Phellinus igniarius and mulberry leaf tea and preparation method thereof

Abstract

The invention relates to phellinus igniarius mulberry leaf tea and a preparation method thereof.The method comprises the following steps that 1, mulberry leaf chippings are blended into a wet material with water, the wet material is inoculated with phellinus igniarius strains for fermentation culture, and phellinus igniarius stock seeds are obtained; (2) blending a mixture containing mulberry leaf debris and a starch material into a culture material with water, and inoculating the phellinus igniarius stock seed for fermentation culture to obtain a first culture material; (3) crushing the first culture material into blocks, and continuously culturing to obtain a second culture material; and (4) pressing and drying the second culture material to obtain the phellinus igniarius and mulberry leaf tea. The invention also provides the phellinus igniarius and mulberry leaf tea prepared according to the method, a novel inonotus hirsutus strain Chengyuan 183-1 and application of the inonotus hirsutus strain Chengyuan 183-1 in preparation of the phellinus igniarius and mulberry leaf tea. The phellinus igniarius and mulberry leaf tea prepared by the method has nutritional and medicinal components of the phellinus igniarius mycelia and the mulberry leaves, and has high nutritional value and health-care efficacy.

Description

Technical Field

[0001] This invention relates to the field of tea-making technology, and in particular to a method for making tea by inoculating and fermenting mulberry leaves with Phellinus linteus, and the resulting Phellinus linteus and mulberry leaf tea. Background Technology

[0002] Sanghuang is a general term for a group of rare medicinal fungi, referring to polypores that grow on broad-leaved trees such as mulberry, poplar, and clove. Modern research has identified it as belonging to the genus *Sanghuang*. Sanghuangpore ) and Fibriophyte ( Unknown Several large fungal species. The latest classification system considers *Phellinus linteus* to belong to the phylum Basidiomycota, class Agaricomycetes, order Hymenochaetales, family Hymenochaetaceae, and genus *Phellinus* (Sanghuang). Sanghuangpore ), representing species such as mulberry tree (Sanghuang). Sanghuangpore sanghuang Common closely related species include *S. vaninii* (the mainstream commercial variety, commonly known as "Yanghuang"), *S. baumii*, and *S. lonicericola*, totaling about 14 species, all belonging to the genus *S. vaninii*. *S. vaninii* is also mentioned. Unknown ) coarse-haired fibrous fungus ( Unknown hairy According to expert research, it is the traditional Chinese medicine "Sanghuang", which has extremely high medicinal value.

[0003] Sanghuang (Phellinus linteus) has been used in traditional Chinese medicine for over two thousand years. Ancient medical texts record various effects: the black variety is used for women's abdominal masses, metrorrhagia, leukorrhea, and sudden breast swelling; the yellow variety treats men's edema, abdominal masses, abdominal pain, and early-stage wounds; if it is yellow, mature, and white, it stops diarrhea, invigorates qi, reduces hunger, and replenishes primordial yang. It treats epistaxis, amenorrhea, postpartum blood stasis, infertility due to yin and yang imbalances, and male lumps. It stops intestinal bleeding, hemorrhoids, blood stasis, weakness, wounds, and sore throat; it is suitable for all blood disorders. It benefits the five internal organs, promotes intestinal and stomach qi, eliminates toxins, and invigorates qi and strengthens the body. Modern medical research has found that Sanghuang has significant anti-cancer and anti-tumor effects, and has significant efficacy in enhancing immunity, antibacterial and antiviral properties, antioxidation, liver protection, lowering blood sugar, and treating gout.

[0004] Besides traditional Chinese medicine, there are also reports of using mulberry leaves as raw material for fermentation with Phellinus linteus to make tea. To date, the Phellinus linteus used in tea making is almost exclusively from mulberry trees (Phellinus linteus). Sanghuangpore sanghuang Among publicly available patents and local enterprise standards, the only one that appears most frequently and is explicitly marked as "preferred" is mulberry mushroom (Sanghuang). Sanghuangpore sanghuang ).

[0005] CN202310703304.3 is the only patent document that mentions *Phellinus cylindrica* in a tea-making formula that uses mulberry leaves as raw material and is fermented with *Phellinus cylindrica*. It provides a method for making *Phellinus cylindrica* mycelium tea bags using mulberry leaf brick tea as fermentation material. The preparation method comprises the following steps: S1: After activating the Sanghuang slant mother culture, it is inoculated into a solid culture medium for quality improvement and optimization under the stimulation of high and low concentration inorganic selenium drop and the stress of steam extract of mulberry leaf Fu brick tea; S2: Under sterile conditions, the Sanghuang mother culture is inoculated into a solid-liquid mixed fermentation agent culture solution for fermentation and culture under the dual effects of low-solidity inorganic selenium stress and the protective stimulation of steam extract of mulberry leaf Fu brick tea; S3: Under sterile conditions, the Sanghuang solid-liquid mixed fermentation agent is inoculated into the optimized fermentation material of bagged pure mulberry leaf Fu brick tea that has been sterilized at high temperature, and then transferred to a solid fermentation chamber for constant temperature, humidity and low carbon dioxide culture; S4: Preparation of Sanghuang mycelium tea bags. This patent claims to fundamentally solve the problems of bitterness, poor taste and difficulty in long-term storage of existing mulberry leaf Fu brick tea, and the prepared Sanghuang mycelium tea bags have better health benefits.

[0006] However, the process steps and raw materials of CN202310703304.3 are relatively complex, requiring pure mulberry leaf Fu brick tea as the base material; it needs to combine the complete extracellular enzyme system of Phellinus linteus to fully degrade the crude fiber of pure mulberry leaf Fu brick tea, thereby converting these polysaccharides into monosaccharides; the solid culture medium used in this method needs to be optimized for quality improvement under the dual effects of high and low concentration differences of inorganic selenium and the stress of steam extraction agent of mulberry leaf Fu brick tea; the liquid culture medium used in this method needs to be fermented and cultured under the dual effects of low concentration inorganic selenium stress and the protective stimulation of steam extraction agent of Phellinus linteus of mulberry leaf Fu brick tea. This method also requires the rational combination of solid fermentation mycelia of mulberry, poplar, and coarse-textured Phellinus linteus, and needs to utilize the different structural components of mulberry, poplar, and coarse-textured Phellinus linteus to enhance complementarity.

[0007] In summary, the following problems still exist when using mulberry leaves as raw material to make tea through inoculation and fermentation with *Follium coccidioides*: (1) *Follium coccidioides* needs to be used in combination with other *Phellinus linteus* strains; (2) *Phellinus linteus* strains need to be subjected to stress and stimulation for quality improvement or cultivation; (3) Specific mulberry leaf raw material forms need to be used; (4) The process is complex, such as the need to improve and optimize the strains; (5) The raw materials are complex, such as the need for multiple strains, inorganic selenium, steam extractant for Fu brick tea, and steam extractant for *Follium chrysogenum* in Fu brick tea; (6) Using *Follium coccidioides* alone for inoculation and fermentation will result in problems such as long fermentation time, low fermentation degree, uneven fermentation, and off-flavors due to lack of oxygen.

[0008] The inventors isolated a new strain of *Fomitopsis coarseness* and discovered that this strain can be used to produce nutrient-rich mulberry leaf tea by inoculating mulberry leaves alone using a simple process. This led to the completion of this invention. Summary of the Invention

[0009] To address the aforementioned problems in the prior art, the first aspect of this invention provides a method for preparing Sanghuang mulberry leaf tea, wherein the method includes the following steps: (1) Mix mulberry leaf fragments with water to form a wet material, and inoculate the wet material with Phellinus linteus for fermentation culture to obtain Phellinus linteus original seed; (2) Use water to prepare a mixture containing mulberry leaf fragments and starchy materials into a culture medium, and inoculate the original mulberry seed for fermentation culture to obtain the first culture medium; (3) The first culture medium is broken into blocks and cultured again to obtain the second culture medium; (4) Press the second culture medium and dry it to obtain Sanghuang Mulberry Leaf Tea.

[0010] The second aspect of the present invention provides a mulberry leaf tea prepared by the method described in the first aspect of the present invention.

[0011] Compared with the prior art, the present invention has the following advantages: (1) This invention does not require the use of coarse-haired fibrous fungus in combination with other Phellinus linteus species.

[0012] (2) This invention does not require stress or stimulation of the Phellinus linteus strain for quality improvement or cultivation; (3) This invention can directly use mulberry leaves as raw material; (4) The process of this invention is simple and does not require quality improvement or optimization of the strain; (5) The raw materials of this invention are simple, and a single strain can be used. Only the strain, mulberry leaf fragments, starchy materials and water are needed; (6) The present invention also has the advantages of short fermentation time, high degree of fermentation, uniform fermentation and no odor. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0014] As described above, the first aspect of the present invention provides a method for preparing Sanghuang mulberry leaf tea, wherein the method includes the following steps: (1) Mix mulberry leaf fragments with water to form a wet material, and inoculate the wet material with Phellinus linteus for fermentation culture to obtain Phellinus linteus original seed; (2) Use water to prepare a mixture containing mulberry leaf fragments and starchy materials into a culture medium, and inoculate the original mulberry seed for fermentation culture to obtain the first culture medium; (3) The first culture medium is broken into blocks and cultured again to obtain the second culture medium; (4) Press the second culture medium and dry it to obtain Sanghuang Mulberry Leaf Tea.

[0015] In some other preferred embodiments, in step (1), the amount of water used is 40% to 60% by mass of the wet material. In some other preferred embodiments, in step (2), the amount of water used is 40% to 60% of the culture material, more preferably 45% to 55% by mass, and even more preferably 50% to 54% by mass.

[0016] In steps (1) and (2), excessive moisture can affect the growth rate of mycelium.

[0017] Preferably, in step (2), the mulberry leaf fragments account for 70% to 100% of the dry weight of the culture material, more preferably 85% to 95%.

[0018] Preferably, in step (2), the starchy material accounts for 0-30% of the dry weight of the culture medium, more preferably 5% to 15%, for example, 10%. Adding a certain amount of starchy material, such as jujube powder or yam powder, promotes fermentation because mulberry leaves have a high nitrogen content, and adding starchy material can increase the carbon-to-nitrogen ratio. If too much starchy material is added, it may increase the viscosity of the culture medium, which is not conducive to mycelial growth. Of course, the original culture made from grains, wheat grains, etc., can also be used to inoculate the mulberry leaf culture medium, but the grains, wheat grains, etc., used for inoculation need to be picked out before the secondary fermentation.

[0019] The present invention does not impose any particular restrictions on the culture container. The wet material mentioned in step (1) can be cultured in the original seed bottle; the culture material mentioned in step (2) can be cultured in the edible fungus cultivation bag, preferably a polypropylene plastic bag, which will not decompose into harmful substances during high-temperature sterilization; after being crushed into blocks in step (3), it can be cultured in a fermentation box (e.g., a food-grade plastic box).

[0020] In step (2), the edible fungus cultivation bags used can be polypropylene plastic bags (which will not decompose into harmful substances during high-temperature sterilization).

[0021] Preferably, the wet material described in step (1) and the culture material described in step (2) are both sterilized before fermentation. The sterilization method can be a conventional method. For example, atmospheric pressure steam sterilization or high pressure steam sterilization can be used. Atmospheric pressure sterilization can be performed at 100°C for 8 hours, and high pressure sterilization can be performed at 126°C for 2 hours.

[0022] In some other preferred embodiments, the culture temperature in steps (1), (2), and (3) is independently 25°C to 28°C. The present invention does not impose a particular limitation on the culture time, as long as the mycelium fully colonizes the culture container. However, in some preferred embodiments, the fermentation time in step (3) can be 3 to 5 days, for example, 4 days. Within this culture time range, the mycelium can fully colonize the culture medium. *Fomitopsis coarseness* mycelium does not prefer light and is aerobic during its development period; therefore, dark culture is recommended. Through comparative experiments, the inventors found that light culture can make the mycelium color deeper yellow, but it does not increase the content of various active ingredients, especially total flavonoids and total polyphenols; in fact, the content is lower than that of dark culture.

[0023] In some other preferred embodiments, the mulberry leaf fragments mentioned in steps (1) and (2) are independently obtained by crushing mulberry leaves after removing the stems, drying, and pressing them into fragments. The mulberry leaves are processed into fragments rather than being pulverized into powder mainly because powdered culture media have poor aeration and are not easy to ferment; excessively large fragments will also affect aeration. Therefore, in some other preferred embodiments, the size of the fragments (here, the size refers to the maximum dimension of the leaf plane, i.e., the maximum length of the fragments) is preferably 0.5 cm to 5 cm, preferably less than 0.5 cm to 2 cm, and more preferably around 1 cm.

[0024] The mulberry leaves used are best picked before the first frost in autumn. At this time, the nutritional value of the mulberry leaves is higher than that of the leaves picked after the first frost, except for a few medicinal components. Moreover, the mulberry tree is less susceptible to cold, and the petioles have basically formed an abscission layer at this time, making it easy to pick the leaves and having minimal impact on the mulberry tree.

[0025] In some other preferred embodiments, the moisture content of the mulberry leaf scraps is less than 15% by weight, for example less than 10% by weight, so that they can be crushed into scraps.

[0026] Based on the characteristic that *Sanghuang* is an aerobic fungus, the mycelium deep inside may develop very slowly due to insufficient oxygen, and the amount of mycelium in the culture medium is small, resulting in incomplete fermentation. Prolonged lack of oxygen will cause an off-odor in the culture medium. Therefore, this invention adopts a two-stage fermentation process, that is, first, the mycelium is fully developed in step (2). Then, in step (3), it is broken into small pieces for secondary fermentation, which allows the *Sanghuang* mycelium in the mulberry leaf culture medium to come into contact with sufficient oxygen, making the fermentation more complete, thereby increasing the content of *Sanghuang*-specific active ingredients in the culture medium; at the same time, first developing the mycelium in the spawn bag and then breaking it into small pieces can greatly reduce the probability of contamination by other bacteria, and even if it is not operated in a sterile environment, there will be very little contamination by other bacteria.

[0027] In step (3), the first culture medium is broken into small pieces with a size of 1cm to 3cm, preferably 2cm to 3cm. If the pieces are too large, fermentation inside the pieces will be slow; if they are too small, it will be more labor-intensive, the lower layer will have poor aeration when stacked, and the probability of contamination by other microorganisms will increase. There is no particular limitation on the thickness of the small pieces stacked in the culture container such as a culture box, as long as fermentation can proceed smoothly. The thickness can be, for example, 4cm to 7cm, such as 5cm or 6cm.

[0028] In this invention, especially in the second stage of fermentation culture in step (3), the culture temperature in the fermentation box can be 25°C to 28°C, and the fermentation time can be 3 to 5 days. Because it is aerated dark culture, the oxygen is sufficient and the environment is suitable, the mycelium develops very quickly. In 3 to 5 days, the newly generated mycelium from the small pieces completely covers the surface of each small piece and further connects the mycelium on the surface of each small piece together. If the time is too long, the culture medium pieces in the entire fermentation box will be reconnected into a whole by the mycelium, and its interior will be in a state of insufficient oxygen.

[0029] In step (4), the second culture medium is pressed and dried to obtain Sanghuang mulberry leaf tea.

[0030] In some preferred embodiments, the second culture medium can be dried and pulverized before pressing, mixed with water, pressed, and then dried a second time to obtain Sanghuang mulberry leaf tea. The drying temperature before pressing can not exceed 60°C. The medium is pulverized into a fine powder with a mesh size greater than or equal to 80 mesh. The water ratio can be 38% to 45% by weight of the second culture medium, ensuring good internal binding of the dough without excessively sticking to the processing equipment. Alternatively, the second culture medium can be left undried before pressing, placed in a dry and ventilated environment to slowly and evenly lose water until the moisture content reaches 38% to 45% by weight, then pulverized into a paste, and finally formed into clumps for pressing. The clumps can be pressed to a thickness of 1mm to 2mm, producing small tea pieces of any shape, with a size not exceeding 5cm. 2 Alternatively, specialized film-making equipment can be used, with the water ratio depending on the equipment requirements.

[0031] After pressing, the mixture can be dried at 100℃ to completely inactivate the mycelium (and any other bacteria, if present). In addition to small tea flakes, it can also be made into tea cakes or tea bricks. In this case, a suitable mold can be selected, the fermented second culture medium can be added, pressed into shape, and then dried at 100℃.

[0032] In addition, the pulverized second culture medium can be used to manufacture tea chips, or it can be directly consumed as fermented mulberry leaf nutrient powder or added to flour or other grain flour as a food additive to make health-functional foods.

[0033] In some preferred embodiments, the *Phellinus linteus* strain is *Phellinus coarsely fibrous* (… Unknown hairy More preferably, the strain of *Sanghuang* is strain Chengyi 183-1, which is preserved at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024295.

[0034] The preservation information for strain Chengyi 183-1 is as follows: Accession number: CCTCC NO: M 2024295; Classification name: Fomitopsis coarse-haired bacteria ( Unknown hairy ); Name of depositary institution: China Center for Type Culture Collection (CCTCC); Address of the depositary institution: Wuhan University, Wuhan, China; Date of preservation: January 30, 2024.

[0035] The inventors have discovered that using *Phellinus linteus* (a type of fungus) is effective. Unknown hairy Using microbial strains, especially the Chengyi 183-1 strain, to ferment mulberry leaves can solve problems such as long fermentation time, low fermentation degree, uneven fermentation, and off-flavors.

[0036] The second aspect of the present invention provides a mulberry leaf tea prepared according to the method described in the first aspect of the present invention.

[0037] Preferably, the Sanghuang mulberry leaf tea has one or more of the following properties: (1) Boiling water extractable content ≥ 40%; (2) Protein content ≥ 23%; (3) Total amino acid content ≥16%; (4) Crude polysaccharide content ≥ 6.5%; (5) Total polyphenol content ≥ 0.4%; (6) Total flavonoid content ≥ 0.57%; (7) Total triterpenoid content ≥1.5%.

[0038] The Sanghuang mulberry leaf tea prepared by the method of this invention is dark brown to black, and the brewed tea is yellow to brown in color. It has a delicious taste and a distinct Sanghuang fungus aroma.

[0039] In a third aspect, the present invention provides a novel coarse-haired fibrous fungus ( Unknown hairy The strain Chengyi 183-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024295, as described above.

[0040] The present invention provides, in a fourth aspect, *Polyporus coarse-hairedii* (… Unknown hairy Application of mulberry leaf tea in the preparation of mulberry leaf tea.

[0041] Preferably, the *Phellinus linteus* strain is *Phellinus coarsely fibrous* (…). Unknown hairy The strain Chengyi 183-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024295.

[0042] The present invention will be further described below by way of examples, but the scope of protection of the present invention is not limited to these embodiments.

[0043] Example 1: Fermentation experiment with different moisture contents in the culture medium 1.1 Preparation of mulberry leaf culture medium Six gradients of mulberry leaf culture medium (the wet material from step (2)) were prepared, with moisture contents of 50%, 52%, 54%, 56%, 58%, and 60% respectively. Two other culture media were prepared: a dry material containing 80% mulberry leaf scraps + 20% jujube powder; and a dry material containing 80% mulberry leaf scraps + 20% yam powder (the wet material from step (2)), both with a moisture content of 52%. Each treatment was packaged into three 480 ml glass inoculum bottles, each containing 70 grams of dry material to a height of 80 mm, and sealed with a plastic cap with a breathable membrane. The bottles were sterilized by steaming at 126°C for 2 hours.

[0044] 1.2 Vaccination Four-point inoculation was performed on the surface of the culture medium in each culture bottle, namely one point in the center and three points near the bottle wall. The culture strain was the original strain of Phellinus coarse-fiber fungus Phellinus linteus "Chengyi 183-1".

[0045] 1.3 Cultivation and Recording After inoculation, draw a line around each culture bottle at the same level as the culture medium. Place the bottles in a 26°C incubator for dark incubation. Investigate the mycelial growth depth on days 13 and 25. For each bottle, take the average of the maximum and minimum depth values ​​(mycelia grow from the top of the culture medium downwards, with the mycelial tip reaching the bottom of the culture medium; the depth is recorded as the culture medium height). Also, investigate the time it takes for the mycelia to fully cover the culture medium (mycelia covering the central area at the bottom of the culture medium is considered "fully covered").

[0046] 1.4 Results and Analysis For the pure mulberry leaf group, data from day 13 in Table 1 shows that in the early stages of cultivation, the mycelial development rate increased with increasing water content; however, once the water content exceeded 56% by weight, the mycelial development rate began to decline. Data from day 25 in Table 1 shows that the mycelial development was fastest in the medium with a water content of 52% by weight, and the development rate decreased significantly with increasing water content. In the treatment group with a water content of 60% by weight, water accumulated at the bottom of the bottle, and the cultured mycelium failed to fully colonize. Overall, mycelial development showed a trend of initially rapid growth followed by a slowdown, which is presumably due to oxygen deficiency in the deeper layers of the medium.

[0047] Adding jujube powder or yam powder to mulberry leaf culture medium significantly accelerated mycelial growth. The average time for mycelial full colonization was 26.3 days for the medium with jujube powder and 23.7 days for the medium with yam powder.

[0048] The inventors further investigated the amount of yam powder added in subsequent experiments. The results showed that within the range of 0-30%, the mycelial growth rate increased with increasing yam powder content. When brewing tea slices, it was found that tea slices with a higher yam powder content tended to disintegrate easily. Therefore, if intended for tea slice production, the amount added should not be excessive, or an edible adhesive can be added before processing. However, if intended for processing into nutritional powder, the proportion can be increased appropriately.

[0049] Table 1. Mycelial development on mulberry leaf culture media with different moisture contents

[0050] Note: In the significance analysis of the data in Table 1, uppercase letters indicate p<0.01 (indicating a significant difference), and lowercase letters indicate p<0.05 (indicating an extremely significant difference). "On day 25, the sidewalls of the culture bottles in the corresponding treatment groups were covered with mycelium, except for the mulberry leaf and jujube powder group, where there was no mycelium in the center of the bottom of the bottle, so the depth was recorded as 80.0 mm."

[0051] Example 2: Comparison of different strains 2.1 Test strains: These included *Phellinus cirrhosa* strains such as Chengyi 183-1, JH-1, 433, 919, N9, SX, and WD. Among them, strains Chengyi 183-1, JH-1, 433, 919, and N9 were extracted from wild *Phellinus cirrhosa* from Chengde, Hebei Province; strain SX was from Nanyang Sanghuang Biotechnology Co., Ltd. in Henan Province; and strain WD was from Shandong Gusang Agricultural Products Co., Ltd.

[0052] 2.2 Preparation of mulberry leaf culture medium The formula is pure mulberry leaf scraps with a moisture content of 52% by weight. The culture flasks are 480 ml glass culture bottles, totaling 24 bottles. Each bottle contains 70 grams of dry substrate, with a short wooden stick (10 mm in diameter) inserted into the center. The bottles are sealed with plastic caps fitted with breathable membranes. The culture is then sterilized by steam at 126℃ for 2 hours.

[0053] 2.2 Inoculation and Culture After cooling, the culture flasks were inoculated under aseptic conditions, with three flasks inoculated for each strain. During inoculation, a short wooden stick was taken from the middle of the culture medium and inoculated at two points: the bottom and the opening of the cavity. Three additional inoculations were made on the surface of the culture medium near the flask wall. After inoculation, the flasks were placed in a 26°C incubator for dark incubation, and the time required for mycelium to fully colonize the culture flasks was observed. After 35 days of incubation, the flasks were removed under aseptic conditions and broken into 2-3 cm square pieces. The mycelial density on the pieces from different parts of the culture flasks for each strain was compared. The criteria were: a grayish-white culture medium indicated dense mycelium; a black and white culture medium indicated relatively dense mycelium; and a culture medium with white mycelium but predominantly black culture medium indicated sparse mycelium. The pieces of each strain were then placed in an incubator for dark incubation for 5 days, and mycelial development and contamination were observed.

[0054] 2.3 Results and Analysis The experimental results (see Table 2) show that the development speed of different strains in mulberry leaf culture medium varies. Among them, the mycelial growth rate of strain Chengyi 183-1 is the fastest. Its mycelial full coverage time is not significantly different from that of strains 919 N9 and WD, but is significantly shorter than that of strains JH-1 and 433. After 35 days of fermentation, the mycelial development status of different parts of the culture bottle of each strain was compared. It was found that the mycelium of strain Chengyi 183-1 was dense in the upper and middle parts of the culture medium and relatively dense in the lower part, which was better than other strains overall. After 5 days of secondary fermentation, the surface of the small pieces of all strains was covered with a large number of fluffy aerial mycelia. Among them, strain Chengyi 183-1 had the most mycelia. The mycelia between the small pieces were connected together, and the culture medium of the whole box was connected into a whole by mycelia.

[0055] Table 2. Comparison of mycelial development of different strains of *Follium coarctatiforme* fermenting mulberry leaves.

[0056] Note: In the significance analysis of the data in Table 2, uppercase letters indicate p<0.01 (this level indicates a significant difference), and lowercase letters indicate p<0.05 (this level indicates a highly significant difference).

[0057] This experiment shows that the fermentation method used is suitable for *Follium coarseum* strains, especially strain Chengyi 183-1. Secondary fermentation can significantly improve the fermentation degree of mulberry leaf culture medium.

[0058] Example 3: Comparative Experiment of Different Fermentation Methods 3.1 Preparation of mulberry leaf culture medium The formula consists of mulberry leaf scraps (95% by weight) and yam powder (5% by weight). The mulberry leaf scraps and yam powder are mixed thoroughly, and an equal amount of water is added (i.e., the water content is 50% by weight). A total of 21 mushroom bags are prepared, each containing an average of 737 grams of material. The bags are sealed with a ring sealer and sterilized in a pressure cooker at 126°C for 2 hours.

[0059] 3.2 Inoculation and Culture After the spawn bags cooled, each spawn bag was inoculated under sterile conditions (three bags were reserved as uninoculated controls). During inoculation, a hole was made in the center of the spawn bag, and inoculation was performed at three points inside the hole: the upper, middle, and lower. The spawn used was the original strain of *Sanghuang* 'Chengyi 183-1', a type of *Flococcus comatus*. After inoculation, the spawn bags were placed in a 26°C incubator for dark incubation.

[0060] 3.3 Different Fermentation Methods After all the culture bags have fully fermented, randomly select 18 bags. Continue to incubate 6 of them in the original incubator. Open the remaining 12 bags in a clean bench, break the culture medium into small pieces of 2-3 cm square, and place them in clean, transparent acrylic fermentation boxes, one box per bag. Then close the lids. Randomly select 6 boxes and place them in a light incubator at 26℃ with a light intensity of about 1300 Lx. Place the other 6 boxes in a dark incubator at the same temperature (26℃) for incubation in the dark.

[0061] 3.4 Sample Preparation After cultivation, all samples were removed, dried, and pulverized. Two boxes (bags) were randomly selected from the six boxes (bags) for each fermentation method and mixed together to form a parallel sample; that is, three parallel samples were set up for each fermentation method. Samples without secondary cultivation were named Straight 1, Straight 2, and Straight 3; samples cultured in light were named Light 1, Light 2, and Light 3; samples cultured in the dark were named Dark 1, Dark 2, and Dark 3; and the three uninoculated bags each served as a control sample, named CK1, CK2, and CK3.

[0062] 3.5 Sample Nutritional Content Test The contents of total flavonoids, total polyphenols, total triterpenes, total amino acids, and 16 kinds of amino acids were tested on the above samples.

[0063] 3.5.1 Test Method (1) The total triterpenes, total amino acids and 15 amino acids were tested by the testing company. The test method for total triterpenes was NY / T 3676-2020, and the test methods for total amino acids and 15 amino acids were GB 5009.124-2016.

[0064] (2) Method for determining total flavonoids Preparation of flavonoid standard curve: Weigh 10 mg of rutin standard and dilute to 100 mL in a volumetric flask with 60% ethanol. Accurately pipette 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the above rutin standard solution into 10 mL colorimetric tubes. Add 60% ethanol to a final volume of 5.0 mL, add 0.3 mL of 5% NaNO2 solution, shake well, and let stand for 6 min. Add 0.3 mL of 10% Al(NO3)3 solution, shake well, and let stand for 6 min. Add 4.0 mL of 4% NaOH, dilute to volume with 60% ethanol, shake well, and let stand for 15 min. Using 0.0 mg / mL of the standard as a reference solution, measure the absorbance at a wavelength of 510 nm. Plot the standard curve.

[0065] Sample determination: Weigh 0.20-0.25g of sample, accurate to 0.001g, and place it in a 10ml colorimetric tube. Add 5ml of 60% ethanol, vortex for 2min, transfer to a 60℃, 500W ultrasonic cleaner for 30min, centrifuge at 12000r for 20min, collect the supernatant into a 10ml colorimetric tube, repeat the extraction once, combine the two supernatants, and dilute to 10ml. Filter the extract through a 0.45μm filter membrane using a syringe, discarding impurities. Then measure according to the method used to prepare the standard curve described above. During measurement, take 2.5ml of the sample solution and add ethanol to 5ml. Calculate using the following formula: Total flavonoid content = Total flavonoid concentration × Total volume of extract × Dilution factor / Mass of dry powder.

[0066] (3) Method for determining total polyphenols Preparation of standard curve: Accurately weigh 0.110 g gallic acid monohydrate, dissolve and dilute to 100 ml with distilled water. Pipette 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 ml of this solution into 10 ml colorimetric tubes, respectively. Dilute to the mark with distilled water and mix well. Pipette 1 ml of the standard solution into each 10 ml colorimetric tube, add 1 ml of Folin reagent to each, mix well, then add 5 ml of distilled water and mix thoroughly. After 1 min, add 2 ml of 12% sodium carbonate solution, mix well, and develop the color at room temperature in the dark for 2 h. Measure the absorbance at 760 nm and plot the standard curve.

[0067] Sample determination: Weigh 0.20-0.25g of sample, accurate to 0.001g, place in a 10ml centrifuge tube, add 5ml of 60% ethanol, vortex for 2min, and centrifuge at 60℃, 400W for 30min, 12000r for 20min in an ultrasonic cleaner. Transfer the supernatant to a 10ml colorimetric tube and repeat the extraction once. Combine the two supernatants, bring the volume to 10ml, and filter through a 0.45μm filter membrane. Then measure according to the method used to prepare the standard curve. During measurement, take 0.25ml of the sample solution, add water to 1ml, and calculate the total polyphenol content in the sample solution according to the gallic acid standard curve. Total polyphenol mass ratio (mg / g) = total polyphenol concentration × total volume of extract × dilution factor / dry powder mass.

[0068] 3.6 Results and Analysis It took 45 days for all the mycelia in each spawn bag to fully develop. After 45 days, the 12 spawn bags were opened and broken into pieces for secondary fermentation. After 4 days of secondary fermentation, the mycelia in both the light and dark culture media connected the entire culture media into one.

[0069] Analysis of the nutrient composition results of samples from different treatment groups revealed that after fermentation with *Phellinus linteus*, the contents of total flavonoids, total polyphenols, total triterpenes, total amino acids, and most amino acids in each treatment group were significantly higher than those in the control group. Furthermore, the contents of most nutrients in the double dark culture group were higher than those in other treatment groups. Specifically, the total flavonoid content in the double dark culture group was significantly higher than that in the direct culture group and the control group, the total polyphenol content was significantly higher than that in the direct culture group and significantly higher than that in the control group, and the total triterpenes content was significantly higher than that in the direct culture group (see Tables 3 and 4).

[0070] Flavonoids, polyphenols, and triterpenes are important active components of Phellinus linteus, which have significant antioxidant, anti-aging, anti-tumor, immunomodulatory, anti-inflammatory, and hepatoprotective effects. These three components are present in higher concentrations in the secondary dark culture group, so secondary fermentation and dark culture are recommended as the preferred method.

[0071] Table 3. Comparison of nutrient content in samples with different treatments

[0072] Note: In the significance analysis of the data in Table 3, uppercase letters indicate p<0.01 (this level indicates a significant difference), and lowercase letters indicate p<0.05 (this level indicates a highly significant difference).

[0073] Table 4. Comparison of the contents of 16 amino acids in samples with different treatments (g / 100g).

[0074] Note: In the significance analysis of the data in Table 4, uppercase letters indicate p<0.01 (this level indicates a significant difference), and lowercase letters indicate p<0.05 (this level indicates a highly significant difference).

[0075] Finally, it should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification is illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A method for preparing a Phellinus igniarius mulberry leaf tea, wherein, The method comprises the following steps: (1) preparing a wet material by mixing mulberry leaf pieces with water, inoculating the wet material with a mulberry leaf fungus strain for fermentation culture to obtain a mulberry leaf fungus original strain; (2) preparing a culture material by mixing a mixture containing mulberry leaf pieces and starch materials with water, inoculating the culture material with the mulberry leaf fungus original strain for fermentation culture to obtain a first culture material; (3) crushing the first culture material into pieces for further culture to obtain a second culture material; (4) compressing and drying the second culture material to obtain a mulberry leaf fungus tea.

2. The method according to claim 1, wherein: in step (1), the amount of water is 40% to 60% by mass of the wet material.

3. The method of claim 1, wherein, in step (2): the amount of water is 40% to 60% by mass of the culture material; the mulberry leaf pieces are 70% to 100% by weight of the dry weight of the culture material; and / or the starch materials are 0% to 30% by weight of the dry weight of the culture material.

4. The method according to claim 1, wherein: the fermentation culture temperature in steps (2) and (3) is independently 25°C to 28°C, the fermentation time in step (2) is 30 days to 50 days, and the fermentation time in step (3) is 3 days to 7 days.

5. The method according to claim 1, wherein: the mulberry leaf pieces in steps (1) and (2) are independently mulberry leaf pieces obtained by crushing mulberry leaves after drying and removing stems.

6. The method according to claim 1, wherein: the moisture content of the mulberry leaf pieces is less than 15% by weight; and / or the size of the mulberry leaf pieces in the mulberry leaf fungus tea is less than 5 cm.

7. The method according to claim 1, wherein: the mulberry leaf fungus strain is a strain of Inonotus hispidus; Preferably, the Phellinus sp. is Inonotus hispidus Inonotus hispidus ) strain Chengyi 183-1, which is deposited with the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2024295.

8. The mulberry leaf fungus tea prepared by the method according to any one of claims 1 to 7; Preferably, the mulberry leaf fungus tea has one or more of the following properties: (1) the content of hot water extract is ≥40%; (2) the total polyphenol content is ≥0.4%; (3) the total flavonoid content is ≥0.57%; (4) the total triterpene content is ≥1.5%; (5) the total amino acid content is ≥16%.

9. A type of coarse-haired cilia ( Inonotus hispidus The strain Chengyi 183-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024295.

10. Use of Inonotus hispidus in the preparation of a mulberry leaf fungus tea; Preferably, the Phellinus sp. is Inonotus hispidus Inonotus hispidus ) strain Chengyi 183-1, which is deposited with the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2024295.

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