a fungus bed. Zone A was not treated at all and served as a control. Then, the yellow color is stimulated by adopting a day and night temperature difference method, namely the temperature in the day is about 28 ℃, and the temperature at night is about 20 ℃. Air humidity is 85% -90% during bud forcing, light is scattered, and ventilation is avoided as far as possible. The cut was observed within 3-5 days, as shown in Table 1. The cut of the area B is basically yellow, and only 30% of the fungus bags in the area A are yellow.

Spraying water after the cut part turns yellow, increasing the air humidity to 90% -95%, increasing the ventilation, illuminating by 800lux, growing the fruit body, wherein the growth vigor of the fruit body in the fungus bag in the B area is better than that in the A area, the agronomic characters are good, and when the fruit body tissue becomes hard, the pileus turns from bright yellow to dark yellow and a small amount of spores are ejected, the collection can be carried out.

TABLE 1

^aThe biological efficiency is that the fresh weight of the fruit body/the dry weight of the culture material is multiplied by 100 percent

(4) Wild-imitating cultivation method of phellinus igniarius QJF-2

Chopping oak or mulberry cut in winter in the current year into blocks, splicing into section wood bundles (3 kg-4.5 kg) with the diameter of 15 cm-17 cm, bundling the section wood bundles by using a plastic rope, soaking the section wood bundles in clear water for 20min, fishing out, controlling water for 20min, filling low-pressure polyethylene material bags with the folding diameter of 19 cm-22 cm, the length of 39 cm-45 cm and the thickness of 0.005 cm-0.008 cm, filling sawdust and wheat bran nutrition materials (the ratio of sawdust to wheat bran is 3: 1) at two ends and gaps of a wood section, adding appropriate amount of water, uniformly stirring until the water content is 55% -66%), binding the section wood and the plastic bag tightly, sterilizing at normal pressure, controlling the temperature to rise to 100 ℃ within 4 h-6 h, keeping the temperature for 18h, and taking the section wood out of the pot after the temperature in the pot naturally drops to below 60 ℃.

Adopting a mode of inoculating at one end, digging strains by an inoculating shovel, quickly feeding the strains into a material bag, covering the strain surface with more than 1/2, immediately sealing by a strain sealing film or a jacket bag after inoculating, and culturing for 60 days at 28 ℃ in a well ventilated indoor dark place until hypha grows over the strain bag.

Before the fungus bags enter the cultivation shed, digging pits with the depth of 6cm and the plant row spacing of 10cm multiplied by 10cm or 12cm multiplied by 12cm, and spraying the chlorothalonil in the pits; transferring the fungus bag with the surface covered with the faint yellow mycelia into a cultivation shed, cutting a cut (cutting off a fungus skin) on the fungus bag by using a sterilization blade, vertically placing the fungus bag in a pit, burying sandy soil, wherein the water content of the sandy soil is 70% -72%, dividing the cultivation shed into A, B two areas, spraying a microbial agent to the cut of the phellinus igniarius fungus bag in the B area, bagging, vertically placing the fungus bag on a fungus bed, and performing no treatment on the A area as a comparison. Then, the yellow color is stimulated by adopting a day and night temperature difference method, namely the temperature in the day is about 28 ℃, and the temperature at night is about 20 ℃. Air humidity is 85% -90% during bud forcing, light is scattered, and ventilation is avoided as far as possible. The cut was observed within 5-10 days, as shown in Table 2. The cut of the area B is basically yellow, the fungus bags in the area A are yellow, and the fungus bags in the area A are not yellow.

Spraying water after the cut part turns yellow, increasing the air humidity to 90% -95%, increasing the ventilation, illuminating by 800lux, and having better agronomic characters than those of the fruiting body in the fungus bag in the area A in the growth period of the fruiting body in the area B.

After the cultivation shed is in winter, the sun-shading net is directly covered on the surface of the fungus bag, the temperature is controlled, the moisture is preserved, and the sun-shading net is uncovered to the top of the cultivation shed in the next 4 months of the year, so that the sun-shading effect is achieved. After the local air temperature is 20 ℃, the phellinus igniarius sporocarp enters a growing period and is managed in a conventional fruiting mode.

Three-year-old phellinus igniarius is stacked in two layers or three layers, is in a horseshoe shape or a fan shape, is woody and hard, and can be harvested when pileus is dark yellow to brown, white growth rings on the edge disappear and a small amount of spores are ejected.

TABLE 2

Example 3QJF-2 research on antitumor effect of fruiting body

QJF-2 fruiting bodies obtained in the embodiment of the invention and the traditional polysaccharide of the phellinus igniarius fruiting bodies of the poplar are respectively extracted by a water extraction and alcohol precipitation method, and the extraction rates are respectively 5.6% and 7.4%. Dissolving the two fruiting body polysaccharides with ultrapure water to a concentration of 1mg/mL, filtering for sterilization, and diluting to 300 μ g/mL with culture solution for use.

Cell strains HepG2, J82 and T24 are laboratory storage strains, after three cells are recovered and passaged, the cells are washed by PBS, pancreatin is added for cell digestion, and the digested cells (5000 cells/well) are inoculated in a 96-well plate and are cultured at 37 ℃ under 5% CO₂The culture box of (1) was cultured overnight. After the cells adhere to the wall, the culture solution is discarded, a drug group, a negative control group and a solvent control group are arranged, 100 mu L of corresponding solutions are respectively added, and each group is provided with 3 multiple wells. And (3) placing the 96-well plate into an incubator for continuous culture for 48h, discarding culture solution, adding 100mL of serum-free culture solution containing 10% of WST-1 reagent into each well, standing for 2h in the incubator, and measuring the light absorption value at 450nm by using an enzyme-labeling instrument. The results were analyzed with statistical software SPSS. The results are shown in Table 3.

As shown in Table 3, QJF-2 polysaccharide is superior to polysaccharide of Phellinus linteus fruiting body in inhibiting tumor cell proliferation, and has strong toxicity to tumor cells.

IC of influence of Phellinus linteus fruiting bodies on 3 kinds of tumor cell viability in Table 32₅₀Value of

Example 4Solid fermentation of Phellinus linteus strain QJF-2

(1) Slicing radix Dauci Sativae and rhizoma Solani Tuber osi, wrapping with corn flour, millet flour, soybean meal powder, etc., placing into polypropylene bags of 17cm × 33cm × 0.05cm, placing 3-5 radix Dauci Sativae or rhizoma Solani Tuber osi into each bag, and keeping at 121 deg.C for 30 min;

(2) cooling the fungus bags to room temperature, inoculating with Phellinus linteus QJF-2 mycelium under aseptic condition, sealing each fungus bag with 3 inoculation points, placing in an incubator at 28 deg.C, and culturing in dark for 30 days;

(3) pulverizing radix Dauci Sativae slices or rhizoma Solani Tuber osi slices full of Phellinus linteus strain QJF-2 mycelium, oven drying, and pulverizing again to obtain Phellinus linteus powder.

Example 5Liquid fermentation of Phellinus linteus strain QJF-2

Seed culture medium: 200g of potatoes, 20g of glucose and 1L of water.

Control medium (integrated PD medium): potato 200g, soybean powder 30g (lixivium), glucose 20g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 0.5g, vitamin B₁10mg, 1L of water.

Experimental groups:

the liquid fermentation medium comprises: 200g of carrot (leachate), 30g of soybean meal (leachate), 20g of glucose, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate and vitamin B₁10mg, 1L of water.

Preparing a liquid fermentation culture medium according to the formula, filling 250mL of the liquid culture medium into a 500mL triangular flask, keeping the temperature at 121 ℃ for 30min, inoculating 3% of seed culture solution according to the volume ratio after the culture medium is cooled, and performing shaking culture at 150rpm and 28 ℃ for 10 days; filtering Phellinus Linteus mycelium pellet, and oven drying; pulverizing to obtain Phellinus Linteus mycelium powder.

Example 6Determination of contents of total flavonoids, total triterpenes, crude polysaccharides and beta-carotene in phellinus linteus powder

The content of total flavonoids, total triterpenes, crude polysaccharides and beta-carotene in phellinus linteus fruit bodies and phellinus linteus powder obtained in example 4 are determined by Beijing Hua detection company, and the content (1171mg/100g) of the total triterpenes in phellinus linteus powder is far higher than that (354mg/100g) of the total triterpenes in the fruit bodies, and the phellinus linteus powder using carrots as a culture medium also has rich beta-carotene (0.073 g/kg). In addition, the content of crude polysaccharide (3.4g/100g) in the phellinus linteus powder is obviously higher than that of crude polysaccharide (1.95g/100g) of fruiting body, but the content of total flavone (65mg/100g) is obviously lower than that of fruiting body (235mg/100 g).

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Zhejiang Qianjian prescription pharmaceutical health Co., Ltd

Institute of Agricultural Resources and Agricultural Division, Chinese Academy of Agricultural Sciences

<120> Phellinus igniarius strain and cultivation method thereof

<130> 2020

<141> 2021-02-05

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 736

<212> DNA

<213> Phellinus linteus QJF-2()

<400> 1

ttccgtaggt taacctgcgg aaggatcatt atcgagtttt gaaagcgaga cctgctgctg 60

gtgcgaaatc gcgcatgtgc acggtcttcg cgctcaaatc caactcaaac ccctgtgcac 120

cttatatatc gcgagtcgaa gttagtagcc tgaggtcttg taagtaatta gtagaagggc 180

gaaagcgagt cttgctcgtt aggtagcctt tcgaaaatga aagcgagtgc gtcgggtgaa 240

gacttcggct tgtcgttaca aaacacctta tattgtcttt gtgaatgtaa tgctccttgt 300

gggcgaaaat aaatacaact ttcaacaacg gatctcttgg ctctcgcatc gatgaagaac 360

gcagcgaaat gcgataagta atgtgaattg cagaattcag tgaatcatcg aatctttgaa 420

cgcaccttgc gccccttggt attccgaggg gcatgcctgt ttgagtgtca tgtttatctc 480

aaaccgctcg tctttcttaa ttgaagggct tgaggtttgg acttggaggt ttactgctgg 540

cgcctttcga ggggtcggct cctcttaaat acattagctg ggctttggct cgcgtttacg 600

gtgtaatagt tgattccatt caccaacgag cgcttgcctg acgagcttgc ttctagccgt 660

ccgcgtcgtc ggacaaggag tcacctcctt cttgacacct ttgacctcaa atcaggtagg 720

attacccgcc gaactt 736

Claims (10)

1. A Phellinus linteus strain is characterized in that,

the Phellinus linteus strain is specifically Phellinus linteus QJF-2 with the preservation number of CGMCC No. 19653.

2. A method for cultivating Phellinus linteus strain according to claim 1, comprising:

spraying microbial agent on the cut of the fungus bag with the Phellinus igniarius mycelium, and bagging to induce the Phellinus igniarius.

3. The method of cultivating Phellinus linteus strain according to claim 2,

the cultivation method comprises bag cultivation or wild-like cultivation.

4. The method of cultivating Phellinus linteus strain according to claim 3,

the bag material cultivation method comprises the following steps:

(a1) uniformly stirring the compost, putting the compost into a fungus bag, sterilizing at high pressure, and cooling;

(a2) adding the cultivated species into the culture material obtained in the step (a1), and culturing at constant temperature in a dark place until hyphae grow over the fungus bags;

(a3) when the outdoor temperature reaches above 25 ℃, transferring the fungus bags obtained in the step (a2) into a cultivation shed for color conversion, cutting a cut on the fungus bags after the color conversion is finished, spraying a microbial agent to the cut, bagging, vertically placing on a fungus bed in the cultivation shed, and keeping the air humidity in the cultivation shed at 80% -85%;

(a4) after the cut part turns yellow, the bag is removed, the water is sprayed, the air humidity is controlled to be 85% -90%, and ventilation is carried out until harvesting.

5. The method of cultivating Phellinus linteus strain according to claim 3,

the wild-imitating cultivation comprises the following steps:

(b1) cutting the wood sections into short wood sections with the length of 17-20 cm, chopping into thin sections, splicing into wood section bundles, putting into clear water for soaking, taking out, putting into a fungus bag, filling culture materials in gaps and on the surfaces of the wood section bundles, tying fungus bag openings, and sterilizing at normal pressure to obtain fungus sticks;

(b2) adding the cultivated species into the fungus sticks obtained in the step (b1), and culturing at constant temperature in a dark place until hyphae overgrow the fungus sticks;

(b3) when the outdoor temperature reaches above 25 ℃, transferring the fungus sticks obtained in the step (b2) into a cultivation shed for color conversion, cutting a cut on the fungus sticks after the color conversion is finished, spraying a microbial agent to the cut, bagging, vertically placing on a fungus bed in the cultivation shed, and keeping the humidity of air in the shed to be 85-90%;

(b4) after the cut part turns yellow, the bag is removed, the water is sprayed, the air humidity is controlled to be 85% -90%, and ventilation is carried out until harvesting.

6. The method for cultivating Phellinus linteus Linteus strain according to claim 5, wherein,

the step (b3) further comprises overwintering management, specifically: after winter, the sunshade net at the top of the cultivation shed is directly covered on the surface of the fungus stick, and the sunshade net is uncovered to the top of the cultivation shed in the next 4 months for fruiting management.

7. A solid-state culture method of Phellinus linteus strain according to claim 1,

the method comprises the following steps:

(c1) inoculating the Phellinus linteus mycelium onto sterilized carrot or potato slices, and culturing at 28 deg.C;

(c2) and (c1) crushing the carrot slices or potato slices full of phellinus igniarius mycelia obtained in the step (c1), drying, and crushing again to obtain phellinus igniarius powder.

8. A liquid fermentation method of Phellinus linteus strain according to claim 1,

the method comprises the following steps:

(d1) preparing a liquid fermentation culture medium, and sterilizing; wherein the liquid fermentation medium comprises carrot extract;

(d2) inoculating phellinus igniarius mycelium into the liquid fermentation culture medium obtained in the step (d1), standing at room temperature, and performing shaking culture to obtain phellinus igniarius mycelium pellets;

(d3) and (d2) filtering the phellinus igniarius mycelium pellets obtained in the step (d2), drying and crushing to obtain phellinus igniarius mycelium powder rich in carotene.

9. Phellinus linteus powder prepared by the solid state culture method according to claim 7.

10. Phellinus linteus mycelium powder rich in carotene and prepared by liquid fermentation method according to claim 8.