A taiwanofungus camphoratus and bitter buckwheat fermented tea and a preparation method thereof

Abstract

The application discloses a preparation method of a Taiwanofungus camphoratus and tartary buckwheat fermented tea, and comprises the following steps: activating Taiwanofungus camphoratus (Taiwanofungus camphoratus) MRC156-2K-11 with a biological preservation number of CCTCC No. M 20261055 and preparing a seed liquid; adding a culture medium containing tartary buckwheat in a 250 mL container, then inoculating the seed liquid of the Taiwanofungus camphoratus strain MRC156-2K-11, and performing solid-state fermentation to prepare the Taiwanofungus camphoratus and tartary buckwheat fermented tea. The Taiwanofungus camphoratus and tartary buckwheat fermented tea prepared by the application realizes the synergistic enrichment of functional components of Taiwanofungus camphoratus and inherent nutrients of tartary buckwheat, is a fermented tea product with higher content of active ingredients such as triterpenes, more definite health care functions and better flavor acceptance, fills the product blank of the Taiwanofungus camphoratus and tartary buckwheat fermented tea, expands the deep processing and high-value application path of the two, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fermented tea made from Antrodia camphorata and buckwheat, and its preparation method. Background Technology

[0002] Buckwheat (Fagopyrum tataricum (L.) Gaertn.) is one of my country's oldest grain crops, with a cultivation history of over 2000 years. Modern research shows that buckwheat is rich in protein, dietary fiber, vitamins, and other nutrients, as well as various bioactive components such as flavonoids, polyphenols, and D-chiroinositol, which possess antioxidant, blood sugar-lowering, and anti-inflammatory health benefits. Buckwheat tea, as a natural grain substitute, is widely popular due to its long history of consumption, unique wheat aroma, and health benefits. However, its relatively rough taste and noticeable bitterness directly affect consumer acceptance. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide an Antrodia camphorata-tartary buckwheat fermented tea and its preparation method.

[0004] The technical solution adopted in this invention is as follows:

[0005] A method for preparing Antrodia camphorata and buckwheat fermented tea includes the following steps:

[0006] S1. Activate Antrodia camphoratus (Taiwanofungus camphoratus) MRC156-2K-11 with biological preservation number: CCTCC No.M 20261055 and prepare seed liquid;

[0007] S2. Add a culture medium containing buckwheat to a container, then inoculate with the seed liquid of Antrodia camphorata strain MRC156-2K-11 and carry out solid-state fermentation to obtain Antrodia camphorata buckwheat fermented tea.

[0008] Preferably, in S1, the Antrodia camphorata strain MRC156-2K-11 is selected and activated on potato dextrose agar medium. The activated strain is cut into 2 cm pieces, crushed with a sterilized flat bamboo stick, and inoculated into 50 mL of liquid seed medium. It is then cultured in the dark at 28 ℃ and 150 r / min for 7 days to obtain the seed liquid.

[0009] Preferably, in step S2, the solid-state fermentation is carried out in the dark for 20 days at a fermentation temperature of 28°C.

[0010] Preferably, in step S2, the volume of tartary buckwheat accounts for 20%-30% of the container volume; the inoculation amount of seed liquid is 10-11% of the tartary buckwheat volume.

[0011] Further preferably, in S2, the culture medium containing tartary buckwheat in the 250mL container has the following formula: 18~27g tartary buckwheat, 0.63~0.94g glucose, 0.21~0.32g lactose, 1.25~1.87g oats, 0.42~0.62g red beans, 5~7.5 mg vitamin B1, 10 mL water, and natural pH.

[0012] The present invention also provides fermented Antrodia camphorata and buckwheat tea prepared by the above preparation method.

[0013] The beneficial effects of this invention are as follows: This invention combines Antrodia camphorata (Taiwanofungus camphoratus) MRC156-2K-11 (biological preservation number: CCTCC M 20261055) with tartary buckwheat, which is both a food and a medicine. Using the high triterpenoid-producing Antrodia camphorata strain MRC156-2K-11 as the fermentation strain and tartary buckwheat as the main substrate, a solid-state fermentation process is used to obtain an Antrodia camphorata-tartary buckwheat fermentation co-culture. This achieves the synergistic enrichment of the functional components of Antrodia camphorata and the inherent nutrients of tartary buckwheat, thereby producing a fermented tea product with higher content of active ingredients such as triterpenes, clearer health benefits, and better flavor acceptance. This fills the product gap of Antrodia camphorata-tartary buckwheat fermented tea, expands the deep processing and high-value application path of both, and is suitable for large-scale industrial production. Attached Figure Description

[0014] Figure 1 This is the standard curve for oleanolic acid.

[0015] Figure 2 This is the standard curve for glucose.

[0016] Figure 3 This is the standard curve for quercetin.

[0017] Figure 4 The effect of fermentation time on the yield of triterpenoids in fermented Antrodia camphorata and buckwheat tea;

[0018] Figure 5 The effect of fermentation temperature on the yield of triterpenoids in fermented Antrodia camphorata and buckwheat tea;

[0019] Figure 6 The effect of inoculum size on the yield of triterpenoids in Antrodia camphorata fermented tea made from buckwheat;

[0020] Figure 7 The effect of sample loading on the yield of triterpenoids in Antrodia camphorata fermented tea made from buckwheat;

[0021] Figure 8 The effect of carbon source on the yield of triterpenoids in Antrodia camphorata fermented tea;

[0022] Figure 9 Effect of nitrogen source on the yield of triterpenoids in Antrodia camphorata and buckwheat fermented tea;

[0023] Figure 10 The effect of carbon source complex ratio on the yield of triterpenes in Antrodia camphorata and buckwheat fermented tea;

[0024] Figure 11 The effect of nitrogen source ratio on the yield of triterpenes in Antrodia camphorata and buckwheat fermented tea.

[0025] Figure 12 The effects of added total carbon and nitrogen content and ratio on the yield of triterpenoids in Antrodia camphorata and buckwheat fermented tea;

[0026] Figure 13 The effect of the interaction between sample loading and inoculum size on the yield of triterpenoids in Antrodia camphorata fermented tea made from buckwheat;

[0027] Figure 14 The effect of the interaction between sample loading and fermentation time on the yield of triterpenoids in fermented Antrodia camphorata and buckwheat tea;

[0028] Figure 15 The effect of the interaction between inoculum size and fermentation time on the yield of triterpenoids in fermented Antrodia camphorata and buckwheat tea;

[0029] Figure 16 This is a curve showing the change in mouse body weight;

[0030] Figure 17 Comparison of major organ coefficients among mice in each group.

[0031] The *Antrodia camphorata* strain MRC156-2K-11 of this invention was deposited at the China Center for Type Culture Collection (CCTCC) on May 22, 2026, with accession number CCTCC No. M 20261055. It is classified as *Antrodia camphorata*, with the Latin scientific name *Taiwanofungus camphoratus*. According to the phylogenetic classification and identification of fungi, this strain belongs to the order Apocynaceae, family Polyporaceae, genus *Philothorax*, and is a perennial fungus. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] The *Antrodia camphorata* strain MRC156-2K-11 in this invention is derived from the original *Antrodia camphorata* strain MRC156 via Co... 60 - Obtained through physical mutation induced by gamma ray irradiation, the specific preparation method is as follows:

[0034] 1. Preparation of spore suspension

[0035] The original strain of *Antrodia camphorata*, MRC156, was inoculated onto PDA plates and cultured at 28 ℃ for 14 days. After mycelial contamination, the conidia were washed away with sterile physiological saline, and the conidia suspension was collected. The concentration of the conidia suspension was adjusted to 5 × 10⁻⁶. 7per mL.

[0036] 2. Co 60 - Gamma-ray irradiation treatment and lethality determination

[0037] Take 10 mL of spore suspension into a sterile centrifuge tube and perform Co. 60 - Gamma-ray irradiation treatment. Irradiation dose gradients were set at 0 (control), 1000, 2000, and 4000 Gy, with three replicates for each dose. Immediately after irradiation, the spore suspension was serially diluted, spread onto PDA plates, and incubated at 28 °C in the dark for 7 days, followed by colony counting. Spore survival gradually decreased with increasing irradiation dose. At a dose of 1000 Gy, the spore lethality reached 95.5%; at 2000 Gy, the lethality reached 99.9%, with only a very small number of spores surviving; at 4000 Gy, no surviving colonies were observed, and the lethality was 100%.

[0038] 3. Screening of mutant strains

[0039] Mutant strains of *Antrodia camphorata* were activated on PDA medium. The activated mycelium was cut into 2 cm pieces, crushed with sterilized flat bamboo sticks, and inoculated into 50 mL of liquid seed culture medium. The mixture was incubated in the dark at 28 ℃ and 150 r / min for 20 days. Mycelium was collected, and its dry weight and triterpenoid content were measured. Using the original *Antrodia camphorata* strain MRC156 as a control, the triterpenoid content growth rate of each strain was calculated. Finally, the *Antrodia camphorata* strain MRC156-2K-11, with superior triterpenoid production capacity, was selected.

[0040] In this invention, the triterpenoid content is determined using the vanillin-glacial acetic acid colorimetric method:

[0041] Accurately weigh 0.50 g of the dried and pulverized fermentation sample, add 10 mL of anhydrous ethanol, and extract by sonication for 30 min, then let stand overnight. After centrifugation at 8000 r / min, take 100 μL of the supernatant and place it in a 25 mL stoppered test tube. Evaporate the ethanol in a boiling water bath until no alcohol odor remains in the tube. Then, add 100 μL of 5% vanillin-acetic acid solution and 800 μL of perchloric acid solution sequentially, stopper and mix well, heat in a 60℃ constant temperature water bath for 20 min, remove and immediately cool in an ice water bath, add 5 mL of acetic acid, shake well and let stand. Using the corresponding reagents as blank controls, measure the absorbance at a wavelength of 550 nm. Plot a standard curve with oleanolic acid mass (mg) as the x-axis (X) and absorbance as the y-axis (Y). Figure 1 The total triterpenoid content in the sample was calculated based on the standard curve.

[0042] The polysaccharide content was determined using the phenol-sulfuric acid method.

[0043] Accurately weigh 0.50 g of the dried and pulverized fermentation sample, add 5 mL of ultrapure water to fully suspend it, then add 20 mL of anhydrous ethanol and sonicate for 30 min. Centrifuge at 4000 rpm for 5 min, discard the supernatant (mainly monosaccharides, oligosaccharides, and small molecule impurities), add 10 mL of 80% ethanol solution to the precipitate for washing, centrifuge again and discard the supernatant. Transfer the obtained precipitate to a stoppered container, add 30 mL of ultrapure water, and extract in a boiling water bath for 2 h. Filter the extract, transfer all the filtrate to a 500 mL volumetric flask, dilute to the mark with ultrapure water, and mix well. Accurately transfer 1.0 mL of the above solution into a 10 mL stoppered test tube, add 1.0 mL of 5% phenol solution, mix well, and then quickly add 7.0 mL of concentrated sulfuric acid. Shake well immediately, and then add ultrapure water to the mark. Place the test tube in a boiling water bath and heat for 30 min. After removing it, cool it to room temperature under running water and measure the absorbance at a wavelength of 490 nm. Plot a standard curve with glucose mass (mg) on ​​the x-axis (X) and absorbance on the y-axis (Y). Figure 2 The polysaccharide content in the sample was calculated based on the standard curve.

[0044] The flavonoid content was determined using the aluminum trichloride colorimetric method.

[0045] Accurately weigh 1.00 g of the dried and pulverized fermentation sample, add 50 mL of methanol, and extract ultrasonically at 40℃ for 30 min. Centrifuge at 8000 rpm for 15 min and collect the supernatant. Repeat the extraction of the precipitate twice using the above method, and combine the supernatants from the three extractions. Concentrate the combined solution under reduced pressure at 40℃ to near dryness, dissolve it in methanol, and transfer it to a 25 mL volumetric flask. Dilute to the mark and mix well. Accurately transfer 0.25 mL of the above solution to a 10 mL brown volumetric flask, add 2.0 mL of 0.1 mol / L aluminum trichloride-methanol solution and 3.0 mL of 1.0 mol / L potassium acetate aqueous solution, dilute to the mark with methanol, mix well, and let stand at room temperature in the dark for 1 h. Measure the absorbance at 420 nm. Plot a standard curve with quercetin mass (mg) on ​​the x-axis (X) and absorbance on the y-axis (Y). Figure 3 The flavonoid content in the sample was calculated based on the standard curve.

[0046] Example 1

[0047] Preparation method of Antrodia camphorata and buckwheat fermented tea.

[0048] 1. The Antrodia camphorata strain MRC156-2K-11 stored in glycerol tubes was inoculated onto PDA solid medium for activation. The activated strain was cut into 2 cm pieces, crushed with a sterilized flat bamboo stick, and inoculated into 50 mL of liquid seed medium (the liquid medium formula is 30 g / L oats, 30 g / L glucose, 0.1 g / L vitamin B1, natural pH). It was cultured at 28 ℃ and 150 rpm for 7 days to obtain the seed liquid.

[0049] 2. Single-factor experiment on fermentation conditions: Solid-state fermentation was carried out on high-yield triterpenoid strains to investigate the effect of solid-state fermentation conditions on the accumulation of total triterpenoid compounds in fermented Antrodia camphorata and buckwheat tea.

[0050] (1) Determination of the optimal fermentation time

[0051] In a 250 mL Erlenmeyer flask, the inoculum was controlled at 10% (10 mL seed culture), containing 36 g of tartary buckwheat (40% of the container volume), 20 mL of water, 3 g of glucose, 3 g of red adzuki bean, and 10 mg of vitamin B1. The flask was incubated in the dark at 28 ℃. Samples were taken at 5, 10, 15, 20, 25, 30, 35, and 40 days to determine the yield of total triterpenoid compounds. The results are as follows: Figure 4 As shown, the triterpenoid yield peaked on day 20 (3.80 mg / g). Therefore, the optimal fermentation time was determined to be 20 days.

[0052] (2) Determination of the optimal fermentation temperature

[0053] In a 250 mL Erlenmeyer flask, the inoculum was controlled at 10% (10 mL seed culture), containing 36 g of tartary buckwheat (40% of the container volume), 20 mL of water, 3 g of glucose, 3 g of red adzuki bean, and 10 mg of vitamin B1. The flask was incubated in the dark at 24, 26, 28, 30, and 32 °C for 20 days, and the yield of total triterpenoids was determined. The results are as follows: Figure 5 As shown, the triterpenoid yield is highest at 28 °C (3.86 mg / g). Therefore, the optimal temperature is determined to be 28 °C.

[0054] (3) Determination of the optimal inoculation amount

[0055] In a 250 mL Erlenmeyer flask, 36 g of tartary buckwheat (40% of the container volume), 20 mL of water, 3 g of glucose, 3 g of red adzuki beans, and 10 mg of vitamin B1 were added and cultured in the dark at 28 °C for 20 days. Inoculum sizes were set at 5%, 10%, 15%, and 20%, respectively, and the yield of total triterpenoid compounds was determined. Results are as follows... Figure 6 As shown, the triterpenoid yield is highest (3.87 mg / g) when the inoculum is 10%. Therefore, the optimal inoculum is determined to be 10%.

[0056] (4) Determination of the optimal sample size

[0057] In 250 mL Erlenmeyer flasks, sample loading amounts (matrix percentage of flask volume) were set at 10%, 20%, 30%, 40%, 50%, and 60%, respectively. The corresponding culture medium formulations are shown in Table 1. The samples were incubated in the dark at 28 °C for 20 days, and the yield of total triterpenoid compounds was determined. Results are as follows: Figure 7 As shown, the maximum value (6.07 mg / g) is reached at a 20% sample loading. Therefore, the optimal sample loading is determined to be 20%.

[0058] Table 1. Culture medium formulations for different sample volumes

[0059]

[0060] (5) Screening of the best carbon source

[0061] In a 250 mL Erlenmeyer flask, 18 g of tartary buckwheat (20% of the container volume) was inoculated with 10% (5 mL of seed culture), 10 mL of water, 1.5 g of adzuki bean, and 5 mg of vitamin B1. The flask was incubated in the dark at 28 ℃ for 20 days. The 1.5 g carbon source was selected from lactose, mannitol, glucose, maltose, and sucrose (substituted with equal carbon content). The yield of total triterpenoids was determined. Results are as follows: Figure 8 As shown, the triterpenoid yield is highest when glucose is the carbon source (5.97 mg / g). Therefore, glucose is determined to be the optimal carbon source.

[0062] (6) Screening of the optimal nitrogen source

[0063] In a 250 mL Erlenmeyer flask, 18 g of tartary buckwheat (20% of the container volume) was inoculated with 10% (5 mL of seed culture), 10 mL of water, 1.5 g of glucose, and 5 mg of vitamin B1. The flask was incubated in the dark at 28 °C for 20 days. 1.5 g of nitrogen source was used from corn flour, tartary buckwheat, oats, red beans, Job's tears, and wheat bran (substituted with equal nitrogen content). The yield of total triterpenoid compounds was determined. Results are as follows: Figure 9 As shown, the highest triterpenoid yield (5.88 mg / g) was observed when oats were the nitrogen source. Therefore, oats were determined to be the optimal nitrogen source.

[0064] (7) Optimization of carbon source composite ratio

[0065] In a 250 mL Erlenmeyer flask, 18 g of tartary buckwheat (20% of the container volume) was inoculated with 10% (5 mL of seed culture), 10 mL of water, 1.5 g of oats, and 5 mg of vitamin B1. The flask was incubated in the dark at 28°C for 20 days. 1.5 g of total carbon source was added at glucose:lactose ratios of 1:1, 2:1, 3:1, and 4:1, respectively, and the yield of total triterpenoids was determined. The results are as follows: Figure 10 As shown, the triterpenoid yield (8.19 mg / g) is highest when the carbon source ratio is glucose:lactose = 3:1. Therefore, the optimal carbon source ratio is determined to be glucose:lactose = 3:1.

[0066] (8) Optimization of nitrogen source composite ratio

[0067] In a 250 mL Erlenmeyer flask, 18 g of tartary buckwheat (20% of the container volume) was inoculated with 10% (5 mL of seed culture), 10 mL of water, 1.5 g of glucose, and 5 mg of vitamin B1. The flask was incubated in the dark at 28 °C for 20 days. 1.5 g of total nitrogen source was added at oat:red bean ratios of 1:1, 2:1, 3:1, and 4:1, respectively, and the yield of total triterpenoid compounds was determined. The results are as follows: Figure 11 As shown, the highest triterpenoid yield (7.69 mg / g) was achieved when the nitrogen source ratio was oat:red bean = 3:1. Therefore, the optimal nitrogen source ratio was determined to be oat:red bean = 3:1.

[0068] (9) Optimization of carbon and nitrogen source combination and total amount

[0069] The effects of different total added carbon and nitrogen sources (2.0, 2.5, 3.0, 3.5, 4.0 g) and carbon-to-nitrogen ratios (1:1, 1:2, 1:3, 2:1, 3:1) on triterpenoid yield were investigated using fixed carbon source combinations (glucose:lactose = 3:1) and nitrogen source combinations (oat:red bean = 3:1). Specific formulations are shown in Table 2. Results are as follows: Figure 12 As shown, it can be seen that when the total amount of added carbon and nitrogen is 2.5 g (i.e., accounting for 12% of the matrix) and the carbon-nitrogen ratio is 1:2, the triterpenoid yield (10.25 mg / g) is the highest. Therefore, the optimal total amount of added carbon and nitrogen is determined to be 2.5 g (i.e., accounting for 12% of the matrix) and the carbon-nitrogen ratio is 1:2.

[0070] Table 2 Experimental formulation for total added carbon and nitrogen and their proportions

[0071]

[0072] Based on the results of single-factor experiments, the optimal culture medium formula for fermenting tartary buckwheat using the Antrodia camphorata strain MRC156-2K-11 is: 18 g tartary buckwheat, 0.63 g glucose, 0.21 g lactose, 1.25 g oats, 0.42 g red beans, 5 mg vitamin B1, 10 mL water, and natural pH; the optimal fermentation conditions are: 28℃ for 20 days.

[0073] Example 2

[0074] Response surface methodology for optimizing key process parameters

[0075] Under optimal culture medium conditions, based on the results of single-factor experiments involving fermentation time, fermentation temperature, inoculum size, and sample loading, fermentation time (A), inoculum size (B), and sample loading (C) were selected as factors significantly affecting triterpenoid yield. A three-factor, three-level response surface methodology (BBD) was designed using Box-Behnken Design (BBD), with the total triterpenoid yield as the response value. A total of 17 experiments (including 5 central experiments) were designed. Factor level codes are shown in Table 3. Experimental results were analyzed using Design-Expert software and are shown in Table 4.

[0076] Table 3. Coding table of experimental factors in response surface methodology for Antrodia camphorata solid-state fermentation.

[0077]

[0078] Table 4. Analysis of Variance Table for Quadratic Regression Model

[0079]

[0080] The fitted equation obtained from the response surface methodology is Y = 10.25 - 0.20A + 2.32B + 0.72C - 1.13AB + 0.40AC + 2.07BC - 0.43A² - 3.49B² - ​​2.57C². Table 4-6 shows that the model F-value is 11.17, and the P-value is 0.0022 < 0.01, indicating that the quadratic regression model is highly significant. The P-value for the lack-of-fit term is 0.0937 > 0.05, which is not significant, indicating that the model fits well and there are no factors indicating lack of fit. The model's coefficient of determination R² = 0.9349, and Adj-R² = 0.8512, indicating that the model fits the experiment well, with small experimental errors, and can be used to analyze and predict the triterpenoid yield from Antrodia camphorata solid-state fermentation.

[0081] Depend on Figure 13 It can be seen that there is a certain interaction trend between the sample loading volume and the inoculum volume, but it does not reach a statistically significant level; Figure 14 It can be seen that the interaction between sample loading and fermentation time is very weak; Figure 15 It can be seen that the interaction between inoculum size and fermentation time is extremely significant. Therefore, using the optimization function of Design-Expert software, with the goal of maximizing triterpenoid yield, the optimal process parameters were obtained as follows: sample loading 30%, inoculum size 11%, fermentation time 20 days, and the model predicted a triterpenoid yield of 9.715 mg / g. The corresponding optimized culture medium formulation is: 27 g buckwheat substrate, 0.94 g glucose, 0.32 g lactose, 1.87 g oats, 0.62 g red beans, and 7.5 mg vitamin B1.

[0082] Three parallel validation experiments were conducted under these optimized conditions, and the average actual triterpenoid yield was measured to be 10.287 mg / g, which is very close to the model prediction. The relative error was only 5.6%, indicating that the response surface model is reliable and the optimization of process parameters has practical value.

[0083] Example 3

[0084] Quality Analysis of Antrodia camphorata and Buckwheat Fermented Tea

[0085] (1) Determination of the content of active ingredients in Antrodia camphorata and buckwheat tea

[0086] The contents of crude polysaccharides, total flavonoids, and total triterpenes in Antrodia camphorata before and after fermentation with tartary buckwheat were determined by the phenol-sulfuric acid method, the aluminum trichloride colorimetric method, and the vanillin-glacial acetic acid colorimetric method, respectively. The data obtained are shown in Table 5.

[0087] Table 5. Comparison of active ingredient content between fermented and unfermented buckwheat tea made with Antrodia camphorata.

[0088]

[0089] The experimental results showed that the content of the above three functional components in the fermented Antrodia camphorata and buckwheat tea was significantly increased after fermentation, indicating that solid-state fermentation improved the nutritional and functional value of buckwheat.

[0090] (2) Sensory evaluation

[0091] To objectively evaluate the sensory quality of Antrodia camphorata-fermented buckwheat tea, this study developed a specific sensory evaluation form (Table 5-1). The evaluation panel consisted of 10 trained food professionals.

[0092] Brewing method: Accurately weigh 5.0 g of granular tea sample, place it in a 150 mL tea tasting cup, pour in boiling water until full, start timing immediately, brew for 5 minutes, then strain the tea for evaluation.

[0093] Evaluation Process: Evaluators independently score each sample. First, they observe the appearance and shape of the dry tea, then evaluate the liquor color, aroma (hot and warm smells), and taste in sequence. Each score is multiplied by its weight and summed to obtain the total score (out of 100). The final result is the average of the scores from 10 evaluators. See Table 6 for detailed sensory evaluation criteria.

[0094] Table 6 Sensory Evaluation Table of Antrodia camphorata and buckwheat fermented tea

[0095]

[0096] The sensory evaluation results are shown in Table 7. The overall score of the fermented tea was 91.3 ± 1.4 points, which is basically the same as that of the unfermented tea (91.5 ± 1.2 points). Although the total scores are similar, the flavor characteristics have undergone a qualitative change, shifting from a single grain aroma to a complex fungal aroma. This indicates that the fermentation of Antrodia camphorata successfully endowed the buckwheat tea with a unique fungal aroma while maintaining its original excellent quality, resulting in a product with good sensory acceptance.

[0097] Table 7 Sensory Evaluation Results of Antrodia camphorata and Buckwheat Fermented Tea

[0098]

[0099] Example 4

[0100] Preliminary safety evaluation of Antrodia camphorata fermented buckwheat tea: The preliminary safety evaluation of Antrodia camphorata fermented buckwheat tea was conducted through an acute oral toxicity test on mice.

[0101] Preparation of the test substance: Weigh 50 g of Antrodia camphorata and buckwheat fermented tea sample, add 500 mL of boiling water and extract for 30 min, filter and collect the filtrate. Repeat the extraction once with the residue under the above conditions. Combine the two filtrates, concentrate under reduced pressure at 60℃, and freeze-dry under vacuum to obtain the water extract powder. Prepare a suspension of the required concentration with pure water before use. Animal grouping and administration: A 14-day acute toxicity experiment was conducted using the limit method. Twenty mice were selected and grouped according to sex, and placed in 4 cages with 5 mice in each cage. Two cages were set up as the control group and two cages as the administration group, with an equal number of male and female mice in each group. Control group: Administered an equal volume of solvent (pure water). Administration group: Administered Antrodia camphorata and buckwheat tea water extract at a dose of 15000 mg / kg·bw. Administered orally by gavage at a volume of 10 mL / kg·bw once daily for 14 consecutive days.

[0102] (1) General clinical observation and mortality

[0103] During the 14-day continuous observation period, the mice's condition at the end of the experiment is shown in Table 8. No abnormalities were observed in either the control group or the 15000 mg / kg bw group. All animals exhibited normal physical characteristics, with smooth fur and no hair loss; they moved freely and gaited normally, without restlessness, lethargy, or convulsions; their breathing was stable with no abnormal secretions; their feces were normal granular, without diarrhea or bloody stools; there were no abnormal secretions from the eyes, nose, or mouth; their eyelids closed normally, and their corneas were transparent. No deaths occurred in either group throughout the experiment, resulting in a 100% survival rate.

[0104] Table 8 General observation records of acute oral toxicity test in mice (15000 mg / kg·bw)

[0105]

[0106] (2) Body weight and organ coefficient

[0107] The body weight of both groups of mice remained stable over 14 days. Figure 13 There were no significant differences in body weight between the treated group and the control group at any time point (P>0.05). There were also no significant differences in food intake between the groups, indicating that continuous administration of the test substance at doses up to 15000 mg / kg·bw for 14 days did not adversely affect the normal growth and appetite of mice.

[0108] (3) Gross anatomical observation

[0109] After the experiment, all mice underwent gross dissection to observe major organs such as the heart, liver, lungs, kidneys, stomach, and intestines. The results showed that no visible abnormalities such as congestion, edema, hemorrhage, necrosis, nodules, or tumors were observed in any of the organs of either group of mice. The position, shape, size, and color of the organs were all normal.

[0110] The calculation results of the major organ coefficients are as follows: Figure 15 As shown in the figure. Compared with the control group, there were no significant differences in the organ coefficients of the heart, liver, lungs and bilateral kidneys in the mice treated with 15000 mg / kg·bw (P>0.05).

[0111] In summary, it can be preliminarily determined that the aqueous extract of Antrodia camphorata and buckwheat tea did not produce significant subacute toxicity in mice under repeated 14-day administration. This provides important toxicological evidence for the safety of this product for consumption and lays the foundation for further in-depth research.

[0112] Ethical Statement: All procedures in this experiment strictly adhered to the principles of laboratory animal welfare ethics and were reviewed and approved by the Laboratory Animal Ethics Committee of Chengdu University.

[0113] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A method for preparing fermented Antrodia camphorata and buckwheat tea, characterized in that, Includes the following steps: S1. Activate and prepare seed liquid from Antrodia camphoratus (Taiwanofungus camphoratus) MRC156-2K-11 with biological preservation number: CCTCC No. M 20261055; S2. Add a culture medium containing buckwheat to a container, then inoculate with the seed liquid of Antrodia camphorata strain MRC156-2K-11 and carry out solid-state fermentation to obtain Antrodia camphorata buckwheat fermented tea.

2. The preparation method according to claim 1, characterized in that, In S1, the Antrodia camphorata strain MRC156-2K-11 was selected and activated on potato dextrose agar medium. The activated strain was cut into 2 cm pieces, crushed with sterilized flat bamboo sticks, and inoculated into 50 mL of liquid seed culture medium. The culture was carried out in the dark at 28 ℃ and 150 r / min for 7 days to obtain the seed liquid.

3. The preparation method according to claim 1, characterized in that, In S2, solid-state fermentation is carried out in the dark for 20 days at a fermentation temperature of 28°C.

4. The preparation method according to claim 1, characterized in that, In step S2, the volume of tartary buckwheat accounts for 20%-30% of the container volume; the inoculation amount of seed liquid is 10-11% of the tartary buckwheat volume.

5. The preparation method according to claim 1, characterized in that, In S2, the culture medium containing tartary buckwheat in a 250mL container has the following formula: 18-27g tartary buckwheat, 0.63-0.94g glucose, 0.21-0.32g lactose, 1.25-1.87g oats, 0.42-0.62g red beans, 5-7.5 mg vitamin B1, 10 mL water, and natural pH.

6. The fermented Antrodia camphorata and buckwheat tea prepared by the preparation method according to any one of claims 1-5.

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