Preparation method of pachyrhizua angulatus and cyperus esculentus compound fermented beverage

Abstract

The invention relates to a preparation method of a pachyrhizua angulatus and cyperus esculentus compound fermented beverage, which is characterized in that pachyrhizua angulatus pulp from which starch is extracted and defatted cyperus esculentus powder are used as raw materials, bread juice is added, and the mixture is subjected to compound fermentation with lactic acid bacteria and saccharomycetes to prepare a compound bacterium fermented beverage, so that the added value of pachyrhizua angulatus and cyperus esculentus is effectively increased.

Description

Technical Field

[0001] The present invention relates to the technical field of beverages, and more particularly to a method for preparing a composite fermented beverage of kudzu vine and cyperus rotundus. Background Art

[0002] Pueraria root, also known as kudzu root or dried kudzu, is the dried root of the leguminous plant Pueraria lobata. Its fleshy root is soft, tender, sweet, and residue-free. It contains a large amount of starch, primarily isoflavones, primarily puerarin, as well as polysaccharides and various trace elements, making it a valuable food and health supplement. Pueraria root is extracted to produce a slurry containing total flavonoids, puerarin, and other substances, and has numerous applications. Pueraria root beverages include kudzu root oral liquids and compound beverages.

[0003] Cyperus oleiferus, also known as tiger nut, sedge fruit, iron water chestnut, underground chestnut, and underground walnut, is a perennial herbaceous plant of the genus Cyperus in the family Cyperaceae. It is a high-quality, high-yielding, and highly valuable multi-purpose crop that combines oil, grain, livestock, and animal feed. Its tubers are used as a grain, medicine, and food ingredient, and can be consumed directly to satisfy hunger or refined into high-quality edible oils, health foods, and biopharmaceuticals. Its fibrous roots can be used to extract essential oils that nourish the mind and improve eyesight. The Xinhua Compendium of Materia Medica states that sedge oleiferus has pungent, sweet, and warm properties, and has the effects of soothing the liver and promoting qi, strengthening the spleen, and invigorating the stomach. It is primarily used to treat flank pain and chest tightness caused by liver qi stagnation; and abdominal distension, stomach pain, poor appetite, spleen deficiency, poor appetite, food stagnation, and indigestion caused by spleen and stomach qi stagnation.

[0004] At present, there are still some deficiencies in the application of kudzu vine and kudzu vine. The present invention aims to propose a preparation method of a kudzu vine and kudzu vine composite fermented beverage, thereby improving the added value of kudzu vine and kudzu vine and providing a scientific basis for industrial production. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for preparing a composite fermented beverage of kudzu vine and cyperus rotundus. The method uses kudzu vine pulp after starch extraction and defatted cyperus rotundus powder as raw materials, adds bread juice, and prepares a composite fermented beverage through composite fermentation with lactic acid bacteria and yeast, thereby effectively improving the added value of kudzu vine and cyperus rotundus.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A method for preparing a composite fermented beverage of kudzu vine and cyperus rotundus is as follows: S1. Raw material pretreatment: S11. Preparation of kudzu vine pulp: ultrafinely crush the dried kudzu vine powder, pass it through a 100-mesh sieve, mix the obtained kudzu vine powder with water at a material-water ratio of 1:30, add citric acid to adjust the pH to 3.7-4.2, soak in a constant temperature water bath at 30-35°C for 3h, let it stand and settle for a period of time, centrifuge to obtain kudzu vine pulp, and refrigerate for later use.

[0007] S12. Prepare bread juice: bake fresh bread until both sides are browned, cool to room temperature, cut into small pieces, wrap with gauze, add pure water, saccharify at a constant temperature of 69-73℃, filter with double-layer gauze, and refrigerate for later use.

[0008] S13. Prepare kudzu nut extract: mix kudzu nut pulp and defatted kudzu nut powder for extraction, add amylase and extract in a water bath at 85-95°C, then cool to 60-65°C, add saccharifying enzyme and extract in a water bath at 58-62°C, filter through double-layer gauze, boil, and refrigerate for later use.

[0009] S2, fermentation: S21. Raw material mixing: uniformly mix the kudzu nut extract, bread juice and pure water in a certain proportion to obtain a raw material mixture.

[0010] S22. Yeast activation: Add water and sucrose to the brewing dry yeast and place it in 22-40℃ for activation until a lot of bubbles appear.

[0011] S23. Activation of lactic acid bacteria: inoculate lactic acid bacteria powder into sterilized skim milk, culture at a constant temperature of 40-45°C until the milk is coagulated, remove from the milk, and refrigerate at 4°C for later use. The lactic acid bacteria powder includes thermophilic Streptococcus and bulgaricus.

[0012] S24, yeast fermentation: inoculate yeast into the raw material mixture for primary fermentation, place the raw material mixture at 20-38°C for fermentation, and then filter through a double-layer gauze sterilized at high temperature to obtain the filtrate.

[0013] S25. Homogenization: Use an adjustable speed homogenizer to mix and homogenize the mixed sample liquid, and continue fermentation and cultivation.

[0014] S26, lactic acid bacteria fermentation: After the yeast fermentation is completed, inoculate the activated lactic acid bacteria for secondary fermentation at 30-42℃.

[0015] S27. Refrigeration: Store the fermented product at 4°C.

[0016] In one embodiment, in step S12, fresh bread is baked in an oven at 150°C for 10 minutes until both sides of the bread are browned. After cooling to room temperature, the bread is cut into small pieces of about 3 cm in size, wrapped with gauze, and pure water is added at a material-to-water ratio of 1:20. The bread is saccharified at a constant temperature of 70°C for 3 hours, filtered through a double layer of gauze, and refrigerated for later use.

[0017] In one embodiment, in step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is (10-20):1.

[0018] In one embodiment, in step S13, the kudzu pulp and defatted cyperus juncea flour are mixed and extracted, 0.05% amylase is added and the mixture is extracted in a water bath at 85°C-95°C for 1 hour, the mixture is cooled to 65°C, 0.05% saccharifying enzyme is added and the mixture is extracted in a water bath at 60°C for 1 hour, the mixture is filtered through double gauze, and the mixture is boiled for 30 minutes and refrigerated for later use.

[0019] In one embodiment, in step S21, the kudzu nut extract, bread juice and pure water are evenly mixed in a ratio of (2-4):(2-4):(3-5).

[0020] In one embodiment, in step 22, brewer's dry yeast is added with 2% sucrose at a material-water ratio of 1:10 (W / V) and activated at 30° C. for 30 minutes until a large number of bubbles are generated.

[0021] In one embodiment, in step S23, lactic acid bacteria powder is inoculated into sterilized skim milk, cultured at 42°C until curdled, and then taken out and refrigerated in a 4°C refrigerator for later use, wherein the lactic acid bacteria powder includes thermophilic Streptococcus and bulgaricus, and the mass ratio of thermophilic Streptococcus to bulgaricus is 1:1.

[0022] In one embodiment, in step S24, yeast is inoculated into the raw material mixture for primary fermentation, and the raw material mixture is placed at 32° C. for fermentation for 4-20 hours.

[0023] In one embodiment, in the two fermentation steps, the volume ratio of yeast to lactic acid bacteria is (1-3):(1-3).

[0024] In one embodiment, in the two fermentation steps, the total inoculation amount of yeast and lactic acid bacteria is 1%-5%.

[0025] A kudzu vine and cyperus rotundus composite fermented beverage is prepared by the above preparation method.

[0026] In summary, the present invention has the following beneficial effects: The invention uses kudzu pulp and defatted cyperus juncea powder after starch extraction as raw materials, adds bread juice, and prepares a composite bacteria fermented beverage through composite fermentation with lactic acid bacteria and yeast, thereby effectively improving the added value of kudzu and cyperus juncea. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the standard curve of total flavonoids content in Pueraria root; Figure 2It is a schematic diagram of the anaerobic culture device; Figure 3 This is a graph showing the effect of the mass ratio of kudzu pulp to defatted cyperus rotundus flour on the DPPH• scavenging capacity of fermented beverages; Figure 4 This is a graph showing the effect of the addition amount of defatted kudzu nut extract on the DPPH scavenging capacity of fermented beverages; Figure 5 This is a graph showing the effect of bread juice addition on the DPPH• scavenging ability of fermented beverages; Figure 6 This is a graph showing the effect of bacterial inoculum size on the DPPH• scavenging ability of fermented beverages; Figure 7 This is a graph showing the effect of bacterial inoculation ratio on the DPPH• scavenging ability of fermented beverages; Figure 8 This is a graph showing the effect of yeast fermentation time on the DPPH scavenging ability of fermented beverages. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0029] It is worth noting that the directional words such as "upper" and "lower" involved in this article are all relative to the perspective of the drawings. They are only for the convenience of description and cannot be understood as limitations on the technical solution.

[0030] The present invention provides a method for preparing a composite fermented beverage of kudzu vine and cyperus rotundus, as follows: S1. Raw material pretreatment: S11. Preparation of kudzu vine pulp: ultrafinely crush the dried kudzu vine powder, pass it through a 100-mesh sieve, mix the obtained kudzu vine powder with water at a material-water ratio of 1:30, add citric acid to adjust the pH to 3.7-4.2, soak in a constant temperature water bath at 30-35°C for 3h, let it stand and settle for a period of time, centrifuge to obtain kudzu vine pulp, and refrigerate for later use.

[0031] S12. Prepare bread juice: bake fresh bread until both sides are browned, cool to room temperature, cut into small pieces, wrap with gauze, add pure water, saccharify at a constant temperature of 69-73℃, filter with double-layer gauze, and refrigerate for later use.

[0032] S13. Prepare kudzu nut extract: mix kudzu nut pulp and defatted kudzu nut powder for extraction, add amylase and extract in a water bath at 85-95°C, then cool to 60-65°C, add saccharifying enzyme and extract in a water bath at 58-62°C, filter through double-layer gauze, boil, and refrigerate for later use.

[0033] S2, fermentation: S21. Raw material mixing: uniformly mix the kudzu nut extract, bread juice and pure water in a certain proportion to obtain a raw material mixture.

[0034] S22. Yeast activation: Add water and sucrose to the brewing dry yeast and place it in 22-40℃ for activation until a lot of bubbles appear.

[0035] S23. Activation of lactic acid bacteria: inoculate lactic acid bacteria powder into sterilized skim milk, culture at a constant temperature of 40-45°C until the milk is coagulated, remove from the milk, and refrigerate at 4°C for later use. The lactic acid bacteria powder includes thermophilic Streptococcus and bulgaricus.

[0036] S24, yeast fermentation: inoculate yeast into the raw material mixture for primary fermentation, and place the raw material mixture at 20-38°C for fermentation.

[0037] S25. Homogenization: Use an adjustable speed homogenizer to mix and homogenize the mixed sample liquid, and place it in an incubator to continue fermentation and cultivation.

[0038] S26. Lactic acid bacteria fermentation: After the yeast fermentation is completed, the activated lactic acid bacteria are inoculated for secondary fermentation, and fermented at 30-42°C for 2-24 hours. Then, the product is filtered through a double-layer gauze sterilized at high temperature, and the filtrate is collected to obtain the fermentation product.

[0039] S27. Refrigeration: Store the fermented product at 4°C.

[0040] Preferably, in step S12, the fresh bread is baked in an oven at 150°C for 10 min until both sides of the bread are browned, and after cooling to room temperature, it is cut into small pieces of bread of about 3 cm, wrapped with gauze, and pure water is added at a material-water ratio of 1:20. The bread is saccharified at a constant temperature of 70°C for 3 h, filtered through double-layer gauze, and refrigerated for later use.

[0041] Preferably, in step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is (10-20):1.

[0042] More preferably, in step S13, the kudzu pulp and defatted cyperus oleifera powder are mixed and extracted, 0.05% amylase (the mass of amylase accounts for the mass of the kudzu pulp-defatted cyperus oleifera powder mixture) is added, and the mixture is extracted in a water bath at 85°C-95°C for 1 hour, the temperature is cooled to 65°C, 0.05% saccharifying enzyme (the mass of saccharifying enzyme accounts for the mass volume ratio w / v of the kudzu pulp-defatted cyperus oleifera powder mixture) is added, and the mixture is soaked in a water bath at 60°C for 1 hour, filtered through double gauze, and then boiled for 30 minutes, and refrigerated for later use.

[0043] Preferably, in step S21, the kudzu nut extract, bread juice and pure water are uniformly mixed in a ratio of (2-4):(2-4):(3-5), and more preferably, the ratio is 3:3:4.

[0044] Preferably, in step 22, brewer's dry yeast is added with 2% sucrose (the mass of sucrose is the percentage of the total mass of the yeast and water mixture) at a material-water ratio of 1:10 (W / V), and activated at 30° C. for 30 minutes until a large number of bubbles appear.

[0045] Preferably, in step S23, lactic acid bacteria powder is inoculated into sterilized skim milk, cultured at 42°C until curdled, taken out and refrigerated at 4°C for later use, wherein the lactic acid bacteria powder includes thermophilic Streptococcus and Lactobacillus bulgaricus, and the mass ratio of thermophilic Streptococcus to Lactobacillus bulgaricus is 1:1.

[0046] Preferably, in step S24, yeast is inoculated into the raw material mixture for primary fermentation, and the raw material mixture is placed at 32° C. for fermentation for 4-20 hours.

[0047] Preferably, in the two fermentation steps, the volume ratio of yeast to lactic acid bacteria is (1-3):(1-3).

[0048] Preferably, in the two fermentation steps, the total inoculation amount of yeast and lactic acid bacteria is 1%-5%.

[0049] The specific solutions of the present invention are described below with reference to specific embodiments. Example 1

[0050] According to the preparation steps of the present invention, a composite fermented beverage of kudzu nut and cyperus rotundus is produced, wherein: In step S13, the mass ratio of kudzu pulp to defatted cyperus chinensis powder is controlled to be 10:1, 12:1, 15:1, 17:1, and 20:1, respectively, to obtain kudzu chinensis extract. In step S21, calculated by mass percentage, the amount of the kudzu nut extract added is 30%, the amount of bread juice added is 30%, and the amount of pure water added is 40%.

[0051] In steps S24-S26, the yeast and lactic acid bacteria inoculation ratio (volume ratio) is 1:1, the total inoculation amount of the two strains is 3% (the percentage of the strain mass to the raw material mixture mass), and the yeast fermentation time is 12 hours.

[0052] After preparing the kudzu nut extract according to step S1, a fermented beverage is prepared according to step S2, and the optimal mass ratio of kudzu nut pulp to defatted kudzu nut powder is determined using DPPH•free radical scavenging ability as an evaluation index. Example 2

[0053] Compared with Example 1, this embodiment differs in that: In step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is 12:1.

[0054] In step S21, the addition amount of bread juice is 30% by mass, and the addition amounts of kudzu nut extract are controlled to be 10%, 20%, 30%, 40%, and 50% respectively, with the remainder being water.

[0055] The remaining steps were the same as those in Example 1. The optimal addition amount of the Cyperus rotundus extract was determined using the DPPH• free radical scavenging ability as the evaluation index. Example 3

[0056] Compared with Example 1, this embodiment differs in that: In step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is 12:1.

[0057] In step S21, calculated by mass percentage, the amount of the kudzu nut extract added is 30%, the amounts of the bread juice added are controlled to be 10%, 20%, 30%, 40%, and 50%, respectively, and the remainder is water.

[0058] The remaining steps are the same as those in Example 2. The optimal amount of bread juice to be added is determined. Example 4

[0059] Compared with Example 1, this embodiment differs in that: In step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is 12:1.

[0060] In steps S24-S26, the bacterial inoculation amount is controlled to be 1%, 2%, 3%, 4%, and 5%, respectively.

[0061] The remaining steps are the same as those in Example 2. Determine the optimal bacterial inoculation amount. Example 5

[0062] Compared with Example 1, this embodiment differs in that: In step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is 12:1.

[0063] In steps S24-S26, the inoculation ratios (volume ratio) of yeast and lactic acid bacteria are controlled to be 3:1, 2:1, 1:1, 1:2, and 1:3, respectively.

[0064] The remaining steps are the same as those in Example 2. Determine the optimal bacterial inoculation ratio. Example 6

[0065] Compared with Example 1, this embodiment differs in that: In step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is 12:1.

[0066] In steps S24-S26, the yeast fermentation time is controlled to be 4 hours, 8 hours, 12 hours, 16 hours, and 20 hours respectively.

[0067] The remaining steps are the same as in Example 2. The optimal yeast fermentation time is determined.

[0068] According to the single factor experimental conclusions of Examples 1-6, a 4-factor 3-level experiment was designed to select the addition amount of kudzu nut extract, the addition amount of bread juice, the inoculation amount of bacteria, and the yeast fermentation time. + •The comprehensive score of free radical scavenging rate, total antioxidant capacity and sensory evaluation was used as the response value. The Box-Behnken experimental design was used, and the levels of each factor in the experiment are shown in Table 1.

[0069] Table 1 Response surface experimental design

[0070] DPPH• free radical scavenging ability, ABTS + • Free radical scavenging ability and total antioxidant capacity are in vitro antioxidant models, with ascorbic acid as a positive control. 50 The value indicates the DPPH• free radical scavenging ability, ABTS + • Free radical scavenging ability, IC 50 The value indicates that the sample solution inhibits DPPH•(ABTS + •) is the concentration of the extract when the absorbance is 50%, IC 50 The smaller the value, the stronger the antioxidant capacity. The larger the absorbance value, the stronger the total antioxidant capacity of the sample solution.

[0071] Determination of DPPH free radical scavenging ability: Take 2 mL of sample solution, add 2 mL of 2×104 mol / L DPPH ethanol solution, shake well, react in the dark at room temperature for 30 min, and measure the absorbance at 517 nm. For the blank group, replace the sample with 2 mL of anhydrous ethanol. Calculate IC based on the absorbance value 50 value: ; In the formula: A1 is the absorbance value of the sample group solution; A2 is the corresponding absorbance value measured when anhydrous ethanol replaces the DPPH•ethanol solution; A0 is the absorbance value of the blank group.

[0072] ABTS + • Determination of free radical scavenging ability: ABTS +• and potassium persulfate were prepared into 7.4 mmol / L and 2.6 mmol / L solutions respectively with distilled water. Take equal volumes of each solution, mix them evenly, and let them stand at room temperature in the dark for 12-16 hours. Dilute them 40-50 times. Dilute the ABTS + • solution with phosphate buffer solution to an absorbance of 0.70 ± 0.02 to obtain the working solution. Add 1.9 mL of ABTS to 0.2 mL of the sample solution. + • Mix the working solution, shake for 10 seconds, let it stand for 6 minutes, and then measure the absorbance A1 at 734 nm. Use distilled water as a blank control and measure the absorbance A0 ; Where: A1 is the absorbance value of the sample solution; A2 is anhydrous ethanol instead of ABTS + •The corresponding absorbance value is measured when the working solution is added; A0 is the absorbance value of the blank group.

[0073] Determination of total antioxidant capacity: aspirate 0.25 mL of sample solution, add 0.625 mL of phosphate buffer (pH 6.6) and 0.625 mL of 1% K3Fe(CN)6 solution, mix and place in a 50°C water bath for 20 min, add 0.625 mL of 10% TCA solution, mix well, aspirate 0.625 mL, add 0.625 mL of water and 0.625 mL of 0.1% FeCl3 solution, mix well, let stand at room temperature for 10 min, and measure the absorbance A at 700 nm. The reagent is used as the blank control A0.

[0074] Total reducing power W A =A-A0; Where: W A is the total antioxidant capacity; A is the absorbance of the sample; A0 is the absorbance of the blank group.

[0075] Determination of total flavonoids content in Cyperus rotundus fermented beverage: Drawing of the standard curve: Accurately weigh 2.0 mg of puerarin standard, dissolve it in 70% ethanol solution and dilute it to a 10 mL volumetric flask, which is called the mother solution. Accurately pipette 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, and 600 μL of solution from the mother solution into 6 10 mL volumetric flasks, and also dilute it with 70% ethanol to prepare a series of control solutions with concentration gradients. Measure the absorbance of the reference solution at a wavelength of 250 nm, use 70% ethanol as the reference solution, the absorbance as the vertical coordinate Y, and the concentration of puerarin reference solution as the horizontal coordinate X (mg / mL) to draw the standard curve, see Figure 1 The regression equation of absorbance and puerarin concentration was y=74.536x+0.0115, and the correlation coefficient R 2 =0.9996, showing a good linear correlation in the range of 0-0.012 mg / mL.

[0076] Determination of total flavonoid content in samples: For fermented beverages produced under certain process conditions, pipette 100 μL of the fermentation sample into a 10 mL volumetric flask with 70% ethanol. Measure the sample absorbance at a wavelength of 250 nm. Using 70% ethanol as the reference solution, calculate the total flavonoid content in the sample (to three decimal places) based on the standard curve. The calculation formula is as follows: Where: C is the puerarin content obtained from the regression equation, mg / mL; V1 is the fixed volume of the sample solution, mL; V2 is the volume of the sample fermentation liquid, mL.

[0077] GC-MS analysis of volatile components Headspace solid-phase microextraction GC-MS conditions for fermented beverages: Accurately measure 6.0 mL of different samples into a 15 mL solid-phase microextraction sample bottle, add 0.375 g of sodium chloride to promote the volatilization of aroma components, seal the bottle with a lid, insert the aged 50 / 30 μm DVB / CAR / PDMS solid-phase microextraction head into the headspace of the sample bottle, equilibrate at 70°C for 30 min, adsorb for 15 min, remove the extraction head and insert it into the GC injection port, start the instrument to collect data at the same time, and desorb for 5 min.

[0078] Gas chromatography-mass spectrometry detection conditions: Chromatographic conditions: injection port temperature 250°C, injection volume 2 μL; temperature program: initial temperature 50°C, hold for 2 min, increase at 5°C / min to 200°C, hold for 5 min, increase at 25°C / min to 250°C, hold for 5 min. The chromatographic column was . The carrier gas flow rate was 1.3 mL / min, nitrogen was used, and split injection was used with a split ratio of 1:1.

[0079] Mass spectrometry conditions were as follows: electron ionization source, electron energy 70 eV, filament energy 0.20 mA, ion source temperature 230 °C, interface temperature 250 °C, and mass scan range 35.00-500.00 m / z.

[0080] Headspace solid-phase microextraction and GC-MS conditions for Pueraria lobata and Pueraria lobata: 3.0 g of Pueraria lobata or Pueraria lobata fragments were weighed and placed in a 20 mL headspace vial. A 50 / 30 μm DVB / CAR / PDMS extraction head was used, equilibrated at 90 °C for 13 min, extracted for 50 min, and finally desorbed at the injection port for 5 min.

[0081] Gas chromatography-mass spectrometry detection conditions Chromatographic conditions: TR-PESTICIDE elastic quartz capillary column (30 m×0.25 mm, 0.25 μm); heating program: initial temperature 40°C, hold for 1.0 min, then increase the temperature to 200°C at a rate of 5°C / min, hold for 2 min, and then increase the temperature to 250°C at a rate of 15°C / min and hold for 2.0 min; split ratio 10:1; inlet temperature 250°C; carrier gas: 99.999% high-purity nitrogen; flow rate 1.2 mL / min.

[0082] Mass spectrometry conditions: electron ionization source; ion source temperature 280°C; transfer line temperature 280°C; electron energy 70 eV; full scan scan mode; mass scan range 35-400 m / z.

[0083] Sensory evaluation: Ten students majoring in food were randomly selected to conduct sensory evaluation based on five aspects: color, aroma, texture, taste and characteristics. The sensory scoring table is shown in Table 2.

[0084] Table 2 Sensory scoring table

[0085] Comprehensive scoring: The comprehensive scoring method was used to determine the optimal fermentation process of the compound fermented beverage of kudzu nut and cyperus rotundus. In the response surface experiment, the weights of the factors were assigned, and the sensory evaluation weight was 0.5. The DPPH•free radical IC 50 The value weight is 0.1, ABTS+• free radical IC 50 The weight of the value is 0.1, and the weight of the total antioxidant capacity is 0.3. That is, the comprehensive score = sensory score * 0.5 + DPPH•free radical IC 50 value*0.1+ABTS + • Free radical IC 50 Value*0.1+total antioxidant capacity*0.3. The following is the grade distribution table 3 and grade score table 4.

[0086] Table 3 Grade distribution table

[0087] Table 4 Grade score table

[0088] Product index testing method Soluble solids content and pH determination: The soluble solids of the sample were determined using a handheld refractometer

[32] . pH determination: The pH value of the sample was determined using a pH and PH value analyzer.

[0089] Method for determining the number of viable lactic acid bacteria: Lactic acid bacteria in fermented beverages were counted according to GB 4789.35-2016

[33] . The viable bacteria were counted by culture observation using the gradient dilution plate coating method, and the number of viable bacteria was expressed as CFU / mL.

[0090] The lactic acid bacteria in the embodiment are powders of Lactobacillus bulgaricus and Streptococcus thermophilus in a ratio of 1:1. Lactobacillus requires anaerobic culture. The anaerobic culture method is as follows: in a larger sealed container, add two small beakers, each containing a mixture of 15% sodium hydroxide solution and 10g of pyrogallic acid, i.e., an oxygen-absorbing solution. Place the small beaker containing the oxygen-absorbing solution in a sealed container, place the prepared MRS oxygen culture medium on top of the oxygen-absorbing solution, cover both the culture medium and the small beaker with a clean large beaker, and add an appropriate amount of water along the beakers, as shown in the schematic diagram. Figure 2 shown.

[0091] Observation of bacterial morphology under a scanning electron microscope: Take a small amount of the fermented beverage for Gram staining and observe the morphology of the bacterial strain under a scanning electron microscope.

[0092] Data statistics and analysis: All experimental measurements were repeated 3 times. The experimental data were organized using Excel software, and the experimental results were routinely processed and analyzed using GraphPad Prism 5 software.

[0093] The results of Examples 1-6 are analyzed as follows: 1. Single factor test results of compound fermentation process of kudzu vine and cyperus rotundus: 1.1 Effect of the mass ratio of kudzu pulp to defatted cyperus chinensis powder on the antioxidant capacity of fermented beverages (Table 5 and Figure 3 shown.

[0094] Depend on Figure 3 As shown in Table 5, beverages fermented with different mass ratios of kudzu pulp and defatted cyperus chinensis powder had varying degrees of scavenging activity against DPPH• free radicals. The scavenging activity increased with increasing concentration, showing a dose-effect relationship. The beverage fermented with a mass ratio of kudzu pulp to defatted cyperus chinensis powder of 10:1 had the strongest DPPH• free radical scavenging ability, with an IC 50 The value was 0.2166 mL / mL. At a concentration of 0.1 mL / mL, its DPPH radical scavenging rate was 41.28%. The results showed that the optimal mass ratio of kudzu pulp to defatted cyperus juncea flour was 10:1, and beverages made with other mass ratios also had good effects.

[0095] Table 5 Effect of the mass ratio of kudzu pulp to defatted cyperus chinensis flour on the antioxidant capacity of fermented beverages

[0096] 1.2 Effects of the amount of Cyperus rotundus extract added on the antioxidant capacity of fermented beverages are shown in Tables 6 and Figure 4 shown.

[0097] Depend on Figure 4 As shown in Table 6, different addition amounts of Cyperus rotundus extract had different degrees of scavenging effects on DPPH• free radicals in fermented beverages, and the scavenging activity increased with increasing concentration, showing a dose-effect relationship. The fermented beverage with 20% Cyperus rotundus extract had the strongest DPPH• free radical scavenging ability, and its IC 50 The value was 0.2041 mL / mL. At a concentration of 0.1 mL / mL, the DPPH radical scavenging rate was 32.39%. The results indicate that the optimal addition level of Cyperus rotundus extract is 20%. Beverages made with other addition levels also showed good results.

[0098] Table 6 Effect of the addition amount of Cyperus rotundus extract on the antioxidant capacity of fermented beverages

[0099] 1.3 Effect of bread juice addition on the antioxidant capacity of fermented beverages (Table 7 and Figure 5 shown.

[0100] Depend on Figure 5 As shown in Table 7, different addition amounts of bread juice have different degrees of scavenging effect on DPPH• free radicals in fermented beverages, and the scavenging activity increases with the increase of concentration, showing a dose-effect relationship. The fermented beverage with 20% bread juice addition has the strongest DPPH• free radical scavenging ability, and its IC 50 The value was 0.1921 mL / mL. At a concentration of 0.1 mL / mL, the DPPH radical scavenging rate was 35.35%. The results indicate that the optimal addition level of bread juice is 20%. Beverages prepared with other addition levels also showed good results.

[0101] Table 7 Effect of bread juice addition on the antioxidant capacity of fermented beverages

[0102] 1.4 Effects of bacterial species (yeast: lactic acid bacteria) inoculation amount on the antioxidant capacity of fermented beverages are shown in Table 8 and Figure 6 shown.

[0103] Depend on Figure 6As shown in Table 8, different inoculum concentrations of different bacterial strains exhibited varying degrees of DPPH radical scavenging activity in fermented beverages, with scavenging activity increasing with increasing concentration, demonstrating a dose-response relationship. Fermented beverages with a 1% inoculum exhibited the strongest DPPH radical scavenging activity, with an IC50 value of 0.1961 mL / mL. At a concentration of 0.1 mL / mL, the DPPH radical scavenging rate was 41.79%. These results indicate that the optimal inoculum concentration for the mixed bacterial strain (yeast + lactic acid bacteria) is 1%. Beverages prepared with other inoculum concentrations also demonstrated good results.

[0104] Table 8 Effect of bacterial inoculation amount on the antioxidant capacity of fermented beverages

[0105] 1.5. Effect of strain (yeast: lactic acid bacteria) inoculation ratio on the antioxidant capacity of fermented beverages (see Table 9 and Figure 7 shown.

[0106] Depend on Figure 7 As shown in Table 9, different bacterial species (yeast: lactic acid bacteria) inoculation ratios have different degrees of scavenging effects on DPPH• free radicals in fermented beverages, and the scavenging activity increases with increasing concentration, showing a dose-effect relationship. The fermented beverage with a yeast: lactic acid bacteria ratio of 3:1 has the strongest DPPH• free radical scavenging ability, and its IC 50 The value was 0.1659 mL / mL. At a concentration of 0.1 mL / mL, its DPPH radical scavenging rate was 38.14%. The results showed that the optimal yeast:lactic acid bacteria inoculation ratio was 3:1. Beverages prepared with other bacterial species also showed good results.

[0107] Table 9 Effect of bacterial inoculation ratio (yeast: lactic acid bacteria) on the antioxidant capacity of fermented beverages

[0108] 1.6 The effect of yeast fermentation time on the antioxidant capacity of fermented beverages is shown in Table 10 and Figure 8 shown.

[0109] Depend on Figure 8 As shown in Table 10, different yeast fermentation times have different degrees of scavenging effects on DPPH• free radicals in fermented beverages, and the scavenging activity increases with the increase of concentration, showing a dose-effect relationship. The fermented beverage with a yeast fermentation time of 8h has the strongest DPPH• free radical scavenging ability, and its IC 50 The value was 0.2179 mL / mL. At a concentration of 0.1 mL / mL, the DPPH radical scavenging rate was 35.47%. The results indicate that the optimal fermentation time for yeast is 8 hours. Beverages made with other fermentation times also performed well.

[0110] Table 10 Effect of yeast fermentation time on the antioxidant capacity of fermented beverages

[0111] 2. Response surface optimization experiment results of the composite fermentation process of kudzu vine and cyperus rotundus Response surface test results: Based on the single factor experiment, a 4-factor 3-level experiment was designed with the addition amount of kudzu nut extract, bread juice addition amount, bacterial inoculation amount, and yeast fermentation time as the investigation factors. The DPPH• free radical scavenging ability, ABTS + • The free radical scavenging ability, total antioxidant capacity, and the comprehensive score of sensory evaluation were used as the response value. The experimental design was carried out using Box-Behnken central combination. Regression model and variance analysis were established.

[0112] In the regression model, the amount of Cyperus rotundus extract (B) significantly affected the overall score of the product (P<0.05), while the yeast fermentation time (A), inoculation size (C), and bread juice addition (D) had no significant impact on the overall score (P>0.05). The order of importance of factors influencing the overall score of fermented beverages was B>A>C>D, indicating that the amount of Cyperus rotundus extract (B) had the greatest impact on the overall score of fermented beverages, followed by yeast fermentation time (A), inoculation size (C), and bread juice addition (D), with the least impact.

[0113] The present invention employs a primary fermentation with yeast followed by a secondary fermentation with lactic acid bacteria to produce the optimal process conditions for a kudzu and cyperus rotundus composite fermented beverage: a primary yeast fermentation time of 6.89 hours, a kudzu and cyperus rotundus extract addition of 28.21%, a bacterial inoculum (yeast: lactic acid bacteria) of 3.26%, and a bread juice addition of 28%. This yielded a comprehensive score of 95.11. Based on the feasibility of the operational process, the optimal fermentation process was modified to a primary yeast fermentation time of 7 hours, a kudzu and cyperus rotundus extract addition of 29%, a bacterial inoculum (yeast: lactic acid bacteria) of 3%, and a bread juice addition of 28%. Validation tests conducted under these optimized conditions yielded a comprehensive score of 93 for the kudzu and cyperus rotundus fermented beverage, very close to the simulated value, further validating the model's practicality.

[0114] DPPH• free radical scavenging ability, ABTS + •The free radical scavenging ability, total antioxidant capacity and comprehensive score of sensory evaluation were used as indicators. The product fermented only by yeast was beverage I, the product fermented with mixed yeast (yeast + lactic acid bacteria) was beverage II, and the product fermented only by lactic acid bacteria was beverage III. The antioxidant capacity of the kudzu nut beverages produced by the three fermentation processes was analyzed.

[0115] The total flavonoids content of kudzu pulp, kudzu nut extract, beverage I, beverage II, and beverage III were also analyzed, and the results are shown in Table 11. The higher the total flavonoids content in kudzu, the stronger its antioxidant capacity.

[0116] From Table 11, we can see that the total flavonoids content of Pueraria lobata pulp is relatively high, and its DPPH• free radical scavenging ability and ABTS + • The free radical scavenging ability and total antioxidant capacity are relatively strong, showing good antioxidant capacity. The total flavonoid content and antioxidant capacity of the Cyperus rotundus extract obtained by the extraction process do not change much, that is, the total flavonoid content lost during the extraction process is not much.

[0117] After yeast fermentation, beverage I had a low total flavonoid content, relatively weak antioxidant capacity, and a moderate taste, with an overall score of 86.5. Beverage II had a relatively moderate total flavonoid content, but relatively strong antioxidant capacity and a good taste, with an overall score of 93. Beverage III had the highest total flavonoid content, but relatively weak antioxidant capacity and a mediocre taste, with an overall score of 83. In summary, the mixed-strain fermented kudzu nut beverage of the present invention has a more refreshing taste.

[0118] Table 11 IC50 values ​​and comprehensive scores of kudzu pulp, extracts and different fermentation processes

[0119] 3. GC-MS analysis of the volatile components of kudzu vine fermented beverages revealed that the flavor of kudzu vine fermented beverages produced using different fermentation processes was primarily composed of alcohols, esters, acids, and hydrocarbons, with the total relative content of these four volatile components ranging from 58% to 85%. In contrast, the volatile flavor compounds of Pueraria lobata and kudzu vine were primarily composed of aldehydes, pyrazines, alcohols, ketones, and furans, with the total relative content of these five volatile components ranging from 80% to 95%.

[0120] Group N, consisting of fermented beverages fermented solely by yeast, contained eight alcohols (16.66%), six esters (30.54%), four acids (18.60%), two aldehydes (0.95%), four hydrocarbons (9.36%), and two other compounds (23.88%). The main volatile compounds (relative content >10%) were benzylhydrazine (22.75%), octanoic acid (14.49%), isoamyl acetate (13.07%), and linalyl acetate (11.92%). Octanic acid has goaty and cheesy aromas, but its threshold is very high. Isoamyl acetate has fruity, raw pear, and banana aromas. Linalyl acetate has a floral and fruity aroma. Ethyl octanoate has wine, brandy, and fruity-floral aromas.

[0121] In group M, a fermented beverage produced by a combination of yeast and lactic acid bacteria, seven alcohols (18.27%), five esters (25.76%), three acids (17.39%), two hydrocarbons (1.55%), and two other compounds (37.03%) were detected. The main volatile compounds (relative content >10%) were benzylhydrazine (36.36%), octanoic acid (12.02%), isoamyl acetate (12.00%), linalyl acetate (10.51%), and 2-ethylhexanol (10.21%).

[0122] In the R group, which was fermented only by lactic acid bacteria, a total of 5 alcohols (7.49%), 5 esters (41.90%), 4 acids (44.91%), 1 hydrocarbon (0.28), and 2 other compounds (5.43%) were detected. Its volatile compounds (relative content > 6%) were mainly isoamyl acetate (30.73%), octanoic acid (32.09%), hexanoic acid (9.29%), and linalyl acetate (6.02%). Hexanoic acid has a cheese-like, sweat-like odor. Volatile flavor analysis of Pueraria lobata revealed four alcohols (4.59%), one ester (1.39%), six aldehydes (33.97%), two ketones (8.59%), six pyrazines (49.32%), and two furans (2.14%). The main volatile compounds (relative content >10%) were 2-methylpyrazine (20.60%), 2,6-dimethylpyrazine (18.53%), 3-furfural (14.66%), and n-octanal (11.98%). 2-Methylpyrazine has a cocoa-like nutty aroma, while n-octanal has a strong fatty and fruity aroma.

[0123] The volatile flavor compounds detected in Pueraria lobata included seven alcohols (13.12%), eight esters (9.91%), three acids (2.86%), nine aldehydes (41.09%), four ketones (14.78%), one hydrocarbon (1.05%), three pyrazines (2.99%), two furans (11.56%), and two other compounds (2.64%). The main volatile flavor compounds (relative content >7%) were nonanal (15.89%), n-octanal (11.99%), 2-n-pentylfuran (7.89%), and (3E,5E)-octa-3,5-dien-2-one (7.71%). 2-Acetylfuran (3.61%) exhibits sweet and caramel aromas and is a flavor compound associated with potato and coffee. 1-Octen-3-ol (3.07%) has a mushroom and herbal aroma.

[0124] An appropriate amount of acids can provide a buffering effect on kudzu nut fermented beverages, harmonizing and enhancing the main flavor components of the kudzu nut fermented beverages. This also helps to generate volatile components and enhance the overall flavor of the fermented beverages. Alcohols can provide kudzu nut fermented beverages with unique flavor components, playing a significant role in the flavor of the fermented beverages. Esters, as a whole, can provide the fermented beverages with delicious fruity aroma components, making them sweet and mellow.

[0125] 4. Analysis of test results of product indicators: 4.1. Main physical and chemical indicators: pH 4.7; soluble solids content 2.8%.

[0126] 4.2. Lactic acid bacteria count: The total number of lactic acid bacteria colonies in the fermented beverages obtained by MRS medium and MC medium was 41.5×10 6 CFU / mL.

[0127] Based on Examples 1-6, single-factor and response surface experiments were used to optimize the fermentation process for a fermented beverage using kudzu pulp, defatted cyperus chinensis powder, and bread juice as raw materials. The results showed that the optimal raw material ratio and fermentation process parameters for the combined bacterial strains (yeast + lactic acid bacteria) were: a 10:1 mass ratio of kudzu pulp to defatted cyperus chinensis powder, 29% kudzu pulp extract, 3% bacterial inoculum (yeast:lactic acid bacteria), 28% bread juice, 40% pure water, 7 hours of fermentation time, and a 3:1 inoculum ratio (yeast:lactic acid bacteria by volume). Under these (yeast + lactic acid bacteria) mixed fermentation conditions, the resulting kudzu pulp-based cyperus chinensis fermented beverage exhibited a bright light yellow color, a sweet and sour taste, a pleasant aroma, and a good flavor.

[0128] The total flavonoids content of the kudzu nut beverage fermented by (yeast + lactic acid bacteria) is 0.201 mg / mL, and the DPPH•free radical IC 50 The value was 0.9716±0.0416mL / mL, ABTS + • Free radical IC 50 The value was 0.0979±0.0016 mL / mL, the total antioxidant capacity was 0.222±0.032 mL / mL, the sensory score was 96 points, and the comprehensive score was 93 points. The aroma and flavor of the beverage were better than those made by yeast fermentation alone and lactic acid bacteria fermentation alone.

[0129] The data were further analyzed using headspace solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry. The results showed that a total of 30 volatile flavor compounds were detected across the three fermentation processes, and a total of 49 volatile flavor compounds were detected across the two kudzu root raw materials. In the kudzu nut fermented beverage, alcohols, esters, and acids were found to be predominant, primarily including octanoic acid, isoamyl acetate, linalyl acetate, 2-ethylhexanol, and hexanoic acid. In the kudzu root raw material, aldehydes, furans, and ketones were found to be relatively prevalent, primarily including nonanal, n-octanal, 2-n-pentylfuran, and (3E,5E)-octa-3,5-dien-2-one.

[0130] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a composite fermented beverage of kudzu vine and cyperus rotundus, characterized in that: as follows: S1. Raw material pretreatment: S11. Preparing kudzu vine pulp: ultrafinely crush the dried kudzu vine powder, pass it through a 100-mesh sieve, mix the obtained kudzu vine powder with water at a material-water ratio of 1:30, add citric acid to adjust the pH to 3.7-4.2, and extract it in a constant temperature water bath at 30-35° C. for 3 h. After standing and settling for a period of time, centrifuge to obtain kudzu vine pulp, and refrigerate it for later use; S12. Prepare bread juice: bake fresh bread until both sides are browned, cool to room temperature, cut into small pieces, wrap with gauze, add purified water, saccharify at a constant temperature of 69-73°C, filter with double-layer gauze, and refrigerate for later use; S13. Preparing a kudzu bean extract: mixing kudzu bean pulp with defatted kudzu bean flour and extracting, adding amylase and extracting in a water bath at 85-95° C., then cooling to 60-65° C., adding saccharifying enzyme and extracting in a water bath at 58-62° C., filtering through double gauze, boiling, and refrigerating for later use; S2, fermentation: S21, raw material mixing: mixing the cyperus rotundus extract, bread juice and pure water in a certain proportion to obtain a raw material mixture; S22, Yeast activation: Add water and sucrose to the brewer's dry yeast and place it in 22-40℃ for activation until a lot of bubbles appear; S23, Activating Lactic Acid Bacteria: Inoculate lactic acid bacteria powder into sterilized skim milk, incubate at a constant temperature of 40-45°C until curdling occurs, remove from the milk, and refrigerate at 4°C for later use. The lactic acid bacteria powder includes Streptococcus thermophilus and Lactobacillus bulgaricus. S24, yeast fermentation: inoculating yeast into the raw material mixture for primary fermentation, and placing the raw material mixture at 20-38°C for fermentation; S25, homogenization: using an adjustable speed homogenizer to mix and homogenize the mixed sample solution, and continue fermentation and cultivation; S26, lactic acid bacteria fermentation: After the yeast fermentation is completed, activated lactic acid bacteria are inoculated for secondary fermentation, fermented at 30-42°C, and then filtered through a double-layer gauze sterilized at high temperature to obtain the filtrate to obtain the fermentation product; S27. Refrigeration: Store the fermented product at 4°C.

2. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In step S12, the fresh bread is baked in an oven at 150°C for 10 min until both sides of the bread are browned. After cooling to room temperature, the bread is cut into small pieces of about 3 cm in size, wrapped with gauze, and pure water is added at a material-water ratio of 1:

20. The bread is saccharified at a constant temperature of 70°C for 3 h, filtered through double-layer gauze, and refrigerated for later use.

3. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In step S13, the mass ratio of kudzu pulp to defatted cyperus juncea flour is (10-20):1; Mix the kudzu pulp and defatted cyperus juncea flour and extract them. Add 0.05% amylase and extract them in a water bath at 85℃-95℃ for 1 hour. Cool the mixture to 65℃, add 0.05% saccharifying enzyme and extract them in a water bath at 60℃ for 1 hour. Filter the mixture through double-layer gauze, boil the mixture for 30 minutes, and refrigerate for later use.

4. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In step S21, the kudzu nut extract, bread juice and pure water are uniformly mixed in a ratio of (2-4):(2-4):(3-5).

5. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In step 22, brewer's dry yeast is added with 2% sucrose at a material-water ratio of 1:10 and activated at 30°C for 30 minutes until a large number of bubbles appear.

6. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In step S23, lactic acid bacteria powder is inoculated into sterilized skim milk, and cultured at 42°C until curdled. The milk is then taken out and refrigerated at 4°C for later use. The lactic acid bacteria powder includes thermophilic Streptococcus and bulgaricus, and the mass ratio of thermophilic Streptococcus to bulgaricus is 1:

1.

7. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In step S24, yeast is inoculated into the raw material mixture for primary fermentation, and the raw material mixture is placed at 32° C. for fermentation for 4-20 hours.

8. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 1, wherein: In the two fermentation steps, the volume ratio of yeast to lactic acid bacteria is (1-3):(1-3).

9. The method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to claim 8, characterized in that: In the two fermentation steps, the total inoculation amount of yeast and lactic acid bacteria is 1%-5%.

10. A composite fermented beverage of kudzu vine and cyperus rotundus, characterized in that: The beverage is prepared by the method for preparing the composite fermented beverage of kudzu vine and cyperus rotundus according to any one of claims 1 to 9.

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