Morus alba strain suitable for making fermented barley tea and application thereof

Abstract

This invention relates to a strain of *Sanghuang* fungus suitable for making fermented barley tea and its applications, belonging to the field of biotechnology. The *Sanghuang* fungus strain described in this invention was deposited on April 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41280. This strain is suitable for solid-state fermentation of barley grains for making barley tea. The mycelium of this strain can penetrate the interior of barley grains, fully converting and utilizing the nutrients in the barley grains, exhibiting characteristics such as rapid growth and good appearance. The roasted *Sanghuang* barley tea has a yellow and lustrous color, overcoming the drawback of traditional barley tea being prone to causing "heatiness," possessing both the mellow aroma of barley tea and the unique refreshing aroma of *Sanghuang* fungus, and containing medicinal components of *Sanghuang* fungus. The *Sanghuang* fungus mycelium formed by fermentation and culture of this strain using barley grains as nutrients effectively solves the shortcomings of *Sanghuang* fungus being difficult to cultivate and expensive, while greatly increasing the added value of traditional barley tea.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of Phellinus linteus from the mulberry tree suitable for making fermented barley tea and its applications. Background Technology

[0002] Barley (Hordeum vulgare L.) is a globally important cereal crop, rich in various carbohydrates, dietary fiber, protein, and vitamins, offering a variety of nutritional benefits. Barley tea, made from barley grains, is widely popular in my country and other East Asian countries for its unique aroma and taste. However, very little of the nutrients from barley actually enters the tea infusion; most are discarded along with the barley flour after brewing, failing to be effectively utilized. Furthermore, barley tea contains the husk, which can irritate the stomach, making it unsuitable for those with weak digestive systems. People with damp-heat or indigestion may experience "heatiness" after drinking barley tea, exacerbating symptoms such as bloating and constipation. Therefore, traditional barley tea urgently needs to improve the utilization rate of barley grain nutrients and address the drawbacks such as the "heatiness" experienced by some individuals after consumption.

[0003] Sanghuang is a rare medicinal fungus that parasitizes the mulberry tree (Morus L.). It is rich in various polyphenols, polysaccharides, flavonoids and their derivatives, sterols and other active substances. It has multiple medicinal effects such as improving immunity, anti-cancer, anti-inflammatory and antioxidant properties. It is known as "forest gold" and "king of anti-cancer", and has great value for industrial development and application.

[0004] There is only one genuine Sanghuang, namely Sanghuang from the mulberry tree (Sanghuangporus sanghuang). It is a parasitic fungus that exclusively parasitizes mulberry trees and has extremely high medicinal value. However, due to its extreme rarity, its extremely demanding growing environment, and the difficulty in artificial cultivation, coupled with the fact that medical books throughout history have regarded Sanghuang from the mulberry tree as the genuine Sanghuang, more than a dozen kinds of so-called "Sanghuang" (imitations) can be found on the market today. The most common ones are Sanghuang from the poplar tree (Sanghuangporus vaninii) (accounting for more than 90% of the market), Sanghuang from the pine tree (phellinus pini), Sanghuang from the baumii tree (Sanghuangporus baumii), Sanghuang from the oak tree (Sanghuangporus quercicola), and Inonotus hispidus; as well as several other less common imitations of Sanghuang: Sanghuangporus lonicericola, Inonotus zonatus, Fuscoporia mori, and Phellinus. Examples of mulberry species include *Sanghuangporus mori*, *Inonotus linteus*, *Sanghuangporus alpinus*, *Sanghuangporus toxicodendri*, and *Sanghuangporus weigelae*. Imitation *Sanghuang* species are often mistakenly called "Sanghuang," but some do not actually parasitize mulberry trees; they merely resemble the genuine *Sanghuang* (mulberry tree *Sanghuang*) in appearance, and some are closely related species to *Sanghuang*.

[0005] Currently, the artificial cultivation techniques for authentic Sanghuang (Mulberry Sanghuang) mainly employ mycelial fermentation culture, mulberry branch bag culture, or branch and trunk culture. However, these cultivation techniques are not yet mature, primarily due to the tendency for fermentation culture to cause mycelial mutations, and mulberry branch culture resulting in slow growth, low yield, and susceptibility to contamination by other microorganisms. Therefore, although authentic Mulberry Sanghuang possesses extremely high medicinal value, its extreme scarcity, difficulty in cultivation, and high price are also significant drawbacks. Thus, the cultivation of Mulberry Sanghuang urgently needs to expand its nutrient sources, shorten the cultivation cycle, and reduce costs while maintaining its medicinal efficacy.

[0006] If mulberry fungus (Sanghuang) can be used in the solid-state fermentation of barley to make mulberry fungus fermented barley tea, it would not only significantly transform and utilize the various nutrients in barley, but also retain the unique medicinal components of mulberry fungus. This mulberry fungus fermented barley tea can effectively solve the shortcomings of mulberry fungus being difficult to cultivate and expensive, while greatly increasing the added value of traditional barley tea.

[0007] To date, there are few reports of using genuine mulberry fungus *Sanghuang* to ferment barley and produce medicinal fungal fermented barley tea. Therefore, it is urgent to select and breed a genuine *Sanghuang* strain suitable for solid-state fermentation of barley, and on this basis, develop a technology for making solid-state fermented barley tea from mulberry fungus, which has very important practical application value. Summary of the Invention

[0008] Technical problem: The technical problem to be solved by the present invention is to provide a strain of Phellinus linteus from the mulberry tree suitable for making fermented barley tea.

[0009] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned mulberry fungus strain in the production of mulberry fungus solid fermented barley tea.

[0010] Technical Solution: A mulberry tree suitable for making fermented barley tea, the mulberry tree *Sanghuangporus sanghuang* strain described herein is classified as *Sanghuangporus sanghuang* (Wild Mulberry No. 3), and was deposited on April 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode: 100101), with accession number CGMCC No. 41280. The *Sanghuangporus sanghuang* strain described in this invention belongs to the family Hymenochaetaceae, genus *Sanghuangporus*, and species *Sanghuangporus*. This strain was isolated from the fruiting body of a wild-type mulberry tree, and after successive generations of screening and domestication, it was named Wild Mulberry No. 3.

[0011] The characteristics of the mulberry strain *Phellinus linteus* No. 3 of this invention are as follows:

[0012] This strain is suitable for solid-state fermentation culture using barley grains as nutrients, producing a large amount of mycelium, and has the following characteristics:

[0013] (1) This strain can use barley grains as nutrients without the need to add other growth aids, and it grows quickly: after 45 days of cultivation, it can fill a 12*24cm (folded width*length) polypropylene bag, completing the process from inoculation to yellowing and maturity for harvesting.

[0014] (2) Good appearance: After the mycelium covers the entire barley grain and matures, it can secrete a large amount of yellow pigment, with a golden color and uniform mycelium shape; the barley grain does not turn black during solid fermentation.

[0015] (4) Firmness: After the mycelium turns yellow and matures, it is highly firm and can tightly wrap each grain of wheat, making it difficult to tear.

[0016] (5) Mycelium can penetrate into the inside of wheat grains and make full use of the nutrients in the wheat grains.

[0017] (6) Fresh and odorless: The barley obtained from solid-state fermentation has no off-odor and possesses the unique fresh aroma of mulberry mushroom. The mulberry mushroom solid-state fermented barley tea made from barley fermented with this strain has the following characteristics:

[0018] (1) It has both the mellow aroma of barley tea and the unique fragrance of mulberry wood mulberry.

[0019] (2) The tea liquor is more yellow and lustrous than commercially available barley tea;

[0020] (3) It can overcome the disadvantage of traditional barley tea being easy to cause "heatiness" and has no side effects such as "heatiness" when consumed for a long time;

[0021] (4) The total flavonoid content of the fermented barley tea (dried powder) obtained from the cultivation of mulberry linteus reached an average of 5.53 mg / g. It contains medicinal substances such as quercetin, naringenin chalcone, and kaempferol, as well as medicinal components of mulberry linteus such as citric acid D, dimer linteus, and pyranoflavone A.

[0022] The application of the aforementioned *Sanghuang* strains suitable for making fermented barley tea in the production of fermented barley tea includes the following steps:

[0023] 1. Preparation of barley culture medium

[0024] (1) Select plump, clean, and impurity-free dry barley grains.

[0025] (2) Add 3000ml of tap water to every 1000g of the above barley, soak at 25℃ for 2 hours, boil in boiling water and keep for 20 minutes, filter out the remaining water and set aside.

[0026] (3) Pack the cooked barley grains into a 12*24cm (folded width*length) polypropylene mushroom bag, insert a 1cm diameter plastic punch in the middle to punch a hole, remove the punch after punching, put on a plastic neck ring of appropriate size, and seal the opening with the mushroom cap.

[0027] (4) Sterilize the polypropylene bags containing barley grains at 121°C for 20 minutes to make barley grain culture medium for large-scale fermentation, and place them in a clean bench to cool for later use.

[0028] 2. Fermentation culture of Sanghuang mushroom from Mulberry Tree on Barley Medium

[0029] (1) The mulberry strain Sanghuang, which has been artificially domesticated for a long time, was inoculated into the polypropylene bag containing barley culture medium and cultured at 28°C in complete darkness.

[0030] (2) After 22 days of complete darkness, the indoor light was changed to 10-12 hours of weak light (50-100 lux) per day, and the rest of the time was dark, and then cultured for another 8 days.

[0031] (3) After 30 days, the pale yellow mycelium can cover the bag. Manually squeeze the bag to increase the gap between the grains inside the bag. At the same time, maintain 10-12 hours of weak light (200-300 lux) per day and darkness for the rest of the time. Continue to cultivate for 15 days.

[0032] (4) On day 45 of cultivation, the mushroom bag visibly turned golden yellow. Upon opening the plastic cap, the surface of the barley was covered with a large amount of golden yellow mycelial film. Breaking apart the fermented barley grains revealed a very tight structure, with the grains connected by mycelium and exhibiting uniform mycelium growth. The gaps between the barley grains were filled with golden yellow *Sanghuang* mycelium. A longitudinal cut with a scalpel revealed that most of the barley grains were embedded with golden yellow *Sanghuang* mycelium after solid-state fermentation with *Wild Mulberry No. 3*. The barley after solid-state fermentation with *Wild Mulberry No. 3* emitted a distinct, fresh aroma characteristic of *Sanghuang*. This indicated that the *Sanghuang* mycelium fermented in the barley culture medium had matured, and fermentation was complete.

[0033] 3. Processing of fermented barley tea made from mulberry leaves and mulberry fungus

[0034] (1) Knead the fermented barley by hand until each grain is separated, and dry it in an oven at 80°C until it reaches a constant weight.

[0035] (2) Place the dried barley grains fermented with mulberry mulberry fungus in an iron pot, stir-fry over low heat for 4 minutes, then stir-fry at 250°C for about 1.5 minutes. After smelling the roasted aroma of barley and the rich aroma of mulberry mulberry fungus, quickly remove it and cool it at room temperature.

[0036] (3) After cooling, the barley tea is packaged in food bags to produce solid fermented barley tea made from mulberry leaves and mulberry mushroom.

[0037] (4) Place the above-mentioned finished mulberry wood mulberry solid fermented barley tea in a cup, steep it with boiling water or boil it in water before drinking.

[0038] Beneficial effects:

[0039] This invention marks the first time a strain of *Sanghuang* fungus suitable for solid-state fermentation using barley as a nutrient has been domesticated and screened. The mycelium of this strain can penetrate the interior of barley grains, fully converting and utilizing the nutrients. The mycelium exhibits rapid growth (matures in just 45 days in a 12*24cm bag) and excellent appearance (golden color, and the fermented barley grains do not turn black). Fermented barley tea made from barley grains fermented with this strain has the following characteristics: the tea liquor is yellow and lustrous, possessing both the mellow aroma of barley tea and the unique refreshing aroma of *Sanghuang*; the fermented barley tea overcomes the "heatiness" problem associated with traditional barley tea, has a significantly higher total flavonoid content than commercially available barley tea, and contains medicinal components of *Sanghuang*. The *Sanghuang* mycelium formed by fermenting barley with this strain effectively solves the problems of difficult cultivation and high price of *Sanghuang*, while greatly increasing the added value of traditional barley tea. Attached Figure Description

[0040] Figure 1 This refers to the state in which the surface of barley grains is covered by the mycelium of Wild Mulberry No. 3.

[0041] Figure 2 The image shows the state of barley in the culture bag after 45 days of solid-state fermentation using Wild Mulberry No. 3.

[0042] Figure 3 It is the golden-yellow mycelium of Phellinus linteus growing between barley grains.

[0043] Figure 4 The image shows the cross-section of barley grains from the solid-state fermentation of Wild Mulberry No. 3.

[0044] Figure 5 A comparison of the liquor color of commercially available barley tea and mulberry wood sanghuang solid-fermented barley tea (left image: commercially available barley tea; right image: mulberry wood sanghuang solid-fermented barley tea). Detailed Implementation

[0045] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0046] Example 1: Preparation of barley culture medium

[0047] 1. Select plump, clean, and dry barley grains that are free of shriveled or impurities.

[0048] 2. Add 3000ml of tap water to every 1000g of the above barley, boil for 20 minutes, then filter out the remaining liquid and set aside.

[0049] 3. Place the cooked barley grains from step 2 above into tissue culture flasks, with 50g of cooked barley grains in each 200ml tissue culture flask, as barley culture medium.

[0050] 4. Sterilize the above culture medium at 121℃ under high temperature and pressure for 20 minutes, and then place it in a clean bench to cool and store for later use.

[0051] Example 2: Preliminary screening of various *Sanghuang* strains suitable for solid-state fermentation culture of barley

[0052] 1. Selection of strains

[0053] Five strains of Phellinus linteus from different origins were selected and grown in PDA medium. At the same time, various commercially available Phellinus linteus imitation strains were also selected, including: Phellinus linteus from poplar, Phellinus linteus from pine, Phellinus linteus from mulberry, Phellinus linteus from oak, and Phellinus coarse-haired fibrous fungus. Several other less common Phellinus linteus imitation strains were also selected, including Phellinus linteus from small-pore honeysuckle, Phellinus ring-zone, Phellinus brown inoculum, Phellinus laevigata, Phellinus fissura, Phellinus alpineus, Phellinus lacquer tree, and Phellinus weigela.

[0054] 2. Inoculation and culture:

[0055] (1) Inoculate the above-mentioned imitation Sanghuang and Sanghuang from mulberry trees into the culture medium of Example 1 respectively, and tighten the bottle cap;

[0056] (2) Incubate in a completely dark environment at 28℃ for 22 days.

[0057] (3) After 22 days, the room was kept in low light (50-100 lux) for 10-12 hours a day and in darkness for the rest of the time, and then cultured for another 8 days.

[0058] (4) After 30 days of cultivation, the growth status was investigated (Table 1). It was found that some of the imitation Sanghuang could not grow or had difficulty growing. Some of the growth failed to form yellow mycelium and had a sour and rotten odor, making them unsuitable as strains for fermenting barley tea. This indicates that all kinds of imitation Sanghuang are not suitable as solid-state fermented barley. Each mulberry tree Sanghuang can cover the surface of barley grains and has no odor, meeting the basic conditions for solid-state fermentation of barley grains. However, since it cannot form a large amount of yellow mycelium and the barley grains turn black and black liquid seeps out, further artificial domestication is needed.

[0059] Table 1. Growth status of various *Sanghuang* strains in barley grain medium

[0060]

[0061] Example 3: Domestication of Mulberry Phellinus linteus strains suitable for barley culture

[0062] 1. Preparation of acclimatization culture medium

[0063] (1) Take plump, clean, and dry barley grains that are not shriveled or impurities, and grind them into 300-mesh barley powder using a Chinese medicine pulverizer (25,000 rpm).

[0064] (2) Take a commercially available lemon, cut it into slices, and use a garlic press to extract the lemon juice. Set aside.

[0065] (3) Take 200g of fresh potatoes, chop them, add 100g of barley powder from step 1 above, add 10ml of lemon juice, boil them together and keep for 20 minutes, filter out the juice and remove the residue with two layers of gauze, add 20g of glucose and 15g of agar powder, add water to 1000ml, and sterilize at 121℃ for 20 minutes.

[0066] (4) After sterilization, place it in a clean bench and pour it into a petri dish. After cooling and solidification, prepare an acclimatization culture medium for later use.

[0067] 2. Inoculation and culture

[0068] (1) Five mulberry trees from different origins in Example 2 were selected, inoculated with the above-mentioned acclimatization culture medium, and cultured in a completely dark environment at 28°C for 22 days.

[0069] (2) After 22 days, the room was kept in low light (50-100 lux) for 10-12 hours a day and in darkness for the rest of the time, and then cultured for another 8 days.

[0070] (3) After 30 days of cultivation, observe the growth status of each strain in the above-mentioned acclimatization medium. Each time, select mycelia with good growth status (yellow color, uniform mycelia, no black spots) and inoculate them in the same acclimatization medium for subculture.

[0071] (4) Following the above method, the offspring are replaced once a month and domesticated for more than 3 years.

[0072] Example 4: Further screening of *Phellinus linteus* strains suitable for barley solid-state fermentation culture

[0073] 1. Prepare barley grain culture medium according to steps 1-4 in Example 1 and place it in a 200ml tissue culture flask.

[0074] 2. The five domesticated mulberry strains of Phellinus linteus from Example 3 were inoculated into the above-mentioned barley culture medium and cultured in a completely dark environment at 28°C for 22 days.

[0075] 3. After 22 days, incubate the cells in low light (50-100 lux) for 10-12 hours daily and in darkness for the rest of the time, and then incubate for another 8 days.

[0076] 4. After 30 days of culture, it was found that all five strains of *Sanghuang* from *Morus alba* selected for screening could grow normally in the above-mentioned barley culture medium. Three strains only formed a small amount of yellow mycelium and were discarded; one strain formed a large amount of yellow mycelium, but a small amount of black liquid oozing from blackened barley grains remained in the culture flask, and it was also discarded; only one strain of *Morus alba* showed outstanding overall characteristics. This strain could use barley grains as its sole nutrient source without the addition of other growth aids, and it grew rapidly. After 30 days of culture in a 200ml tissue culture flask, the surface of the barley grains was covered with a large amount of yellow mycelium. Figure 1 The barley grains did not turn black during solid-state fermentation, and no black liquid seeped out of the culture bottle. When the bottle cap was opened, the unique fragrance of authentic mulberry mushroom could be smelled. This strain was selected as the target strain after domestication and used for subsequent solid-state fermentation culture of barley grains.

[0077] Example 5: Identification and Nomenclature of the Target Strain

[0078] The target strain was derived from the trunk of a wild-type mulberry tree (non-cultivated). DNA from the *Sanghuangporus sanghuang* strain screened in Example 4 was extracted and amplified by PCR using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') for the internal transcribed spacer (ITS) sequence of the ribosomal gene rDNA. The amplified product was gel-recovered, purified, and sequenced. The sequence was completely identical to that of *Sanghuangporus sanghuang* with GenBank accession number MZ424788.1, confirming that all tested strains belong to the family Hymenochaetaceae, genus *Sanghuangporus*, and species *Sanghuangporus*. This strain was named "Wild Mulberry Yuan No. 3" (CGMCC No. 41280).

[0079] Example 6: Large-scale fermentation culture of Phellinus linteus in barley culture medium

[0080] The purpose of this embodiment is to enable large-scale, efficient fermentation culture. The small container (200ml tissue culture bottle) used for screening mulberry fungus strains in Example 4 is replaced with a polypropylene bag with a folded width * length of 12 * 24cm. The specific steps are as follows:

[0081] 1. Prepare cooked barley grains according to steps 1-2 in Example 1.

[0082] 2. Pack the cooked barley grains into the polypropylene spawn bag, filling it to about 2 / 3 of the bag's opening height. Insert a 1cm diameter plastic punch into the middle to make a hole. After punching the hole, remove the punch, then put on a plastic neck ring of appropriate size, and seal the bag with the spawn cap.

[0083] 3. Sterilize the polypropylene bags containing barley grains at 121°C for 20 minutes to prepare barley grain culture medium for large-scale fermentation. Place the medium in a clean bench to cool and store for later use.

[0084] 4. The domesticated mulberry strain Sanghuang No. 3 was inoculated into the polypropylene bags containing barley culture medium and cultured in a completely dark environment at 28°C.

[0085] 5. After culturing in complete darkness for 22 days, the indoor light was changed to 10-12 hours of low light (50-100 lux) per day, with darkness for the rest of the time, and then cultured for another 8 days.

[0086] 6. Because the weight of the barley grains in the spawn bag is higher than that in the tissue culture flask, the cultivation time is correspondingly longer. After 30 days, the pale yellow mycelium can fully cover the spawn bag. Manually squeeze the spawn bag to increase the gaps between the barley grains inside. At the same time, maintain 10-12 hours of weak light (200-300 lux) per day and darkness for the rest of the time, and continue cultivation for 15 days.

[0087] 7. On day 45 of cultivation, the substrate bags were visibly turned completely golden yellow. Figure 2 This confirmed that the strain can use barley grains as a single nutrient source and grows rapidly without the addition of other growth aids. After 45 days of cultivation, the mycelium can fully colonize a 12*24cm (folded width*length) polypropylene bag. Opening the plastic cap and removing the neck ring reveals that the surface of the barley is covered with a large amount of golden-yellow mycelial film, and the barley grains do not turn black during solid-state fermentation. At this point, breaking apart the fermented barley grains reveals that the grains are connected by mycelium, resulting in a very tight and firm structure with good appearance. The gaps between the barley grains are filled with golden-yellow, uniform mulberry mycelium (…). Figure 3 The mycelium tightly wraps each grain of barley, making it difficult to tear. When barley fermented with Wild Mulberry No. 3 is cut longitudinally with a scalpel, golden-yellow mulberry mycelium is visible embedded in most grains, indicating that Wild Mulberry No. 3 mycelium can fully utilize and transform nutrients on the surface and inside of the grains, producing a large amount of golden-yellow mycelium. Figure 4Meanwhile, the barley after solid-state fermentation has no off-odor, but clearly emits the unique, fresh aroma of mulberry fungus (Sanghuang). This indicates that the mulberry fungus mycelium fermented in the barley medium has matured and fermentation is complete. Because barley is inexpensive, and the fermentation method using barley as a nutrient is simple, the resulting mulberry fungus mycelium effectively solves the problems of difficult cultivation and high price associated with mulberry fungus. Furthermore, since the fermented barley tea imparts medicinal components to mulberry fungus, it greatly enhances the added value of traditional barley tea.

[0088] Example 7: Frying of barley tea fermented with mulberry wood and Phellinus linteus

[0089] 1. Pour out the barley grains that have been fermented in the polypropylene bags grown in Example 5, knead them by hand until each grain is dispersed, and place them in an oven to dry at 80°C until constant weight.

[0090] 2. Place the dried barley grains fermented with mulberry and mulberry fungus in an iron pot, stir-fry over low heat for 4 minutes, then stir-fry at 250℃ for about 1.5 minutes. After smelling the roasted aroma of barley and the rich aroma of mulberry and mulberry fungus, quickly remove it and cool it to room temperature.

[0091] 3. After cooling, package in food bags to produce mulberry wood mulberry solid fermented barley tea.

[0092] 4. Place the above-mentioned solid-fermented barley tea made from mulberry leaves and mulberry fungus in a cup, steep with boiling water for 5 minutes, or boil in boiling water for 2 minutes. You will smell the aroma of barley and the unique, refreshing fragrance of mulberry leaves and mulberry fungus. Let it cool slightly before drinking. Compared to commercially available barley tea, the solid-fermented barley tea made from mulberry leaves and mulberry fungus has a more yellow and lustrous color. Figure 5 It has a richer and more mellow flavor. Long-term consumption does not cause stomach discomfort or other adverse reactions such as "internal heat".

[0093] Example 8: Determination of medicinal component content in fermented barley tea made from mulberry leaves and Phellinus linteus

[0094] 1. Take commercially available barley tea and the solid fermented barley tea product obtained from mulberry linden barley tea in step 3 of Example 6, and pulverize it into 300-mesh powder using a traditional Chinese medicine pulverizer (25,000 rpm). Use this powder as a sample to determine the medicinal component content of the fermented barley tea.

[0095] 2. A standard curve equation was established using rutin as a standard, and the total flavonoid content in the samples was determined by the NaNO2-Al(NO3)3 colorimetric method. The average total flavonoid content of mulberry barley tea was 5.53 mg / g, which was much higher than the average total flavonoid content of commercially available barley tea (0.79 mg / g).

[0096] 3. The medicinal components in step 1 of this embodiment were determined by HPLC. Quercetin, naringenin chalcone, and kaempferol were detected at a wavelength of 365 nm. The contents of quercetin, naringenin chalcone, and kaempferol in mulberry barley tea were found to be 0.562 mg / g, 0.0041 mg / g, and 0.039 mg / g, respectively. Quercetin, naringenin chalcone, and kaempferol were not detected in commercially available barley tea, presumably due to their low content.

[0097] 4. Using ultra-high performance liquid chromatography-tandem quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS), we were able to rapidly identify and characterize the monomeric compounds in the components. We discovered that the mulberry barley tea contains active substances found only in mulberry barley, such as citricin D, dimeric morrizoin, and pyranoflavone A, which are not present in commercially available barley tea.

Claims

1. A strain of *Sanguisorba officinalis* suitable for making fermented barley tea, wherein the strain is classified and named *Sanguisorba officinalis*. Sanghuangporus sanghuang The strain was deposited on April 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 41280.

2. The application of the *Sanghuang* strain of *Mulberry Tree* suitable for making fermented barley tea as described in claim 1 in the production of fermented barley tea made from *Mulberry Tree*.

3. A method for preparing fermented barley tea made from mulberry leaves and Phellinus linteus, characterized in that, Using barley as the fermentation substrate and the *Sanghuang* strain of mulberry tree as the fermentation strain according to claim 1, fermentation and culture are carried out, followed by roasting, to obtain fermented barley tea made from *Sanghuang* mulberry tree.

4. The method for preparing fermented barley tea using mulberry and Phellinus linteus according to claim 3, characterized in that, Includes the following steps: (1) After cooking, the barley grains are drained and placed into polypropylene bags for sterilization. After cooling, the barley grains are inoculated with the mulberry fungus strain suitable for making fermented barley tea as described in claim 1 and cultured at 28°C in complete darkness for 22 days. (2) After 22 days of culture, the indoor light was changed to 10-12 hours and 50-100 lux per day, with darkness for the rest of the time, and then cultured for another 8 days. (3) After 30 days of cultivation, when the pale yellow mycelium has covered the bag, manually squeeze the bag to increase the gap between the grains inside the bag. At the same time, maintain 10-12 hours and 200-300 lux per day, and keep the rest of the time in darkness. Continue cultivation for 15 days. (4) After a total of 45 days of cultivation, when the mushroom bags have turned golden yellow to the naked eye, open the mushroom bags and collect the barley fermented from the mulberry trees. (5) Take out the fermented barley grains and knead them until each grain is separated. Place them in an oven and dry them to a constant weight. (6) Place the dried barley grains fermented with mulberry mulberry in an iron pot and stir-fry them. After smelling the roasted aroma of barley and the rich aroma of mulberry mulberry, quickly remove them and cool them at room temperature to make the finished product of mulberry mulberry fermented barley tea.

5. The method for preparing fermented barley tea from mulberry trees according to claim 4, characterized in that, In step (1), the barley grains are cooked as follows: add 3000ml of tap water to every 1000g of barley grains, soak at 25℃ for 2 hours, boil in boiling water and keep for 20 minutes, filter out the residual water, and obtain the boiled barley grains.

6. The method for preparing fermented barley tea using mulberry and Phellinus linteus according to claim 4, characterized in that, In step (1), after the barley grains are put into the polypropylene spawn bag, a plastic punch with a diameter of 1cm is inserted into the middle of the barley grains to make a hole. After the hole is made, the punch is removed, a plastic neck ring is put on, and the spawn bag is sealed with a cap.

7. The method for preparing fermented barley tea using mulberry and Phellinus linteus according to claim 4, characterized in that, In step (1), the sterilization process is carried out under the following conditions: 121°C for 20 minutes.

8. The method for preparing fermented barley tea from mulberry trees according to claim 4, characterized in that, In step (5), the drying temperature is 80°C.

9. The method for preparing fermented barley tea using mulberry and Phellinus linteus as described in claim 4, characterized in that, In step (6), the stir-frying conditions are as follows: first stir-fry over low heat for 4 minutes, then stir-fry at 250℃ for about 1.5 minutes.

10. Barley tea prepared by the method of fermenting barley tea from mulberry trees as described in any one of claims 3 to 9.

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