Preparation method of multi-strain asparagus fermented beverage

Abstract

The invention relates to the field of beverage fermentation, in particular to a preparation method of a multi-strain asparagus fermented beverage. According to the asparagus fermented beverage, nutrient substance macromolecules in asparagus are converted into micromolecular components which can be absorbed by a human body through a multi-strain fermentation technology, and the asparagus fermented beverage capable of reducing blood sugar, blood pressure and blood fat and dispelling the effects of alcohol is prepared. According to the invention, starch in corn grits and sweet potato flour is subjected to enzymolysis by using saccharifying enzyme, so that the starch is converted into dextrin and oligosaccharide, and the liquid viscosity in a solution is reduced; lactic acid bacteria and saccharomycetes are used for fermenting asparagus concentrated juice mixed liquor, the technical problems of acid production and aroma production are solved, then acetic acid bacteria are used for decomposing alcohol in the liquid, and finally the asparagus stock solution fermented beverage with BRIX larger than or equal to 10.5, PH of 3.6-3.8 and light transmittance T larger than or equal to 85 is obtained.

Description

Technical Field

[0001] The invention relates to the field of beverage fermentation, and in particular to a method for preparing a multi-strain asparagus fermented beverage. Background Art

[0002] Asparagus is a high-end and precious vegetable. It contains a variety of vitamins and trace elements of better quality than ordinary vegetables. Its protein composition contains various amino acids necessary for the human body, and the content ratio is appropriate. Among the inorganic salt elements, there are more trace elements such as selenium, molybdenum, magnesium, and manganese. It also contains a large amount of non-protein nitrogenous substances and asparagine, mainly asparagine. Regular consumption has a certain effect on heart disease, hypertension, tachycardia, fatigue, edema, cystitis, dysuria and other diseases. At the same time, asparagus is beneficial for cardiovascular disease, vascular sclerosis, nephritis, gallstones, liver dysfunction and obesity. In addition, asparagus also has a good effect on lymph node cancer, bladder cancer, lung cancer, kidney stones, skin cancer, other cancers, leukemia, etc. However, asparagus is a seasonal vegetable and is planted once a year. Only by transforming and processing products can people meet the needs of eating it all year round.

[0003] However, the nutrients in the deep-processed asparagus products are large molecules and are difficult for the human body to absorb. How to convert the large nutrients in the deep-processed asparagus products into small molecules that can be absorbed by the human body has become a research and development topic in the industry. The current market solution usually uses a single strain fermentation to prepare related foods, but single strain fermentation has limitations and can only convert a certain type of substance. The retention of nutrients after conversion and the taste of the finished product are often unsatisfactory. Therefore, the present invention adopts multi-strain fermentation technology to prepare asparagus fermented beverages, enhance the nutrients that can be absorbed by the human body, improve the drinking quality, and expand the consumer market. Summary of the invention

[0004] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0005] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a multi-species asparagus fermented beverage. The present invention is achieved through the following technical solutions: A method for preparing a multi-strain asparagus fermented beverage comprises the following steps: Step 1: Wash the asparagus raw material with chlorine dioxide, crush and pulp it after washing, and squeeze out the juice; wherein, the chlorine dioxide (ClO2) is 50-80ppm and the soaking time is ≥30s; the raw material after juicing is sterilized and centrifuged by butterfly centrifuge, and after centrifugation, it is low-temperature vacuum concentrated, degassed, and ultra-high temperature instantaneous sterilization (UHT) is performed to prepare 18°Bx asparagus concentrated juice for use; The sterilization temperature of fresh asparagus juice is controlled within the range of 85℃-95℃, and the discharge temperature is controlled within the range of 50℃-55℃; the insoluble solids after separation by the disc separator are controlled at 1.5-4% (4BIX); Step 2: proportioning the raw materials, adding liquefaction enzyme, liquefying at high temperature, cooling in a tubular manner after liquefaction, saccharifying after cooling, adding saccharifying enzyme, malt extract, and asparagus concentrate, mixing the saccharified liquid, asparagus juice, and malt extract evenly, and then performing lactic acid fermentation; after the lactic acid bacteria fermentation is completed, cooling is performed, yeast is added and stirring is maintained, after the yeast fermentation is completed, separation is performed by a horizontal centrifuge, and centrifugation is performed by a disc centrifuge; the liquid after centrifugation is sterilized, and secondary fermentation is performed after sterilization; acetic acid bacteria fermentation decomposes the alcohol produced by yeast fermentation; Step 3: After the acetic acid bacteria fermentation is completed, membrane filtration, diatomaceous earth filtration, degassing, UHT sterilization, packaging and storage are carried out.

[0006] Furthermore, in step 1, the raw material requirements for asparagus concentrate are as follows: pH 4.0-5.8, concentrated juice soluble solids 16-28Bx.

[0007] Furthermore, in step 2, the formula ratio is calculated by mass: corn grits: 25-40 parts, sweet potato flour: 0.1-1 parts, malt extract: 5-8 parts, asparagus concentrate: 100 parts, liquefaction enzyme: 0.05-0.1 parts, saccharification enzyme: 0.05-0.15 parts, lactic acid bacteria 0.01-0.1 parts, yeast 0.01-0.05 parts, acetic acid bacteria 0.01-0.1 parts.

[0008] Furthermore, in step 2, the lactic acid bacteria fermentation comprises the following steps: (1) Activation of lactic acid bacteria: Take 1L of liquefied liquid and sterilize it in a sterilizer at 121 degrees for 20 minutes. After cooling to 42 degrees, add lactic acid bacteria and perform aseptic operation. (2) Activate the culture medium in a 42-degree incubator for 3 hours before inoculating the fermenter; add the activated lactic acid bacteria solution into the fermenter, seal the inoculation port, and ferment at 38-42 degrees for 7-12 hours. When the pH value of the solution drops to between 4.0 and 5.0, the fermentation is completed.

[0009] Furthermore, in step 2, after the lactic acid bacteria fermentation is completed, the temperature is lowered to 28°C, and then the asparagus yeast is added to the fermentation tank through the inoculation port, the inoculation port is sealed, and stirring is kept to mix the yeast evenly. The fermentation temperature is maintained at 25-32°C, the yeast fermentation time is 8-12 hours, and the fermentation end point is that the pH of the fermentation liquid is 3.5-4.5, there is an obvious yeast aroma, and trace bubbles.

[0010] Further, in step 2, the acetic acid bacteria fermentation steps are as follows: (1) Fermentation tank disinfection: After cleaning, steam disinfection is performed at 121 degrees for 20 minutes; (2) After the fermentation liquid is pumped into the secondary fermentation tank, add the acetic acid bacteria into the secondary fermentation tank through the inoculation port, seal the inoculation port, and keep stirring to ensure that the acetic acid bacteria are evenly mixed. Maintain the fermentation temperature at 25-32°C for 15-24 hours, and the fermentation is completed when the time is up.

[0011] Furthermore, in step three, ultrafiltration membrane filtration: the transmittance is detected to be greater than 85 at 625 nm.

[0012] Furthermore, in step three, UHT sterilization: sterilization temperature is 116±2°C, sterilization time is 15-18s, and outlet temperature is 90°C.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses a multi-strain fermentation technology to convert the macromolecules of nutrients in asparagus into small molecules that can be absorbed by the human body, and prepares an asparagus fermented beverage that can lower blood sugar, blood pressure, blood lipids, and sober up. The present invention uses saccharifying enzymes to hydrolyze the starch in corn grits and sweet potato flour materials, converting the starch into dextrin and oligosaccharides, and reducing the liquid viscosity in the solution; lactic acid bacteria and yeast are used to ferment the asparagus concentrated juice mixture to solve the technical problems of acid production and aroma production, and then acetic acid bacteria are used to decompose the alcohol in the liquid, and finally an asparagus stock fermented beverage with BRIX≥10.5, PH3.6-3.8, and transmittance T≥85 is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below in conjunction with the accompanying drawings.

[0015] Figure 1 Figure 1 is a diagram of lactic acid bacteria cultured according to the present invention; Figure 2 This is a picture of microscopic examination of lactic acid bacteria of the present invention; Figure 3 Screening of acetic acid bacteria images for the present invention; Figure 4 This is a diagram showing the moving distance results in Example 6 of the present invention; wherein Figure 4 A in the middle is the moving distance result table; Figure 4 Middle B is the moving distance result picture; Figure 5 This is a phenotype diagram of the efficacy experiment results in Example 6 of the present invention; Figure 6 It is a drawing of the standard curve in Example 6 of the present invention; Figure 7 The standard curve of the concentration and peak area of ​​rutin, quercetin and kaempferol in the sample determined by HPLC in Example 6 of the present invention; Figure 8 This is a diagram of the detection results in Example 7 of the present invention; Fig. 9 This is the triglyceride result table in Example 7 of the present invention; Fig.10 This is a bar graph of the efficacy test results in Example 7 of the present invention; Fig.11 This is a diagram of the detection results in Example 8 of the present invention; Fig.12 This is a bar graph of the efficacy test results in Example 8 of the present invention; Fig.13 This is a diagram of the detection results in Example 9 of the present invention; Fig.14 This is a bar graph of the efficacy test results in Example 9 of the present invention; Fig.15 This is a phenotype diagram of the efficacy experiment results in Example 9 of the present invention. DETAILED DESCRIPTION

[0016] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0017] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0018] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0019] Unless otherwise specified, the raw and auxiliary materials used in the present invention are all food grade and commonly available on the market.

[0020] Example 1 Preparation of Asparagus Juice Concentrate Asparagus and asparagus peel and segments are selected, cleaned and disinfected (40-90ppm chlorine dioxide (ClO2) is used for disinfection for ≥30s); the raw materials are crushed into pulp after cleaning; the crushed raw materials are squeezed for juice; the raw materials are thoroughly disinfected by pasteurization technology (the sterilization temperature is within the range of 75℃-105℃, and the discharge temperature is adjusted within the range of 40℃-65℃; butterfly separation (control the insoluble solids at 1.0-3% (4Brix), and the comprehensive content is less than 3%); low-temperature vacuum concentration (using three-effect concentration, the first effect temperature is 70℃-90℃, the second effect temperature is 50℃-70℃, and the third effect temperature is 40℃-60℃); degassing; ultra-high temperature instantaneous sterilization (temperature control 115℃-137℃, time 15-45s, outlet temperature is 30-40℃); filling preparation.

[0021] The process of asparagus concentrated juice is optimized through the patent number: ZL200910016153.4 of Shandong Juxinyuan Agricultural Technology Co., Ltd. "Concentrated turbid juice made from asparagus waste and its processing technology".

[0022] Example 2 Selection and preparation of corn grits, sweet potato flour, liquefying enzyme and saccharifying enzyme (1) Corn grits are purchased from the market, with fineness requirements of: no more than 5% passing through an 18w sieve and no more than 10% passing through a 40w sieve.

[0023] Quality requirements .

[0024] Physical and chemical requirements .

[0025] (2) Sweet potato flour is purchased from the market, and the fineness requirement is: the mass fraction passing through a 150μm sieve is not less than 99.5%. Sensory requirements .

[0026] Physical and chemical requirements .

[0027] (3) Screening and preparation of lactic acid bacteria Materials and Methods Experimental materials: Asparagus was collected from the asparagus experimental base of Shandong Juxinyuan Agricultural Technology Co., Ltd.

[0028] Other materials: sucrose, lactic acid bacteria standard solution, MRS medium.

[0029] Test instruments: DHP-9012 constant temperature box, DZF oven, YYS-200 microscope, 723N spectrophotometer, LC3300 high performance liquid chromatograph, TDL-4000C centrifuge, Leiji PHS-3C precision pH meter, YL.23-JH electronic balance.

[0030] Test methods The strain samples were collected from the natural fermentation of asparagus, and 10 test samples were collected.

[0031] Isolation, purification and identification of lactic acid bacteria The 10 samples collected were separated and purified using MRS medium. Each sample corresponded to a culture medium, which was uniformly numbered and inverted at 37°C for 48 hours. Select strains with good growth and inoculate them in MRS flat medium, and purify and culture them according to the above method. Purify multiple times until purified (the individual colonies are of the same size and the bacteria are consistent under Gram staining microscopy). The purified strains were analyzed with the lactic acid bacteria standard solution, and their chromatograms were compared. The tolerance test was performed using low pH MRS flat medium and alkaline MRS flat medium to screen out the lactic acid bacteria strains. The lactic acid bacteria strains obtained after screening were preserved and amplified respectively.

[0032] Preservation method: inoculate the lactic acid bacteria strain into a sterile distilled water test tube with a small amount of soluble sugar and seal it for live preservation; amplification method: place the inoculated lactic acid bacteria strains in turn into McIntosh-Fildes jars, first evacuate the air and replace it with hydrogen to atmospheric pressure, and then turn on the power to make the remaining oxygen and hydrogen in the jar react with platinum to form water. Place the entire anaerobic jar in a constant temperature incubator and culture it at 37°C for 48 hours.

[0033] The standard plate colony counting method for lactic acid bacteria colony determination is to take 1 mL of the test bacterial solution for gradient dilution, and pour the diluted bacterial solution into MRS plate culture medium. The plate is placed at 37°C and cultured for 48 hours. When countable colonies grow, count them, and multiply the counting result by the dilution multiple to obtain the number of viable bacteria in the test bacterial solution.

[0034] Microbial flora analysis like Figure 1 , Figure 2 As shown in the figure, the lactic acid bacteria were screened by separation and purification by MRS plate streaking method, tolerance test (acid resistance, alkali resistance) and Gram staining method. A total of 7 lactic acid bacteria were screened from the 10 strains of bacteria collected. The culture characteristics of the strains are that the colonies are milky yellow, small, round and convex, smooth surface, neat edges, opaque or translucent, turbid bacterial liquid after liquid culture, and no bacterial film. The microscopic characteristics of the strains are rod-shaped and no spore-positive bacteria. Lactic acid bacteria with a large number of viable bacteria are selected for use.

[0035] Live cell count of lactic acid bacteria strains .

[0036] (4) Screening and preparation of acetic acid bacteria Materials and Methods Isolation samples: Asparagus was collected from the asparagus experimental base of Shandong Juxinyuan Agricultural Technology Co., Ltd., washed, and cultured at 36°C for 48 hours to isolate naturally formed acetic acid bacteria.

[0037] Main culture medium Isolation and purification plate culture medium: 1g glucose, 1g yeast extract, 2g calcium carbonate, 2g agar, 100mL water, sterilize at 121℃ for 20 min, cool to 70℃, add 4mL anhydrous ethanol and 0.5ml 0.02% crystal violet glacial acetic acid solution.

[0038] Slant test tube preservation medium; the same as the separation plate medium except that crystal violet solution is not added.

[0039] Basic culture: 1 g yeast extract, 1 g glucose, pH 4.5, 100 mL water, sterilize at 121 °C for 20 min, add 3 mL anhydrous ethanol before use.

[0040] Liquid seeds and fermentation medium: 1 g glucose, 1.5 g yeast extract, 0.05 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 100 mL water, pH 6.5. After sterilization, cool to below 60 °C and add 3.5 mL anhydrous ethanol.

[0041] method The process of acetic acid bacteria isolation and purification: culture asparagus sample → dilution and separation → purification → slant test tube storage → qualitative test of acetic acid production → test of acetic acid production rate.

[0042] Method for determination of acid production Take 1mL of fermentation broth, add 10mL of distilled water, 3-5 drops of 0.02% phenolphthalein alcohol solution, and titrate with a standardized 0.1mol / L NaOH solution until it turns light pink. Calculate the acid content (in acetic acid) in the sample based on the amount of NaOH solution consumed.

[0043] Acid production (%) = (V-V0) × C NaOH ×0.06×100% Where, V--the amount of NaOH consumed in the titration of the fermentation broth sample (mL); V0--the amount of NaOH consumed in titration with blank culture medium as control (mL); C NaOH -Concentration of NaOH standard solution (mol / L); 0.06--The mass of acetic acid equivalent to consuming 1mL 0.1mol / L NaOH solution (g); 100%--Converted to percentage The coefficient of content.

[0044] Separation and purification Take the unsterilized asparagus concentrate and inoculate 10% of it into the acetic acid bacteria liquid fermentation medium for culture. At the same time, measure the acid production, pick out the culture medium with a stable, fast and high acidity rise, take 1mL of the culture medium and dilute it to 10 with sterile water. -1 --10 -8 , respectively, take 0.2mL of the last three dilutions onto the acetic acid bacteria isolation and purification plate medium, immediately spread the plate with a sterile coating rod, invert it in a 30℃ constant temperature incubator, and culture it for 2 days. Repeat the above operation until a single colony appears on the plate. Pick a single colony with a large diameter of the color-changing transparent circle, abundant budding, and dominant growth for purification. Inoculate the purified strain on the slant preservation medium, culture at 30℃ for 48h, and then store it in a 4℃ refrigerator.

[0045] Qualitative test of acetic acid The above-mentioned slant strains were inoculated into the basic culture medium, and cultured in a constant temperature incubator at 30°C for 72 hours. The bacteria in the acetic acid bacteria fermentation broth were removed by centrifugation, and 5 mL was neutralized to pH 7.0 with 2.5 mol / L NaOH solution. 5-6 drops of 5% FeCl3 solution were added. The one that formed a reddish-brown precipitate was acetic acid bacteria.

[0046] Acid production test in liquid fermentation medium Take three rings of the activated slant strains and inoculate them into a 250mL Erlenmeyer flask containing 50mL of liquid fermentation medium. Incubate them in a constant temperature shaker at 30℃ and 160r / min for 48h. Inoculate them into a 250mL Erlenmeyer flask containing 50mL of liquid fermentation medium at a 6% inoculation rate (each sample is replicated in triplicate). During inoculation, the strains need to be shaken fully to make the bacterial liquid uniform. Incubate them in a constant temperature shaker at 30℃ and 160r / min. Take the fermentation liquid at different incubation times to measure its acid production.

[0047] Results and Discussion Isolation and purification of acetic acid bacteria Crystal violet is an excellent precision acid indicator. The color of crystal violet solution is shown in the table below.

[0048] Color table of crystal violet solution .

[0049] Adding a small amount of crystal violet solution to the acetic acid bacteria separation and purification plate culture medium will not affect the growth of acetic acid bacteria, and can quickly separate the acetic acid bacteria with high acid production according to the size of the color change circle. Figure 3As shown, a strain of acetic acid bacteria with an obvious discolored transparent circle and a larger diameter was obtained through separation and purification and was set aside.

[0050] Example 3: Selection of Asparagus Yeast The asparagus yeast selected was the LSNO1 yeast from Shandong Juxinyuan Agricultural Technology Co., Ltd., patent number: ZL202410294571.4 for "A kind of asparagus yeast and its application". The yeast is currently deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit number: CGMCC No.29806.

[0051] Example 4: Preparation of multi-strain asparagus fermented beverage 1. The asparagus raw material is washed with chlorine dioxide, and then crushed, pulped and juiced after washing; wherein, the chlorine dioxide (ClO2) is 50-80ppm and the soaking time is ≥30s; the raw material after juicing is sterilized and centrifuged by butterfly centrifuge, and after centrifugation, low-temperature vacuum concentration, degassing and ultra-high temperature instantaneous sterilization (UHT) are performed to prepare 18°Bx asparagus concentrated juice for use.

[0052] 2. The sterilization temperature of fresh asparagus juice is controlled within the range of 85℃-95℃, and the discharge temperature is controlled within the range of 50℃-55℃; after separation by the disc separator, the insoluble solids are controlled at 1.5-4% (4BIX).

[0053] 3. Carry out raw material proportioning, add liquefaction enzyme, liquefy at high temperature, cool down in a tube after liquefaction, saccharify after cooling, add saccharifying enzyme, malt extract, asparagus concentrate juice, mix the saccharification liquid, asparagus juice and malt extract evenly, and then carry out lactic acid fermentation; after the lactic acid bacteria fermentation is completed, cool down, add yeast and keep stirring, after the yeast fermentation is completed, separate by horizontal centrifuge and centrifuge by disc centrifuge; sterilize the liquid after centrifugation, and then carry out secondary fermentation, in which acetic acid bacteria ferment to decompose the alcohol produced by yeast fermentation.

[0054] Raw material requirements: 1. Asparagus concentrate: pH 4.0-5.8, soluble solids in concentrate 16-28 Bx; 2. Malt extract color measurement: (Take 20g malt extract and add it to 100ml water and treat it at 121℃ for 20min. Use filter paper to filter the liquid. The color should be ≥10.5EBC.); 3. Corn grits and sweet potato flour: keep clean, free of dirt, color difference and corruption.

[0055] Recipe ratio: corn grits: 25-40 parts, sweet potato flour: 0.1-1 part, malt extract: 5-8 parts, asparagus concentrate: 100 parts, liquefaction enzyme: 0.05-0.1 parts, saccharification enzyme: 0.05-0.15 parts, lactic acid bacteria 0.01-0.1 parts, yeast 0.01-0.05 parts, acetic acid bacteria 0.01-0.1 parts.

[0056] Liquefaction enzyme (500-1000g) is added in 3 times: front, middle and back; saccharification enzyme (750-1500g) is added in 3 times.

[0057] Lactic acid bacteria fermentation: Activation of lactic acid bacteria: Take 1L of liquefied liquid, sterilize it in a sterilizer at 121 degrees * 20 minutes, cool it to 42 degrees and add lactic acid bacteria (7.5-15g), pay attention to aseptic operation. Activate it in a 42-degree incubator for 3 hours before inoculating the fermenter. Add the activated lactic acid bacteria liquid to the fermenter, seal the inoculation port, and ferment it at 38-42℃ for 7-12 hours. The fermentation ends when the pH value of the liquid drops to between 4.0 and 5.0.

[0058] After the lactic acid bacteria fermentation is completed, cool down to 28℃, then add asparagus yeast (250-750g) into the fermentation tank through the inoculation port, seal the inoculation port, and keep stirring to mix the yeast evenly. Maintain the fermentation temperature at 25-32℃ and the yeast fermentation time for 8-12 hours. The fermentation end point is that the fermentation liquid pH is 3.5-4.5, with obvious yeast aroma and trace bubbles.

[0059] Acetic acid bacteria fermentation: ① Disinfection of fermentation tank: After cleaning, use steam sterilization at 121 degrees * 20 minutes; ② After the fermentation liquid is pumped into the secondary fermentation tank, add acetic acid bacteria (80-120g) into the secondary fermentation tank through the inoculation port, seal the inoculation port, and keep stirring to make the acetic acid bacteria mixed evenly, maintain the fermentation temperature at 25-32℃ for 15-24 hours, and the fermentation is completed when the time is up; After the acetic acid bacteria fermentation is completed, membrane filtration, diatomaceous earth filtration, degassing, UHT sterilization, packaging, and storage are carried out.

[0060] Ultrafiltration membrane filtration: The transmittance is greater than 85 at 625nm.

[0061] UHT sterilization: sterilization temperature is 116±2℃, sterilization time is 15-18s, outlet temperature is 90℃.

[0062] Example 5 In addition, the present invention has comparative examples 1, 2, and 3, in which less saccharifying enzyme, lactic acid bacteria, and yeast are added, respectively, and other steps are the same as those in the embodiment, and therefore, they are not repeated here.

[0063] Example 6: Alcohol sobering effect test Detection principle Since zebrafish have a similar digestive system to humans, such as liver and intestines, digestion and nutrient absorption and transportation are highly similar to humans. When ethanol is consumed excessively, a large amount of acetaldehyde is produced through metabolism, causing acetaldehyde to accumulate in the body and causing drunkenness. The total movement distance is tested to evaluate the hangover effect of the sample. The movement trajectory of zebrafish is analyzed using a behavioral analyzer. The total movement distance of drunk zebrafish is significantly shorter than that of normal zebrafish.

[0064] The positive control drug in the experiment was RU21.

[0065] Experimental instruments and reagents.

[0066] Zebrafish behavioral trajectory tracking system (DanioVision) (BOM-ZDAN-OBS, Noldus (Beijing) Information Technology Co., Ltd.), anhydrous.

[0067] Ethanol (CAS: 64-17-5), RU21 (imported from the United States).

[0068] Testing system and samples: (1) Wild AB zebrafish; (2) Zebrafish age: 3 days after fertilization; experimental sample size per group: 30 fish per well, per group; (3) Adult fish rearing and breeding methods: follow standard rearing and breeding methods.

[0069] Experimental Procedure Healthy 5dpf zebrafish were randomly assigned to 96-well plates, and one zebrafish was treated in each well. There were 10 wells in each group. 5dpf wild-type AB strain zebrafish were immersed in 4% anhydrous ethanol dissolved in zebrafish culture water for 1 hour to establish a zebrafish drunkenness model. The behavior analyzer was used to record the movement of zebrafish within 1 hour after the administration of anhydrous ethanol. The blank group was given different concentrations of samples in water on the basis of the model. The positive control group was RU-21 diluted in zebrafish culture water, and a model control group was set up at the same time. After continuing to treat at 28℃ for 1 hour, the movement of zebrafish in each well was recorded with a behavior recorder. The two movement behavior data were recorded and the total movement distance of the zebrafish was analyzed and counted. The statistical analysis results of the total movement distance were used to evaluate the preventive and sobering effects of the test product on the two drunkenness models of zebrafish induced by anhydrous ethanol.

[0070] Data processing methods SPSS26 software was used for analysis, GraphPadPrism8 was used for drawing, and data statistics were expressed as mean ± standard deviation. SPSS software was used to test the normal distribution of the experimental results. If the data conformed to the normal distribution, the T parameter test or analysis of variance (ANOVA) was used for significance. If it did not conform to the normal distribution, a non-parametric test was used (the result P value <0.05 indicated a statistically significant difference).

[0071] Judgment basis On the basis of the effectiveness of the experiment, the total movement distance of the model control group was significantly reduced compared with the blank control group (P<0.05), which means that the modeling was successful.

[0072] Compared with the model control group, the total movement distance of the sample treatment group was significantly increased (P<0.05), indicating that the sample has the effect of sobering up.

[0073] Preliminary test results Under the experimental conditions, there was no significant difference between the 5% concentration sample group and the blank control group, so the 5% sample concentration was selected as the formal experimental concentration.

[0074] Official test results Figure 4 , Figure 5 .

[0075] In order to explore the hangover mechanism, this example detected saponins and free amino acids in asparagus fermented beverages, and used rutin as a reference to determine the total flavonoid content. The standard curve of total flavonoids is shown in Figure 6 .

[0076] Determination of total flavonoids content in samples Determination of total flavonoids in asparagus fermentation broth using rutin as a standard .

[0077] In addition, HPLC was used to determine the contents of rutin, quercetin and kaempferol in the samples. The standard curves of concentration and peak area are shown in Figure 7 .

[0078] Contents of three flavonoids in samples .

[0079] From the analysis of liquid chromatography, the contents of three flavonoid components, namely rutin, quercetin and kaempferol, were lower than the total flavonoid content, indicating that it may contain other flavonoid components.

[0080] In addition, chromatographic separation technology was used to determine the content of free amino acids in the fermented asparagus beverage. The test results showed that the content of alanine and leucine in the fermented asparagus beverage was significantly increased compared with Comparative Example 1, Comparative Example 2, and Comparative Example 3, which were 2.4 / 2.0, 1.5 / 1.7, and 3 / 2.6 times of those in Comparative Example 1, Comparative Example 2, and Comparative Example 3, and the total free amino acid content was 2.1 times, 1.9 times, and 2.7 times of those in Comparative Example 1, Comparative Example 2, and Comparative Example 3. In addition, studies have shown that the present invention uses saccharifying enzymes to hydrolyze starch in corn grits and sweet potato flour materials, converting starch into dextrin and oligosaccharides, and reducing the liquid viscosity in the solution.

[0081] Example 7: Blood lipid lowering effect Detection principle 60% of egg yolk powder is lipids. Feeding zebrafish with egg yolk powder can rapidly increase the fat content in the zebrafish blood, thereby establishing a zebrafish hyperlipidemia model. A blood lipid tester was used to detect the triglyceride content in different groups.

[0082] Experimental instruments and reagents Centrifuge (AG22331, Eppendorf Life Sciences), ultrasonic cell disruptor (UP-250, Ningbo Xinzhi Biotechnology Co., Ltd.), PFS-30A (Guilin Youlite Medical Electronics Co., Ltd.), egg yolk powder (CAS: 23383-11-1), glucose (CAS: 50-99-7).

[0083] Testing system and sample size Experimental system: Wild AB strain zebrafish; Zebrafish age: 5 days after fertilization; Sample size for each experimental group: 30 strips per well, 3 wells per group; Adult fish rearing and breeding methods: in accordance with the company's standard rearing and breeding methods.

[0084] Experimental Procedure The zebrafish were divided into four groups: normal control group, model control group, positive control group and sample group. The normal control group did not take high-sugar and high-fat feed, while the model control group, positive control group and sample group all took in the same amount of high-sugar and high-fat feed (the high-sugar and high-fat feed was dissolved in the water used for fish farming and then taken into the zebrafish). The sample group took in the sample while taking the high-sugar and high-fat feed. After taking the sample for a period of time, we tested the blood lipids of the zebrafish.

[0085] Data processing methods SPSS26 software was used for analysis, GraphPad Prism8 was used for drawing, and data statistics were expressed as mean ± standard deviation. SPSS software was used to test the normal distribution of the experimental results. If the data conformed to the normal distribution, the T parameter test or analysis of variance (ANOVA) was used for significance. If it did not conform to the normal distribution, a non-parametric test was used (the result P value <0.05 indicated a statistically significant difference).

[0086] Judgment basis On the basis of the effectiveness of the experiment, the total movement distance of the model control group was significantly reduced compared with the blank control group (P<0.05), which means that the modeling was successful.

[0087] Compared with the model control group, the triglyceride value in the sample treatment group was significantly reduced (P<0.05), indicating that the sample has the function of lowering blood lipids.

[0088] Preliminary test results There was no significant difference between the sample group with a concentration of 0.5% and the blank control group, so the concentration of 0.5% is the maximum safe concentration.

[0089] Formal test results can be found at Figure 8-10 The above experiments have confirmed that it has the effect of lowering blood lipids.

[0090] Example 8: Auxiliary hypoglycemic effect Experimental instruments and reagents Centrifuge (AG22331, Eppendorf Life Sciences), ultrasonic cell disruptor (UP-250, Ningbo Xinzhi Biotechnology Co., Ltd.), Roche Zhihang blood glucose meter (Roche, Germany), glucose (CAS: 50-99-7), metformin.

[0091] Testing system and sample size Experimental system: Wild AB strain horsefish; Zebrafish age: 1 day after fertilization; Sample size for each experimental group: 30 strips per well, 3 wells per group; Adult fish rearing and breeding methods: in accordance with the company's standard rearing and breeding methods.

[0092] The experimental process divided the tested zebrafish into four groups, namely the normal control group, model control group, positive control group and sample treatment group. The normal control group did not receive any treatment, the model control group established a high-sugar environment, the positive control group added metformin while establishing a high-sugar environment, and the sample treatment group added the sample to be tested while establishing a high-sugar environment. After soaking for a period of time, the four groups of zebrafish were crushed and centrifuged to obtain the supernatant, blood sugar was measured, and the data analysis results of the four groups were compared.

[0093] Data processing methods SPSS26 software was used for analysis, GraphPadPrism8 was used for drawing, and data statistics were expressed as mean ± standard deviation. SPSS software was used to test the normal distribution of the experimental results. If the data conformed to the normal distribution, the T parameter test or analysis of variance (ANOVA) was used for significance. If it did not conform to the normal distribution, a non-parametric test was used (the result P value <0.05 indicated a statistically significant difference).

[0094] Judgment basis On the basis of the effectiveness of the experiment, the blood glucose level in the model control group increased significantly compared with the blank control group (P<0.05), which means that the model was successfully established.

[0095] Compared with the model control group, the blood glucose level in the sample treatment group was significantly reduced (P<0.05), indicating that the sample has the effect of assisting in lowering blood glucose.

[0096] Preliminary test results There was no significant difference between the sample treatment group with 0.5% concentration and the blank control group, so 0.5% concentration is the maximum safe concentration.

[0097] Formal test results can be found at Figure 11-12 ,,The above results prove that it has the effect of assisting in lowering blood sugar.

[0098] Example 9: Auxiliary blood pressure lowering effect Experimental instruments and reagents Stereo fluorescence microscope (SZX7, Olympus, Japan).

[0099] Testing system and sample size Experimental system: Transgenic vascular fluorescent zebrafish; Zebrafish age: 3 days after fertilization; Sample size for each experimental group: 30 strips per well, 1 well per group; Adult fish rearing and breeding methods: According to standard rearing and breeding methods.

[0100] Experimental Procedure The zebrafish were divided into two groups: a blank control group and a sample treatment group. The blank control group was not treated with any treatment, and the sample treatment group was cultured with samples for a period of time. The two groups of zebrafish were observed and photographed using a fluorescence microscope. After the software analyzed the pictures, the data analysis results of the two groups were compared.

[0101] Data processing methods SPSS26 software was used for analysis, GraphPad Prism8 was used for drawing, and data statistics were expressed as mean ± standard deviation. SPSS software was used to test the normal distribution of the experimental results. If the data conformed to the normal distribution, the T parameter test or analysis of variance (ANOVA) was used for significance. If it did not conform to the normal distribution, a non-parametric test was used (the result P value <0.05 indicated a statistically significant difference).

[0102] Judgment basis Compared with the blank control group, the blood vessel diameter of the sample-treated group increased significantly and P<0.05, indicating that the sample has an auxiliary blood pressure-lowering effect.

[0103] Preliminary experimental results There was no significant difference between the 1% concentration sample group and the blank control group, so 1% concentration is the maximum safe concentration.

[0104] Test results are shown in Figure 12-15 The results showed that the sample has the effect of assisting in lowering blood pressure.

Claims

1. A method for preparing a multi-strain asparagus fermented beverage, characterized in that: The following steps are involved: Step 1: Wash the asparagus raw material with chlorine dioxide, crush and pulp it after washing, and squeeze out the juice; wherein, the chlorine dioxide (ClO2) is 50-80ppm and the soaking time is ≥30s; the raw material after juicing is sterilized and centrifuged by butterfly centrifuge, and after centrifugation, it is low-temperature vacuum concentrated, degassed, and ultra-high temperature instantaneous sterilization (UHT) is performed to prepare 18°Bx asparagus concentrated juice for use; Step 2: proportioning the raw materials, adding liquefaction enzyme, liquefying at high temperature, cooling in a tubular manner after liquefaction, saccharifying after cooling, adding saccharifying enzyme, malt extract, and asparagus concentrate, mixing the saccharified liquid, asparagus juice, and malt extract evenly, and then performing lactic acid fermentation; after the lactic acid bacteria fermentation is completed, cooling is performed, yeast is added and stirring is maintained, after the yeast fermentation is completed, separation is performed by a horizontal centrifuge, and centrifugation is performed by a disc centrifuge; the liquid after centrifugation is sterilized, and secondary fermentation is performed after sterilization; acetic acid bacteria fermentation decomposes the alcohol produced by yeast fermentation; Step 3: After the acetic acid bacteria fermentation is completed, membrane filtration, diatomaceous earth filtration, degassing, UHT sterilization, packaging and storage are carried out.

2. The preparation method according to claim 1, characterized in that: In step 1, the raw material requirements for asparagus concentrate are as follows: pH 4.0-5.8, concentrated juice soluble solids 16-28 Bx.

3. The preparation method according to claim 1, characterized in that: In step 1, the sterilization temperature of fresh asparagus juice is controlled within the range of 85°C-95°C, and the discharge temperature is controlled within the range of 50°C-55°C; after separation by a disc separator, the insoluble solid content is controlled to be 1.5-4% 4BIX.

4. The preparation method according to claim 1, characterized in that: In step 2, the formula ratio is calculated by mass: corn grits: 25-40 parts, sweet potato flour: 0.1-1 parts, malt extract: 5-8 parts, asparagus concentrate: 100 parts, liquefaction enzyme: 0.05-0.1 parts, saccharification enzyme: 0.05-0.15 parts, lactic acid bacteria 0.01-0.1 parts, yeast 0.01-0.05 parts, acetic acid bacteria 0.01-0.1 parts.

5. The preparation method according to claim 1, characterized in that: In step 2, the lactic acid bacteria fermentation includes the following steps: (1) Activation of lactic acid bacteria: Take 1L of liquefied liquid and sterilize it in a sterilizer at 121 degrees for 20 minutes. After cooling to 42 degrees, add lactic acid bacteria and perform aseptic operation. (2) Activate the culture medium in a 42-degree incubator for 3 hours before inoculating the fermenter; add the activated lactic acid bacteria solution into the fermenter, seal the inoculation port, and ferment at 38-42 degrees for 7-12 hours. When the pH value of the solution drops to between 4.0 and 5.0, the fermentation is completed.

6. The preparation method according to claim 1, characterized in that: In step 2, after the lactic acid bacteria fermentation is completed, the temperature is lowered to 28°C, and then the asparagus yeast is added to the fermentation tank through the inoculation port, the inoculation port is sealed, and stirring is kept to mix the yeast evenly. The fermentation temperature is maintained at 25-32°C, the yeast fermentation time is 8-12 hours, and the fermentation end point is that the pH of the fermentation liquid is 3.5-4.5, there is an obvious yeast aroma, and trace bubbles.

7. The preparation method according to claim 1, characterized in that: In step 2, the acetic acid bacteria fermentation includes the following steps: (1) Fermentation tank disinfection: After cleaning, steam disinfection is performed at 121 degrees for 20 minutes; (2) After the fermentation liquid is pumped into the secondary fermentation tank, add the acetic acid bacteria into the secondary fermentation tank through the inoculation port, seal the inoculation port, and keep stirring to ensure that the acetic acid bacteria are evenly mixed. Maintain the fermentation temperature at 25-32°C for 15-24 hours, and the fermentation is completed when the time is up.

8. The preparation method according to claim 1, characterized in that: In step 3, ultrafiltration membrane filtration: the transmittance is detected to be greater than 85 at 625 nm.

9. The preparation method according to claim 1, characterized in that: In step three, UHT sterilization: sterilization temperature is 116±2℃, sterilization time is 15-18s, and outlet temperature is 90℃.

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