Preparation method of cordyceps militaris culture medium sheet zicao grass fermentation wine and product thereof

Abstract

This invention discloses a method and product for preparing Pien Tze Huang herbal wine fermented on Cordyceps militaris culture medium, belonging to the field of health wine brewing technology. This method uses Cordyceps militaris culture medium (SCM) and Pien Tze Huang herbal wine (GFE) as raw materials, and prepares a health wine with hypoglycemic and antioxidant effects through a complex fermentation process including raw material pretreatment, enzymatic hydrolysis, yeast activation, anaerobic fermentation, and post-treatment. It aims to solve the problems of low raw material utilization, low active ingredient content, and insufficient antioxidant activity in existing technologies. Pretreatment employs ultrasonic extraction. Fermentation conditions are anaerobic fermentation at 28℃ for 18 days, with 3% sugar supplementation every 2 days, controlling the alcohol content at 6%-10% vol, and preferably using an SCM:GFE ratio of 2:1. The resulting product has an alcohol content of 8% vol, is rich in total phenols and total flavonoids, has an α-glucosidase inhibition rate of 82.6%, a DPPH free radical scavenging rate of 78.3%, and a FRAP value of 82.6 mM Fe. 2+ Equivalents / mL, with a harmonious flavor and significant health benefits, provide an effective way for the comprehensive development of Cordyceps militaris and Pien Tze Huang.

Description

I. Technical Field

[0001] This invention relates to the field of health wine brewing technology, specifically to a fermented wine with health benefits such as hypoglycemic and antioxidant effects, prepared by compound fermentation using Cordyceps militaris culture medium and Pien Tze Huang grass as raw materials. II. Background Technology

[0002] Cordyceps militaris, a fungus used in both medicine and food, is rich in various active ingredients such as cordycepin, cordyceps polysaccharides, flavonoids, and polyphenols, exhibiting significant antioxidant and immunomodulatory effects. Pien Tze Huang (also known as White Phoenix Herb) contains natural antioxidants such as flavonoids and phenolic acids, and is widely used in traditional medicine. Combining these two herbs to develop fermented wine can utilize microbial metabolism to enhance the dissolution and transformation of active ingredients, while also imparting a unique flavor and health benefits to the wine. Currently, existing technologies for preparing fermented wines using Cordyceps militaris or Pien Tze Huang mostly focus on single-raw material utilization, and suffer from problems such as unclear process parameters, low utilization rate of active ingredients, and insufficient antioxidant activity. For example, some Cordyceps militaris fermented wines use only Cordyceps militaris as a single raw material, resulting in a limited range of functional components; direct infusion of Pien Tze Huang has drawbacks such as low dissolution rate of active ingredients and poor bioavailability. Therefore, there is an urgent need to develop a process-optimized method for preparing Pien Tze Huang wine using a Cordyceps militaris culture medium rich in active ingredients and with strong antioxidant activity. III. Summary of the Invention

[0003] (I) Purpose of the Invention

[0004] The present invention aims to provide a method for preparing Pien Tze Huang Cao wine fermented on Cordyceps militaris culture medium, in order to solve the problems of low raw material utilization, low content of active ingredients and insufficient antioxidant activity in the prior art, and at the same time provide a fermented wine product prepared by the method.

[0005] (II) Technical Solution

[0006] To address the above problems, this invention first provides a formula for a functional cat treat with kidney-protecting and diuretic properties based on Cordyceps militaris culture medium, effectively processing the wet Cordyceps militaris culture medium to ensure its proper combination with other ingredients. Furthermore, this invention also provides a manufacturing method that uses enzymatic hydrolysis technology to adjust the flavor and texture of the cat treat, while simultaneously improving its absorption by cats.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Based on the first aspect of the present invention, a formulation for a wet culture medium of Cordyceps militaris is provided:

[0009] Cordyceps militaris wet culture medium formula: per 100ml contains 27g oats, 28g low-temperature soybean meal, 0.5g magnesium sulfate, 0.8g potassium dihydrogen phosphate, 1.58g yeast extract, 2g peptone, 8g glucose, 0.6g selenium-enriched yeast, and 10mg vitamin B1. The processing method is as follows:

[0010] Based on a second aspect of the present invention, as a preferred process condition, the fermentation process and production flow of the wine are as follows:

[0011] (I) Raw material pretreatment

[0012] 1. Preparation of Cordyceps militaris culture medium (SCM)

[0013] (1) Crushing and sieving: Crush the dried Cordyceps militaris culture medium and pass it through a 60-mesh sieve. Collect the sieve material for later use.

[0014] (2) Ultrasonic extraction: Add deionized water to the pulverized culture medium at a material-to-liquid ratio of 1:10 (g / mL), and extract ultrasonically at 50℃ and 200w for 30 min. Repeat the extraction twice, combine the two filtrates, and obtain the Cordyceps militaris culture medium extract.

[0015] 2. Preparation of Pien Tze Huang Herb Extract (GFE)

[0016] (1) Crushing and sieving: Crush the dried Pien Tze Huang herbal powder, pass it through a 40-mesh sieve, and collect the sieve material for later use.

[0017] (2) Ultrasonic extraction: Add 50% ethanol at a material-to-liquid ratio of 1:10 (g / mL), and ultrasonically extract at 50℃ and 200w for 30 min. Repeat twice and combine the filtrates.

[0018] (3) Concentration and volume adjustment: The filtrate is concentrated under reduced pressure (45℃, vacuum degree -0.08MPa) to a crude drug content of 1g / mL, and stored at 4℃.

[0019] 3. Yeast activation

[0020] Add 0.2% Lactobacillus plantarum MSJK0048 (CGMCC No. 28238) to a 2% sucrose aqueous solution and activate at 37℃ for 12h. Then add Saccharomyces cerevisiae (1g / 100mL) and activate for 15min.

[0021] 4. Enzymatic hydrolysis

[0022] (1) Preliminary enzymatic hydrolysis: Cordyceps militaris liquid culture medium and Pien Tze Huang grass extract were dispensed in a ratio of 2:1. Then, a mixture of α-amylase, cellulase and pectinase (1:1:1) was added at a material-to-liquid ratio of 0.3% (w / v). The mixture was enzymatically hydrolyzed for 7 hours at 60℃ and 110 rpm.

[0023] (2) Second enzymatic hydrolysis: After 7 hours of enzymatic hydrolysis, add papain at a ratio of 0.5% (w / v) and hydrolyze for 2-3 hours at 60℃ and 110 rpm. After the enzymatic hydrolysis is completed, the hydrolysate is subjected to enzyme inactivation treatment (90℃, 10 min) and cooled to room temperature for later use.

[0024] (II) Fermentation

[0025] (1) Fermentation broth preparation: The enzymatically hydrolyzed Cordyceps militaris liquid culture medium and Pien Tze Huang grass extract were mixed at a ratio of 2:1, and the initial pH was controlled at 5.0 and the initial sugar content was controlled at 10%-12%.

[0026] (2) Inoculation: Inoculate the activated yeast into the above fermentation liquid at a yeast inoculation ratio of 6% and stir evenly.

[0027] (3) Fermentation process control: The inoculated fermentation broth was placed at 28℃ for fermentation. During fermentation, white sugar was added every 2 days to increase the sugar concentration of the fermentation broth by 3%.

[0028] (4) Determination of fermentation endpoint: Continuously monitor the alcohol content of the fermentation broth. When the alcohol content is controlled at 6% vol-10% vol, the fermentation is terminated.

[0029] (III) Post-processing

[0030] (1) Centrifugation: The fermented liquid after fermentation is completed is placed in a high-speed centrifuge and centrifuged at 8000 r / min for 10 min to remove the precipitate and collect the supernatant.

[0031] (2) Filtration: The supernatant after centrifugation is filtered through a 0.45μm filter membrane to further remove impurities and obtain a clear wine.

[0032] (3) Aging: Place the filtered wine in a sealed container and age it for 1-2 months at room temperature and in the dark to make the flavor of the wine more harmonious.

[0033] (4) Packaging: After aging, the wine is packaged into sterile glass bottles and sealed for storage. IV. Description of the attached drawings

[0034] Figure 1 Antioxidant capacity analysis

[0035] The charts show the antioxidant activity of the four groups of samples, including two key indicators: FRAP value (reducing power, left vertical axis) and DPPH free radical scavenging rate (right vertical axis), which intuitively demonstrate the impact of different processes on the antioxidant capacity of the samples.

[0036] analyze:

[0037] Example 2 (18 days, 2:1) showed that both DPPH scavenging rate and FRAP value were at a high and balanced level (DPPH 78.3%, FRAP 82.6%), confirming the conclusion in the instructions that "the antioxidant activity is optimally balanced". The 2:1 raw material ratio allows Cordyceps militaris polysaccharides to synergistically interact with Pien Tze Huang flavonoids and phenolic acids. 18 days of fermentation promotes the dissolution of active ingredients while avoiding degradation caused by long-term fermentation. Microbial metabolism enhances the synergistic effect of antioxidants. In Example 1 (30 days, 2:1), the DPPH scavenging rate was slightly higher than in Example 2, but the FRAP value decreased, which is consistent with the description in the instructions that "long-term fermentation accumulates secondary antioxidant products (increasing DPPH), but heat-sensitive reducing components (such as flavonoids) degrade (reducing FRAP)." In Example 3 (18 days, 3:1), both indicators were lower than in Example 2. This was because the proportion of Cordyceps militaris culture medium was too high (3:1), reducing the proportion of highly antioxidant flavonoids and phenolic acids in Pien Tze Huang, leading to a decrease in antioxidant activity. Comparative Example 1 (unfermented) showed the lowest antioxidant activity (DPPH 48.6%, lowest FRAP value), proving that "microbial fermentation is the key to improving antioxidant activity." The unfermented group lacked enzymatic hydrolysis, resulting in a low dissolution rate of active ingredients and failing to exert a synergistic antioxidant effect.

[0038] Figure 2 Blood glucose lowering experiment: α-glucosidase inhibition rate

[0039] The chart uses a bar graph to show the α-glucosidase inhibition rate (a core indicator of blood sugar reduction) of the four groups of samples, directly reflecting the impact of different processes on blood sugar reduction efficacy.

[0040] analyze:

[0041] Example 2 (18 days, 2:1) showed the highest inhibition rate (82.6%), far exceeding other groups. Example 3 (18 days, 3:1) saw its inhibition rate drop to 65.4%. This was because the ratio of raw materials changed to 3:1, reducing the proportion of Pien Tze Huang herb and resulting in insufficient levels of key hypoglycemic components such as flavonoids. Consequently, the Cordyceps militaris component could not maintain high inhibitory activity independently, confirming the instruction manual's claim that "an imbalance in the raw material ratio leads to a decrease in efficacy." Example 1 (30 days, 2:1) showed an inhibition rate of 58.2%, consistent with the instruction manual's conclusion that "30-day fermentation leads to the degradation of key hypoglycemic components (such as specific flavonoids)." While long-term fermentation increases alcohol content, it damages the active structure of the components. Comparative Example 1 (unfermented) showed an inhibition rate of only 32.3%, far lower than the fermented group, proving that "fermentation is a necessary condition for enhancing hypoglycemic activity"—the unfermented group lacked the microbial metabolism to transform the components, preventing the effective release of active ingredients and making it difficult to inhibit α-glucosidase.

[0042] Figure 3 Nutritional analysis: Total phenols and total flavonoids content

[0043] The chart shows the total phenol (left vertical axis) and total flavonoid (right vertical axis) content of the four groups of samples, revealing the material basis for the differences in efficacy and supporting the "correlation between active ingredients and efficacy".

[0044] analyze:

[0045] Example 2 (18 days, 2:1) had the highest total phenol and total flavonoid content, consistent with the instructions that "the content of active ingredients is significantly higher than that of other groups," providing material support for its high antioxidant and high hypoglycemic activity. Example 3 (18 days, 3:1) had lower total phenol and total flavonoid content than Example 2. Due to the reduced proportion of Pien Tze Huang Cao (1:3), the dissolution of its rich flavonoids and phenolic acids decreased, confirming the instructions that "the reduced proportion of GFE leads to insufficient active ingredients." Example 1 (30 days, 2:1) had a slightly lower total flavonoid content than Example 2. The total phenol content was partially supplemented by small molecule phenols produced by microbial metabolism, resulting in a smaller decrease. Comparative Example 1 (unfermented) had the lowest content of both components. Because it did not undergo enzymatic hydrolysis and fermentation, the structures of cellulose, pectin, etc. in the raw materials were not destroyed, making it difficult for phenols and flavonoids to dissolve, directly leading to low efficacy indicators (hyperglycemic and antioxidant).

[0046] Summary of Attached Figures

[0047] The three charts systematically validated the core conclusions from three dimensions: "antioxidant activity," "blood sugar lowering effect," and "content of active ingredients."

[0048] Example 2 (SCM:GFE = 2:1, fermentation for 18 days) showed the best performance in terms of active ingredient content, hypoglycemic activity, and antioxidant balance, and is the optimal process;

[0049] Fermentation time (18 days is better than 30 days), raw material ratio (2:1 is better than 3:1), and whether fermentation is performed (fermented group is better than unfermented group) are key factors affecting product performance, which are completely consistent with the process optimization logic in the instruction manual. V. Detailed Implementation Methods

[0050] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.

[0051] Unless otherwise specified, the raw materials and reagents used in the following embodiments of the present invention can be obtained commercially.

[0052] The analytical and testing methods used in the following embodiments of the present invention are as follows:

[0053] 1. Nutritional composition testing

[0054] (1) Determination of total phenol content

[0055] The Folin-phenol method was used. Take 0.5 mL of sample, add 2.5 mL of Folin-phenol reagent diluted 1:10, mix well, let stand for 5 minutes, then add 2 mL of 7.5% Na₂CO₃, and bring the volume to 25 mL. Incubate in the dark for 30 minutes, then measure the absorbance at 765 nm. A standard curve was plotted using gallic acid (Y = 0.012X + 0.05, R₀ = 0.012X + 0.05). 2 =0.998), calculate the total phenol content in the sample based on the standard curve.

[0056] (2) Protein content determination

[0057] Using the Coomassie Brilliant Blue G-250 method, take 0.1 mL of sample (or standard solution), add 5 mL of Coomassie Brilliant Blue solution, mix well, let stand at room temperature for 5 min, and measure the absorbance at 595 nm.

[0058] Standard protein solution: Prepare a 0.1 mg / mL standard solution using bovine serum albumin (BSA).

[0059] A standard curve was plotted with BSA concentration on the x-axis and absorbance on the y-axis, and the protein content (mg / mL) was calculated based on the sample absorbance.

[0060] (3) Determination of total flavonoid content

[0061] The sodium nitrite-aluminum nitrate method was used. Take 1 mL of sample, add 0.3 mL of 5% NaNO₂, then add 0.3 mL of 10% Al(NO₃)₃ after 5 minutes, and then add 4 mL of 1M NaOH after 6 minutes. The volume was then adjusted to 10 mL, and the absorbance was measured at 510 nm. A standard curve was plotted using rutin as a standard (Y = 0.008X + 0.02, R₀ = 0.008X + 0.02). 2 =0.999), based on which the total flavonoid content was calculated.

[0062] 2. Microbiological index detection

[0063] (1) Total bacterial count determination

[0064] Method: Plate count method (GB 4789.2-2016 National Food Safety Standard for Microbiological Examination of Food - Determination of Total Colony Count).

[0065] Procedure: Dilute the sample 10-fold serially, take 1 mL and inoculate it onto a nutrient agar plate, incubate at 37℃ for 48 h, and count the number of colonies (standard: ≤1000 CFU / g).

[0066] (2) Coliform count

[0067] Method: MPN method (GB 4789.3-2016 National Food Safety Standard for Microbiological Examination of Food - Coliform Count).

[0068] Procedure: After diluting the sample, inoculate it into lactose bile salt fermentation tubes and incubate at 37℃ for 24 hours. Observe the gas production and calculate the MPN value (standard: ≤10MPN / g) by referring to the table.

[0069] (3) Detection of pathogenic bacteria (Salmonella, Staphylococcus aureus)

[0070] Methods: GB 4789.4-2016 (Salmonella), GB 4789.10-2016 (Staphylococcus aureus).

[0071] Procedure: After sample enrichment culture, isolate the bacteria using selective culture medium, perform biochemical tests and serological identification to determine whether pathogenic bacteria are detected (standard: not detectable).

[0072] 3. Detection of active ingredients

[0073] (1) Determination of cordycepin content

[0074] Method: High performance liquid chromatography (HPLC).

[0075] Conditions: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase methanol-water (20:80, v / v), flow rate 1.0 mL / min, detection wavelength 254 nm, injection volume 10 μL.

[0076] Procedure: The sample was extracted with methanol by ultrasonic extraction (50℃, 30 min), centrifuged and filtered, and then analyzed by an instrument. The cordycepin content (≥50 μg / g) was calculated by external standard method.

[0077] (3) Determination of adenosine content

[0078] Method: High performance liquid chromatography (HPLC).

[0079] Chromatographic conditions: Column: C18 column (4.6×250mm, 5μm); Mobile phase: methanol-water (10:90, v / v); Flow rate: 1.0mL / min; Detection wavelength: 260nm; Injection volume: 10μL.

[0080] Procedure: Sample preparation: Take the supernatant of the fermented wine, filter it through a 0.22 μm filter membrane, and set aside. Prepare a series of adenosine standards (10–100 μg / mL), inject them for determination, and plot a standard curve with concentration on the x-axis and peak area on the y-axis. Inject the filtrate, and calculate the adenosine content (μg / mL) based on the peak area in the standard curve.

[0081] 4. In vitro antioxidant assay

[0082] (1) DPPH free radical scavenging rate determination

[0083] Dilute the sample to an appropriate concentration. Take 1 mL of the diluted sample and add 2 mL of 0.2 mM DPPH ethanol solution. Mix well and incubate in the dark for 30 minutes. Measure the absorbance at 517 nm (Sample A). For the blank group, use anhydrous ethanol instead of DPPH to measure the absorbance (Blank A). For the control group, use ethanol instead of the sample to measure the absorbance (Control A). The clearance rate (%) is calculated using the formula [1 - (Sample A - Blank A) / Control A] × 100%.

[0084] (2) FRAP method for determination

[0085] FRAP reagent is prepared by mixing 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ solution, and 20 mM FeCl3 in a volume ratio of 10:1:1, and preheating to 37°C before use. Add 980 μL of FRAP reagent to 20 μL of sample, mix well, and incubate at 37°C for 4 minutes. Measure the absorbance at 593 nm, using FeSO4 as a standard. The result is expressed as mMFe. 2+ Equivalents / mL

[0086] 5. Efficacy Testing

[0087] (1) Blood glucose lowering efficacy test

[0088] The α-glucosidase inhibition experiment was conducted, and the experimental method is as follows:

[0089] Reagents: 0.1 U / mL α-glucosidase solution (prepared with 0.02 M phosphate buffer, pH 6.8), 5 mM PNPG substrate, 0.1 M Na2CO3 stop solution, and acarbose positive control.

[0090] Steps: Take 50 μL of sample + 50 μL of enzyme solution, pre-incubate at 37℃ for 10 min, add 50 μL of PNPG, incubate for 30 min, then add 100 μL of Na2CO3 to terminate the reaction, and measure the absorbance at 405 nm.

[0091] Calculate: Inhibition rate (%) = [1 - (Absorbance of sample group - Absorbance of blank group) / (Absorbance of enzyme control group - Absorbance of blank group)] × 100%, and calculate IC50. 50 .

[0092] (2) Alcohol content determination (alcohol meter method)

[0093] Take 50 mL of the supernatant from the fermented wine and place it in a 100 mL graduated cylinder. After the temperature stabilizes at 20°C, place it in an alcohol meter and read the scale after it has settled. This is the alcohol content (% vol) of the sample.

[0094] (3) Sugar content determination (DNS method, residual sugar content detection)

[0095] Reagents: DNS colorimetric reagent (weigh 10g DNS, 2g NaOH, 200g potassium sodium tartrate, and dilute with water to 1000mL), glucose standard solution (1mg / mL).

[0096] Procedure: Take 0.5 mL of sample, add 1.5 mL of water and 2 mL of DNS, boil in water for 5 min, cool and then add water to make up to 25 mL, and measure the absorbance at 540 nm.

[0097] Plot a standard curve using glucose standard solution and calculate the residual sugar content (mg / mL) in the sample.

[0098] (4) Sensory evaluation

[0099] Select 10-12 individuals with experience in sensory evaluation of alcoholic beverages (aged 20-50, male-to-female ratio 1:1). These individuals must abstain from spicy and stimulating foods, and avoid smoking and drinking alcohol for 24 hours prior to the evaluation to ensure sensory sensitivity.

[0100] Fermented wines (after post-treatment clarification) from different process optimization groups were dispensed into identical transparent glass cups (20 mL / cup), numbered (random three-digit numbers), and placed in a 25℃ constant temperature water bath to equilibrate the temperature.

[0101] The sensory evaluation was conducted in an odorless, softly lit room, with each group of samples evaluated 10 minutes apart, during which time purified water was provided for rinsing the mouth.

[0102] The evaluators observed the color and clarity of the sample in sequence, swirled the glass gently to smell the aroma, tasted it in small sips to experience the flavor, and then gave a comprehensive evaluation of the overall acceptability, scoring it independently according to the standards.

[0103] SPSS 26.0 statistical analysis was used to calculate the mean score ± standard deviation of each index for each group of samples. One-way ANOVA was used to compare the differences between groups (p<0.05 was considered significant) to clarify the impact of the optimal process on the sensory quality of the wine.

[0104] (I) Example 1: Fermented wine fermented for 30 days (SCM:GFE = 2:1)

[0105] Fermentation conditions:

[0106] 1. Raw material pretreatment

[0107] SCM preparation: Take dried Cordyceps militaris culture medium, pulverize it and pass it through a 60-mesh sieve. Add deionized water at a material-to-liquid ratio of 1:10 (g / mL), and extract it by ultrasonication at 50℃ and 200W for 30 min. Repeat the extraction twice, and combine the filtrates to obtain Cordyceps militaris culture medium extract (SCM).

[0108] GFE preparation: Take dried Pien Tze Huang grass powder, pulverize it and pass it through a 40-mesh sieve. Add 50% ethanol at a material-to-liquid ratio of 1:10 (g / mL), and extract it by ultrasonic extraction at 50℃ and 200W for 30 min. Repeat the extraction twice. Combine the filtrates and concentrate them under reduced pressure at 45℃ and a vacuum degree of -0.08MPa to a crude drug content of 1g / mL. Store at 4℃ to obtain Pien Tze Huang grass extract (GFE).

[0109] Preparation of the mixture: Mix SCM:GFE at a ratio of 2:1 (volume ratio), adjust the initial pH to 5.0, and control the initial sugar content at 10%-12%.

[0110] 2. Enzymatic hydrolysis

[0111] Mix SCM:GFE = 2:1, add α-amylase:cellulase:pectinase (1:1:1) mixed enzyme at a material-to-liquid ratio of 0.3% (w / v), and enzymatically hydrolyze at 60℃ and 110rpm for 7h.

[0112] Add 0.5% (w / v) papain, continue enzymatic hydrolysis at 60℃ and 110 rpm for 2.5 h, inactivate enzyme at 90℃ for 10 min, cool to room temperature, and adjust the initial pH of the hydrolysate to 5.0.

[0113] 3. Fermentation: Mix SCM:GFE = 2:1, and anaerobic ferment at 28°C for 30 days. The sugar supplementation strategy is the same as in Example 2. Fermentation is ended when the alcohol content reaches 10% vol.

[0114] 4. Post-processing: Centrifuge at 8000 r / min for 10 min, filter the supernatant through a 0.45 μm filter membrane, age at room temperature in the dark for 1 month, and then dispense.

[0115] Performance:

[0116] Active ingredients: The content of heat-sensitive components such as total flavonoids may be slightly lower than that in Example 2 due to partial degradation caused by long-term fermentation.

[0117] Hypoglycemic activity: The α-glucosidase inhibition rate was lower than that in Example 2, due to the inactivation of the key hypoglycemic component caused by excessive fermentation.

[0118] Antioxidant capacity: The DPPH scavenging rate was increased, which is presumably related to the accumulation of secondary antioxidant products produced by yeast metabolism.

[0119] Physicochemical properties and flavor: The alcohol content is 10% vol (quite irritating), and the residual sugar content is low (sugar is fully consumed), but long-term fermentation leads to a strong flavor and reduced palatability.

[0120] (II) Example 2: Fermented wine fermented for 18 days (SCM:GFE = 2:1)

[0121] 1. Raw material pretreatment: SCM and GFE were prepared in the same manner as in Example 1.

[0122] 2. Enzymatic hydrolysis

[0123] Mix SCM:GFE = 2:1, add α-amylase:cellulase:pectinase (1:1:1) mixed enzyme at a material-to-liquid ratio of 0.3% (w / v), and enzymatically hydrolyze at 60℃ and 110rpm for 7h.

[0124] Add 0.5% (w / v) papain, continue enzymatic hydrolysis at 60℃ and 110 rpm for 2.5 h, inactivate enzyme at 90℃ for 10 min, and cool to room temperature.

[0125] 3. Yeast activation

[0126] Add 0.2% Lactobacillus plantarum MSJK0048 (CGMCC No.28238) to a 2% sucrose aqueous solution and activate at 37℃ for 12h; add 1g / 100mL of Saccharomyces cerevisiae and continue activation for 15min.

[0127] 4. Fermentation

[0128] The initial pH of the enzymatic hydrolysate was adjusted to 5.0, and the sugar content was 10%-12%. Activated yeast was inoculated at a rate of 6%, and anaerobic fermentation was carried out at 28°C for 18 days. White sugar was added every 2 days to increase the sugar concentration by 3%.

[0129] Fermentation is stopped when the alcohol content reaches 8% vol.

[0130] 5. Post-processing

[0131] Centrifuge at 8000 r / min for 10 min, filter the supernatant through a 0.45 μm filter membrane, age at room temperature in the dark for 1 month, and then bottle.

[0132] Performance:

[0133] Active ingredients: Detected by the Folin-phenol method and the sodium nitrite-aluminum nitrate method, the total phenol and total flavonoid content was significantly higher than that of the unfermented group and other proportion groups.

[0134] Hypoglycemic activity: In the α-glucosidase inhibition experiment, its inhibition rate was the highest in the fermentation group, indicating that 18 days of fermentation at a 2:1 ratio can promote the synergistic effect of hypoglycemic active ingredients (such as flavonoids and polyphenols).

[0135] Antioxidant capacity: The DPPH free radical scavenging rate and FRAP reducing capacity are balanced, reaching 78.3% and 82.6% respectively, confirming the promoting effect of fermentation on the conversion of active ingredients (microbial metabolism enhances the antioxidant synergistic effect of the ingredients).

[0136] Physicochemical properties and flavor: The alcohol content is 8% vol (good palatability). After aging, the wine becomes clear and the flavor is harmonious, meeting the needs of health wine consumption.

[0137] (III) Fermented wine from Example 3, fermented for 18 days (SCM:GFE = 3:1)

[0138] 1. Raw material pretreatment: Prepare SCM and GFE in the same manner as in Example 1, and mix them at a ratio of SCM:GFE = 3:1.

[0139] 2. Enzymatic hydrolysis: Same as in Example 2 (enzyme type, ratio, temperature, and time are the same).

[0140] 3. Yeast activation: Same as in Example 2.

[0141] 4. Fermentation: Except for the raw material ratio of 3:1, the fermentation temperature (28℃), inoculum amount (6%), sugar supplementation strategy, and fermentation time (18 days) are the same as in Example 2. Fermentation is ended when the alcohol content reaches 6% vol.

[0142] 5. Post-processing: Same as Example 2 (centrifugation, filtration, and aging for 1 month).

[0143] Performance:

[0144] Active ingredients: The content of total phenols and total flavonoids is slightly lower than that in Example 2 (due to the reduced proportion of GFE, the dissolution of herbal active ingredients is reduced).

[0145] Blood sugar reduction and anti-oxidation: The α-glucosidase inhibition rate and antioxidant indicators (DPPH, FRAP) were both lower than those in Example 2. It is speculated that the high proportion of Cordyceps militaris culture medium masked the efficacy of active ingredients such as flavonoids in Pien Tze Huang Cao, and the insufficient fermentable sugars led to a lower alcohol content (6% vol) and reduced fermentation metabolites.

[0146] Flavor characteristics: The Cordyceps militaris has a stronger fungal aroma and a weaker herbal fragrance, and its sensory harmony is not as good as in Example 2.

[0147] (iv) Comparative Example 1: Non-fermented mixed extract (uninoculated control group)

[0148] Fermentation conditions:

[0149] 1. Raw material pretreatment

[0150] SCM preparation: Take dried Cordyceps militaris culture medium, pulverize it and pass it through a 60-mesh sieve. Add deionized water at a material-to-liquid ratio of 1:10 (g / mL), and extract it by ultrasonication at 50℃ and 200W for 30 min. Repeat the extraction twice, and combine the filtrates to obtain Cordyceps militaris culture medium extract (SCM).

[0151] GFE preparation: Take dried Pien Tze Huang grass powder, pulverize it and pass it through a 40-mesh sieve. Add 50% ethanol at a material-to-liquid ratio of 1:10 (g / mL), and extract it by ultrasonic extraction at 50℃ and 200W for 30 min. Repeat the extraction twice. Combine the filtrates and concentrate them under reduced pressure at 45℃ and a vacuum degree of -0.08MPa to a crude drug content of 1g / mL. Store at 4℃ to obtain Pien Tze Huang grass extract (GFE).

[0152] Preparation of the mixture: Mix SCM:GFE at a ratio of 2:1 (volume ratio), adjust the initial pH to 5.0, and control the initial sugar content at 10%-12%.

[0153] Mix SCM:GFE = 2:1, add α-amylase:cellulase:pectinase (1:1:1) mixed enzyme at a material-to-liquid ratio of 0.3% (w / v), and enzymatically hydrolyze at 60℃ and 110rpm for 7h.

[0154] Add 0.5% (w / v) papain, continue enzymatic hydrolysis at 60℃ and 110 rpm for 2.5 h, inactivate enzyme at 90℃ for 10 min, cool to room temperature, adjust the initial pH of the hydrolysate to 5.0, and anaerobic ferment at 28℃ for 18 days.

[0155] 2. Post-processing

[0156] Centrifuge directly at 8000 r / min for 10 min, take the supernatant and filter it through a 0.45 μm filter membrane, and store it in the dark (without fermentation or aging).

[0157] Performance:

[0158] Active ingredients: The content of total phenols and total flavonoids was the lowest among all groups, significantly lower than that of the fermentation group. Due to the lack of microbial enzymatic hydrolysis, the dissolution rate of active ingredients was low.

[0159] Blood sugar reduction and antioxidant effects: The α-glucosidase inhibition rate was only 32.3%, and the DPPH clearance rate was 48.6%, which was much lower than that of the fermentation group, proving that fermentation is a necessary condition for enhancing efficacy.

[0160] Physicochemical characteristics: alcohol content 0% vol, residual sugar content as high as 156 mg / mL (sweet and cloying with no alcoholic aroma), which does not conform to the product positioning of fermented wine.

[0161] (V) Summary of Implementation Examples

[0162] 1. Fermentation vs. non-fermentation: The content of active ingredients, hypoglycemic and antioxidant activities of Examples 2-4 (fermented group) were significantly higher than those of Comparative Example 1 (non-fermented group), proving that microbial fermentation can promote the dissolution and transformation of active ingredients through enzymatic hydrolysis, which is the key to improving efficacy.

[0163] 2. Effect of raw material ratio: The total phenol and total flavonoid content and α-glucosidase inhibition rate of Example 2 (2:1) were higher than those of Example 3 (3:1). This is because the synergistic effect of the active ingredients (such as cordyceps polysaccharides and flavonoids) of Cordyceps militaris and Pien Tze Huang Cao was strongest at the 2:1 ratio. However, the high proportion of Cordyceps militaris in the 3:1 ratio would dilute the herbal efficacy ingredients and lead to a decrease in activity.

[0164] 3. Effect of fermentation time: The hypoglycemic activity of Example 2 (18 days) is better than that of Example 4 (30 days) because 18 days is the optimal period for the accumulation of active ingredients; although 30-day fermentation improves the DPPH clearance rate, too long a time will lead to the degradation of key hypoglycemic components (such as specific flavonoids), and the high alcohol content will affect palatability.

[0165] 4. Optimal process: Example 2 (SCM:GFE = 2:1, fermentation for 18 days) showed the best performance in terms of active ingredients, hypoglycemic activity, antioxidant balance and physicochemical flavor, and is the best preparation process for this fermented wine.

Claims

1. A method for preparing Pien Tze Huang Cao wine fermented on a Cordyceps militaris culture medium, characterized in that, Includes the following steps: Raw material pretreatment: Preparation of Cordyceps militaris culture medium extract (SCM): The dried Cordyceps militaris culture medium was pulverized and passed through a 60-mesh sieve. The sieve residue was collected. Deionized water was added to the sieve residue at a material-to-liquid ratio of 1:10 (g / mL). The mixture was ultrasonically extracted at 50℃ and 200W for 30 min. The extraction was repeated twice. The two filtrates were combined to obtain the Cordyceps militaris culture medium extract. Preparation of Pien Tze Huang Herb Extract (GFE): The dried Pien Tze Huang Herb powder was pulverized and passed through a 40-mesh sieve. The sieve residue was collected. 50% ethanol was added to the sieve residue at a material-to-liquid ratio of 1:10 (g / mL). The extract was ultrasonically extracted at 50℃ and 200W for 30 min. The extraction was repeated twice. The filtrates were combined. The filtrate was concentrated under reduced pressure at 45℃ and a vacuum degree of -0.08MPa to a crude herb content of 1 g / mL. The extract was stored at 4℃ to obtain Pien Tze Huang Herb Extract. Yeast activation: Add 0.2% Lactobacillus plantarum to a 2% sucrose aqueous solution and activate at 37℃ for 12h; then add 1g / 100mL of Saccharomyces cerevisiae and continue activation for 15min to obtain activated yeast. Enzymatic hydrolysis: The Cordyceps militaris extract and Pien Tze Huang herb extract were mixed at a volume ratio of 2:1 to obtain a mixture. A mixture of α-amylase, cellulase, and pectinase (1:1:1) was added to the mixture at a material-to-liquid ratio of 0.3% (w / v), and the mixture was hydrolyzed at 60℃ and 110 rpm for 7 h. Then, papain was added at a material-to-liquid ratio of 0.5% (w / v), and the mixture was hydrolyzed at 60℃ and 110 rpm for 2-3 h. After the hydrolysis was completed, the enzymes were inactivated at 90℃ for 10 min, and the mixture was cooled to room temperature to obtain the hydrolysate. Fermentation: Adjust the initial pH of the enzymatic hydrolysate to 5.0 and control the initial sugar content at 10%-12%; inoculate the enzymatic hydrolysate with activated yeast at a rate of 6%, stir well, and place it at 28℃ for anaerobic fermentation; during fermentation, add white sugar every 2 days to increase the sugar concentration of the fermentation broth by 3%; continuously monitor the alcohol content of the fermentation broth, and stop fermentation when the alcohol content reaches 6%-10% vol. Post-processing: Place the fermented liquid after fermentation into a high-speed centrifuge and centrifuge at 8000 r / min for 10 min to remove the precipitate and collect the supernatant; filter the supernatant through a 0.45 μm filter membrane to obtain the clarified wine; place the clarified wine in a sealed container and age it at room temperature and in the dark for 1-2 months; after aging, dispense it into sterile glass bottles and seal them for storage.

2. The preparation method according to claim 1, characterized in that, In the fermentation process, the fermentation time is 18 days, and fermentation ends when the alcohol content reaches 8% vol.

3. A product of Pien Tze Huang herbal wine fermented on a Cordyceps militaris culture medium, characterized in that, It is obtained by the preparation method described in claim 1 or 2.

4. The product according to claim 3, characterized in that, The product contains 25-38 mg / 100 mL of total phenols and 140-190 mg / L of total flavonoids. The total phenol content is determined by the Folin-phenol method and the total flavonoid content is determined by the sodium nitrite-aluminum nitrate method.

5. The product according to claim 3, characterized in that, The product exhibits an α-glucosidase inhibition rate ≥65%, a DPPH free radical scavenging rate ≥70%, and a FRAP value ≥0.65mM Fe. 2+ Equivalents / mL; the α-glucosidase inhibition rate was determined by the α-glucosidase inhibition experiment, the DPPH free radical scavenging rate was determined by the DPPH method, and the FRAP value was determined by the FRAP method.

6. The product according to claim 3, characterized in that, The product has an alcohol content of 6%-10% vol and a residual sugar content of ≤60 mg / mL; the alcohol content is determined by an alcohol meter and the residual sugar content is determined by the DNS method.

7. The product according to claim 3, characterized in that, The product contains cordycepin at a content ≥50 μg / g, and the adenosine content is determined by high performance liquid chromatography (HPLC). The HPLC conditions for determining the adenosine content are as follows: C18 column (4.6 × 250 mm, 5 μm), mobile phase methanol-water (10:90, v / v), flow rate 1.0 mL / min, detection wavelength 260 nm, and injection volume 10 μL.

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