Ultrasonic-assisted radix puerariae enzyme and production process thereof

Abstract

The production technology comprises the following steps that S1, diced kudzuvine roots serve as a raw material, and after smashing, pulping, homogenizing, centrifuging and cooking are conducted, kudzuvine root juice is obtained; s2, adjusting the pH value of the radix puerariae juice, then inoculating Z38 # lactobacillus plantarum and yellow rice wine yeast, and then performing ultrasonic treatment and primary fermentation to obtain primary fermentation liquor; s3, adding a carbon source into the primary fermentation liquor, adjusting the pH value, inoculating Z38 # lactobacillus plantarum and yellow rice wine yeast, and performing secondary fermentation to obtain secondary fermentation liquor; s4, a stabilizer and a flavoring agent are added into the secondary fermentation liquid, the pH value is adjusted, and the ultrasonic-assisted radix puerariae enzyme is obtained; according to the preparation method disclosed by the invention, mixed bacteria of yellow wine yeast and Z38 # lactobacillus plantarum are subjected to secondary fermentation, so that the activity of producing acetaldehyde dehydrogenase, pueraria flavones and physicochemical components of the pueraria enzyme are greatly increased, and various essential amino acids of a human body are generated, so that the alcoholism-relieving pueraria enzyme has better nutrition and flavor.

Description

Technical Field

[0001] The invention relates to the field of food technology, and in particular to an ultrasound-assisted pueraria enzyme and a production process thereof. Background Art

[0002] Pueraria root is rich in nutrients such as starch, isoflavones, organic acids, polysaccharides, saponins and essential amino acids for the human body. Among them, Pueraria root polysaccharides have multiple physiological functions such as anti-oxidation, immune regulation, lipid-lowering and blood sugar-lowering, alcohol detoxification and liver protection; puerarin has anti-inflammatory and anti-cancer capabilities; Pueraria root ethanol extract has a strong hydroxyl free radical scavenging ability and has certain in vitro antioxidant activity; Pueraria root flavonoids and puerarin were treated in zebrafish liver injury models, and it was found that both can alleviate alcohol-induced liver fatty degeneration. Therefore, using Pueraria root as a raw material to prepare enzymes has a positive significance for health care. Enzymes are biological macromolecules with catalytic activity produced by organisms. Edible plant enzymes are products that are made from plants that can be used for food processing, with or without auxiliary materials, and are produced by microbial fermentation and contain specific biologically active ingredients for human consumption. Edible plant enzymes are rich in polyphenols, vitamins, minerals, amino acids, organic acids and other nutrients and bioactive substances, and have main functional effects such as antioxidant effects, digestive system health, and immune system enhancement. The extract of Pueraria root has certain alcohol-resolving properties. The reason is that the extract of Pueraria root in the related art contains a certain amount of acetaldehyde dehydrogenase, which mainly acts on the metabolism of alcohol. Under the action of this enzyme, alcohol is decomposed into CO2 and H2O and excreted from the body, thereby reducing the body's absorption of alcohol.

[0003] However, in the related art, the acetaldehyde dehydrogenase obtained from Pueraria extract has a small content and low enzyme activity, and the types and contents of other physical and chemical components are limited, resulting in poor alcohol-resolving performance of Pueraria enzyme, as well as poor stability and taste.

[0004] Therefore, a technical solution is needed to improve the alcohol-resolving performance and stability of Pueraria enzyme. Summary of the invention

[0005] In view of this, the present application provides an ultrasound-assisted pueraria enzyme and a production process thereof, which are used to solve the problem of how to improve the alcohol-resolving performance and stability of pueraria enzyme.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions: In a first aspect, the present application provides an ultrasound-assisted production process of pueraria enzyme, comprising the following steps: S1. Pueraria root juice is obtained by crushing, beating, homogenizing, centrifuging and steaming Pueraria root diced as raw material; S2. Adjusting the pH value of the Pueraria juice, inoculating Lactobacillus plantarum and rice wine yeast Z38, and then sonicating and fermenting once to obtain a fermentation broth; S3. Adding a carbon source to the primary fermentation broth, adjusting the pH, and then inoculating Lactobacillus plantarum and rice wine yeast Z38, and then performing a secondary fermentation to obtain a secondary fermentation broth; S4. Add stabilizers and flavoring agents to the secondary fermentation broth, and adjust the pH value to obtain the ultrasound-assisted pueraria enzyme.

[0007] Preferably, the specific operations of step S1 are as follows: kudzu root diced → selection → crushing → sieving → adding water → beating → filtering → homogenizing → centrifugation → cooking → kudzu root juice.

[0008] Preferably, in step S1, after the kudzu root is crushed and sieved, the kudzu root powder and distilled water are evenly mixed at a solid-liquid ratio of 1:15 (g / mL), and then centrifuged to obtain the supernatant, which is boiled for 2 hours and cooled to room temperature to obtain kudzu root juice.

[0009] Preferably, in step S2, the time of the primary fermentation is 1-7 days, and the temperature of the primary fermentation is 28-36°C.

[0010] Preferably, in step S2, the ultrasonic time is 30-120 min, and the ultrasonic frequency is 20 KHz - 50 KHz.

[0011] Preferably, in step S2, the pH value of the Pueraria juice is adjusted to 4.5-6.5.

[0012] Preferably, in steps S2 and S3, the inoculation amount of Lactobacillus plantarum Z38 and rice wine yeast is 3%, and the inoculation ratio of Lactobacillus plantarum Z38 and rice wine yeast is 1-3:1-3; the preservation number of Lactobacillus plantarum Z38 (Lactobacillus plantarum Z38) is CCTCC M 20242392, and it was preserved in China Center for Type Culture Collection, Wuhan, China on October 31, 2024.

[0013] Preferably, Lactobacillus plantarum Z38 has DPPH activity, BSH activity, reducing activity, and OH activity.

[0014] Preferably, Lactobacillus plantarum Z38 has tolerance to artificial gastric juice and artificial intestinal juice.

[0015] Preferably, Lactobacillus plantarum Z38 is used to produce acetaldehyde dehydrogenase, and the ability to produce acetaldehyde dehydrogenase is greater than or equal to 135.54 U / mL.

[0016] Preferably, in step S3, the carbon source includes one or more of brown sugar and lemon slices.

[0017] Preferably, in step S3, the pH value is adjusted to 4.5-5.5.

[0018] Preferably, in step S4, the stabilizer includes one or more of xanthan gum, sodium alginate, and pectin, and the flavoring agent includes white sugar.

[0019] Preferably, in step S4, the pH value is adjusted to 4.0-5.0.

[0020] In a second aspect, the present application provides an ultrasound-assisted pueraria enzyme.

[0021] The beneficial effects of the present application are as follows: compared with the traditional single fermentation of Pueraria enzyme, the present application greatly increases the acetaldehyde dehydrogenase activity and physical and chemical components of Pueraria enzyme through the combination of primary fermentation and secondary fermentation, and produces a variety of essential amino acids for the human body, making the alcohol-detoxifying Pueraria enzyme more nutritious and flavorful; the present application utilizes rice wine yeast and Z38 plant lactobacillus mixed bacteria to secondary ferment Pueraria to prepare a new alcohol-detoxifying health drink-enzyme, thereby improving the comprehensive development and utilization of Pueraria, a plant with medicinal and edible homology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of fermentation time on the activity of acetaldehyde dehydrogenase produced by the first fermentation liquid; Figure 2 The effect of fermentation temperature on the activity of acetaldehyde dehydrogenase produced by the first fermentation liquid; Figure 3 The effect of initial pH value on the activity of acetaldehyde dehydrogenase produced by the first fermentation broth; Figure 4 The effect of the ratio of bacterial strains on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth; Figure 5 The effect of ultrasound time on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth; Figure 6 Effect of ultrasound intensity on the activity of acetaldehyde dehydrogenase produced by the first fermentation broth Figure 7 This is the result of the change of flavonoid concentration before and after the first fermentation; Figure 8 These are the test results of the effects of different stabilizers on the stability of pueraria enzyme. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] The present solution is further described below through specific embodiments.

[0025] Source of raw materials Lactobacillus plantarum Z38 is obtained by the following steps: Take the fermented liquid of Northeast kimchi in the fermentation period, number the centrifuge tubes 1, 2, 3, and 4 respectively, use a pipette to take the microecological preparation sample into the centrifuge tube, centrifuge it in a high-speed desktop centrifuge at 10,000 rpm for 1 minute, and discard the supernatant. Add physiological saline to wash, put the centrifuge tube into a vortex mixer and vortex to mix. Then, centrifuge again and discard the supernatant.

[0026] Use a pipette to obtain saline in a centrifuge tube, mix it upside down several times, and the resulting bacterial solution is diluted to 10-1. Add 900uL of saline to each of the other centrifuge tubes. Take 100uL of bacterial solution from centrifuge tube No. 1 and put it in tube No. 1, mix it upside down several times, and the resulting bacteria is diluted to 10-2. Similarly, by analogy, tube No. 3 is diluted to 10-3, and tube No. 4 is diluted to 10-4. Take 0.2mL of dilution from tubes 1, 2, 3, and 4, and inoculate it into the lactic acid bacteria isolation medium by coating method. Cultivate in a constant temperature and humidity incubator at 37℃ for 48h, observe its morphology after the colony is formed, pick the colonies around the color change circle, and perform multiple plate streaking separations to obtain purified single colonies. The single colony obtained by screening is stored on a slant, which is plant lactobacillus No. Z38.

[0027] like Figure 1 As shown, the morphological characteristics of Lactobacillus plantarum Z38 of the present application are: all light green, irregular edges, sticky surface, and small and medium-sized colonies.

[0028] The performance of Lactobacillus plantarum Z38 of the present application was tested: The rDNA-ITS2 sequence of Lactobacillus plantarum Z38 obtained by rDNA-ITS2 sequence sequencing was compared with the homology NCBIBLAST analysis, and the similarity between the rDNA-ITS2 sequence of Lactobacillus plantarum Z38 and the rDNA-ITS2 sequence of Lactobacillus in GenBank reached 99%. The activity properties of Lactobacillus plantarum Z38 are shown in Table 1.

[0029] Table 1 Activity properties of Lactobacillus plantarum Z38

[0030] The plant lactobacillus Z38 of the present application was isolated from Northeast kimchi. The plant lactobacillus Z38 grew slowly in the first 4 h of culture, entered the logarithmic growth phase after 6 h of culture, reached the maximum OD600nm of 1.591 at 18 h of culture, and then entered the stable phase.

[0031] Artificial gastric juice tolerance test: 0.2 mL of cell suspension (10 8cfu / mL) was inoculated into 1.0 mL of artificial gastric juice (pH 2.0) and 0.3 mL of NaCl that had been sterilized by membrane filtration (0.22 μm), mixed thoroughly, and treated in a constant temperature water bath at 37°C. Samples were taken 1 min, 90 min, and 180 min after treatment with artificial gastric juice, and the samples were counted by the standard plate method (dilution factor of 10-10) after gradient dilution. The counts were repeated three times. The results are shown in Table 2.

[0032] Table 2 Results of tolerance of Lactobacillus plantarum Z38 to artificial gastric juice

[0033] Artificial intestinal fluid tolerance test: 0.2 mL of cell suspension (10 8 cfu / ml) were inoculated into membrane-filtered sterilized (0.22 μm) artificial intestinal fluid (pH 8.0) and 0.3 mL NaCl, mixed thoroughly, and treated in a constant temperature water bath at 37°C. Samples were taken at 1 min and 240 min after treatment with artificial intestinal fluid, and counted by the standard plate method (dilution factor of 10-10) after gradient dilution. The counts were repeated three times. The results are shown in Table 3.

[0034] Table 3 Results of tolerance to artificial intestinal fluid of Lactobacillus plantarum Z38

[0035] Fermentation enzyme production ability test: The cultured lactic acid bacteria seed liquid was inoculated into the culture medium at an inoculation rate of 5%, and cultured at a constant temperature of 37°C for 18 hours. After the fermentation liquid of the strain was centrifuged at 5000rpm and 4°C, the precipitate was washed twice with sterile saline, the bacterial mud was collected, and then the bacterial mud and phosphate buffer were made into a bacterial suspension at a ratio of 1:4.5, and ultrasonically broken at 100W power for 20min in an ice bath, and then centrifuged at 5000rpm and 4°C, the supernatant was collected, and the precipitate was ultrasonically treated once again, and the supernatant was combined to obtain a crude enzyme solution. The activity of acetaldehyde dehydrogenase was tested, and strains with enzyme production ability were selected for subsequent experiments. The results are shown in Table 4.

[0036] Table 4 Fermentation enzyme production capacity test results

[0037] Example 1 An ultrasound-assisted production process of pueraria enzyme comprises the following steps: S1. After pulverizing and sieving the kudzu root powder, the kudzu root powder and distilled water were mixed evenly at a solid-liquid ratio of 1:15 (g / mL), and then centrifuged to obtain the supernatant, which was boiled for 2 h and cooled to room temperature to obtain kudzu root juice; S2. The pH value (initial pH value) of the Pueraria juice was adjusted to 4.5, and then 3% of the total amount was inoculated with Lactobacillus plantarum Z38 and rice wine yeast, and the inoculation ratio of Lactobacillus plantarum Z38 and rice wine yeast was 1:2, and then ultrasonically treated with 60% ultrasonic intensity for 60 min and fermented once, the temperature of the fermentation was 34 ° C, the fermentation time was 6d, and a fermentation liquid was obtained; S3. Add 15wt% brown sugar and 9wt% lemon slices to the primary fermentation broth, adjust the pH to 4.5, and then add 3% of the total amount of inoculated Lactobacillus plantarum Z38 and rice wine yeast, the inoculation ratio of Lactobacillus plantarum Z38 and rice wine yeast is 1:2, and then perform secondary fermentation, the secondary fermentation temperature is 32 ° C, the secondary fermentation time is 6d, and the secondary fermentation broth is obtained; S4. Add 0.05wt% sodium alginate and 6% white sugar to the secondary fermentation broth, and adjust the pH value to 5.0 with sodium bicarbonate to obtain the ultrasound-assisted pueraria enzyme.

[0038] Embodiment 2-8 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S2, the primary fermentation temperature is 32°C, and the fermentation time is 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d, respectively.

[0039] Examples 9-12 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S2, the temperatures of the primary fermentations are 28°C, 30°C, 32°C, and 36°C, respectively.

[0040] Examples 13-17 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S2, the pH value (initial pH value) of the pueraria juice is adjusted to 4.5, 5.0, 5.5, 6.0, and 6.5, respectively, and the ultrasound time is 30 minutes.

[0041] Examples 18-22 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S2, the inoculation ratios of Lactobacillus plantarum Z38 and rice wine yeast are 1:1, 1:2, 1:3, 2:1, and 3:1, respectively, and the ultrasound time is 30 min.

[0042] Examples 23-26 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S2, the ultrasound time is 0 min, 30 min, 90 min, and 120 min, respectively.

[0043] Examples 27-30 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S2, with a rated power of 50KHz, the intensities of ultrasound are 0, 60%, 90%, and 120%, respectively.

[0044] Examples 31-39 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that the initial pH value in step S2, the ultrasound time and the ultrasound intensity in step S2 are set according to the primary fermentation process conditions in Table 5.

[0045] Table 5 Details of the setting of the primary fermentation process conditions

[0046] Examples 40-48 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that the parameters in step S3 are set according to the secondary fermentation process conditions in Table 6.

[0047] Table 6 Details of the setting of secondary fermentation process conditions

[0048] Examples 49-63 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those in Example 1, except that 0.15wt% of sodium alginate is replaced by 0wt% xanthan gum, 0.05wt% xanthan gum, 0.1wt% xanthan gum, 0.15wt% xanthan gum, 0.2wt% xanthan gum, 0wt% sodium alginate, 0.05wt% sodium alginate, 0.1wt% sodium alginate, 0.2wt% sodium alginate, 0wt% pectin, 0.05wt% pectin, 0.1wt% pectin, 0.15wt% pectin, and 0.2wt% pectin in sequence.

[0049] Examples 64-72 A production process of pueraria enzyme assisted by ultrasound, the other contents are the same as those of Example 1, except that in step S4, the parameters for pueraria enzyme flavor preparation are set according to Table 7.

[0050] Table 7 Pueraria enzyme flavor preparation parameters

[0051] Test and Evaluation Testing of physical and chemical indicators before and after Pueraria enzyme fermentation: Pueraria juice samples with different fermentation times (Pueraria juice, primary fermentation, secondary fermentation) were taken, and each indicator was measured three times in parallel.

[0052] (1) Determination of flavonoid concentration: A rutin standard curve was drawn with rutin concentration (x) as the horizontal coordinate and absorbance (y) as the vertical coordinate. The regression equation of the rutin standard curve was y=21.601x+0.0256 (R2=0.9985). Using 30% ethanol solution as a blank, the absorbance was measured at 500 nm by UV-visible spectrophotometry, and the concentration of kudzu flavonoids in kudzu enzyme was finally calculated according to the regression equation of the rutin standard curve.

[0053] (2) Determination of pH value: refer to the national standard GB 5009.237-2016 "Determination of pH of Foods".

[0054] (3) Determination of reducing sugar content: refer to the national standard GB 5009.7-2016 "Determination of reducing sugar in food".

[0055] (4) Determination of total acid content: Calculate the coefficient of citric acid as appropriate acid, referring to the national standard GB 12456-2021 "Determination of total acid in food".

[0056] (5) Determination of ethanol content: refer to the national standard GB / T 12143-2018 "Determination of ethanol in food".

[0057] (6) Determination of lactic acid bacteria content: refer to the national standard GB 4789.35-2016 "Food Microbiology Testing - Lactic Acid Bacteria Testing".

[0058] (7) Determination of vitamin B2 and vitamin B6 content: Vitamin B2 shall refer to the national standard GB 5009.85-2016 "Determination of vitamin B2 in foods", and vitamin B6 shall refer to the national standard GB 5009.154-2023 "Determination of vitamin B6 in foods".

[0059] (8) Determination of amino acid types: refer to the national standard GB 5009.124-2016 "Determination of amino acids in food".

[0060] (9) Determination of acetaldehyde dehydrogenase production in fermentation broth: Add 2.52 ml Tris-HCL buffer (0.05 mol / L, pH=8.0), 0.1 ml EDTA solution (0.05 mol / L), potassium chloride solution (3 mol / L), 0.1 mol NAD+ solution (0.02 mol / L), 0.05 ml acetaldehyde solution (0.1 mol / L), and 0.03 ml β-mercaptoethanol solution (0.05 mol / L) into a cuvette, mix well, then add 0.1 ml sample solution, mix quickly, measure the absorbance at 340 nm, and calculate the change in absorbance within 5 min (△A340).

[0061] Figure 1 The effect of fermentation time on the activity of acetaldehyde dehydrogenase produced by the first fermentation broth. Figure 1 It can be seen that when the fermentation time is 1 day, the acetaldehyde dehydrogenase activity in the Pueraria fermentation liquid is low, and when the fermentation time is 6 days, the acetaldehyde dehydrogenase activity in the Pueraria fermentation liquid reaches the highest; with the increase of fermentation time, the acetaldehyde dehydrogenase activity in the Pueraria fermentation liquid is steadily increasing. Due to the insufficient fermentation time, the fermentation liquid processing is seriously affected, making it unable to grow to the conditions that can promote bacterial cells. It can be seen that the appropriate fermentation time can have a positive effect on bacterial proliferation and benefit the fermentation of Pueraria enzymes, but as time goes on, the usefulness of nutrients will decline. Therefore, the optimal fermentation time for Pueraria fermentation liquid is 6 days.

[0062] Figure 2 The effect of fermentation temperature on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth. Figure 2 It can be seen that when the fermentation temperature is 28 ℃, the activity of acetaldehyde dehydrogenase in Pueraria fermentation liquid is low. When the temperature rises to 34 ℃, the activity of acetaldehyde dehydrogenase in Pueraria fermentation liquid reaches the highest; with the increase of temperature, the activity of acetaldehyde dehydrogenase in Pueraria fermentation liquid decreases. At a suitable fermentation temperature, the metabolic activities of yeast and lactic acid bacteria are promoted to a certain extent, and the enzymatic reaction is relatively active, which is conducive to the production of acetaldehyde dehydrogenase; when the fermentation temperature is too high or too low, it is not conducive to the metabolism of yeast and lactic acid bacteria, and affects the enzymatic reaction. Therefore, 34 ℃ is selected as the optimal fermentation temperature for Pueraria fermentation liquid.

[0063] Figure 3 The effect of initial pH value on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth. Figure 3 It can be seen that when the pH is 6.5, the activity of acetaldehyde dehydrogenase is at a relatively low level, but when the environmental conditions become pH 5.0, the measured enzyme activity reaches the maximum value; then the pH value is continuously increased, and the overall trend of enzyme activity measurement shows an increase first and then a decrease. The appropriate pH can promote the growth of yeast and lactic acid bacteria, which is beneficial to the production of acetaldehyde dehydrogenase by yeast and lactic acid bacteria. However, as the lactic acid bacteria produce acid, the acidity of the fermentation broth may be too high, affecting the growth of yeast and lactic acid bacteria and the production of acetaldehyde dehydrogenase, resulting in a decrease in acetaldehyde dehydrogenase in the fermentation broth. Therefore, the optimal initial pH value of Pueraria lobata fermentation broth is 5.0.

[0064] Figure 4 The effect of the ratio of bacterial strains on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth. Figure 4It can be seen that when the strain ratio is 3:1, the acetaldehyde dehydrogenase activity in the Pueraria fermentation liquid is low, and when the strain ratio is 1:2, the acetaldehyde dehydrogenase activity in the Pueraria fermentation liquid reaches the highest value; with the increase of the strain ratio, the acetaldehyde dehydrogenase activity in the Pueraria fermentation liquid decreases. It is possible that the increase in the strain ratio affects the osmotic pressure of the fermentation liquid, which is not conducive to the accumulation of products, and thus affects the ability of Lactobacillus plantarum Z38 and rice wine yeast to produce acetaldehyde dehydrogenase. Therefore, the optimal strain ratio of Pueraria fermentation liquid is 1:2.

[0065] Figure 5 The effect of ultrasound time on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth. Figure 5 It can be seen that when the ultrasonic time is 120 min, the activity of acetaldehyde dehydrogenase produced by the first fermentation broth is low, and when the ultrasonic time is 60 min, the activity of acetaldehyde dehydrogenase produced by the first fermentation broth reaches the highest value; with the increase of fermentation temperature, the activity of acetaldehyde dehydrogenase produced by the first fermentation broth decreases. Too high or too low ultrasonic time will negatively affect the metabolism of the two strains of Lactobacillus plantarum Z38 and rice wine yeast discussed in this paper, and affect their enzymatic reactions, thereby leading to prolonged ultrasonic time, blocked release of metabolites, and unfavorable product accumulation and other negative effects. Therefore, the optimal ultrasonic time for Pueraria fermentation broth is 60 min.

[0066] Figure 6 The effect of ultrasound intensity on the activity of acetaldehyde dehydrogenase produced in the first fermentation broth. Figure 6 It can be seen that when the ultrasonic intensity is 0, the activity of acetaldehyde dehydrogenase produced by the first fermentation broth is low, and when the ultrasonic intensity is 90%, the activity of acetaldehyde dehydrogenase produced by the first fermentation broth reaches the highest value; with the increase of ultrasonic intensity, the activity of acetaldehyde dehydrogenase in the first fermentation broth decreases. Lower ultrasonic power can promote the production of acetaldehyde dehydrogenase by yeast and lactic acid bacteria, and higher density ultrasound will cause damage to the cell membranes of yeast and lactic acid bacteria, affecting the normal growth, reproduction and metabolism of microorganisms. Therefore, the optimal ultrasonic intensity of Pueraria fermentation broth is 90%.

[0067] Table 8 shows the test results of acetaldehyde dehydrogenase activity under different primary fermentation process conditions.

[0068] Table 8 Test results of acetaldehyde dehydrogenase activity under different primary fermentation process conditions

[0069] As shown in Table 8, the optimal process combination of pueraria enzyme prepared by ultrasound-assisted fermentation of Lactobacillus plantarum Z38 and rice wine yeast is an initial pH of 4.5, an ultrasound time of 60 min, and an ultrasound intensity of 60%. Under the optimal process conditions, the acetaldehyde dehydrogenase activity is 6.59 U / g.

[0070] Table 9 is the test results of acetaldehyde dehydrogenase activity under the secondary fermentation process conditions.

[0071] Table 9 Test results of acetaldehyde dehydrogenase activity under different secondary fermentation process conditions

[0072] As shown in Table 9, the optimal process combination for preparing pueraria enzyme by ultrasound-assisted fermentation of Lactobacillus plantarum Z38 and rice wine yeast is 15% brown sugar addition, 9% lemon slice addition, pH 4.5, and fermentation temperature 32 °C, with acetaldehyde dehydrogenase activity as the evaluation index. Under the optimal process conditions, the acetaldehyde dehydrogenase activity was 31.19 U / g.

[0073] Figure 7 The results are the changes in flavonoid concentration before and after the first fermentation. Figure 7 It can be seen that with the end of the first fermentation, the concentration of pueraria flavonoids in the first fermentation solution (15.66 µg / mL) increased by 24.18% compared with the concentration of pueraria flavonoids in pueraria juice (12.61 µg / mL). At the end of the second fermentation, the concentration of pueraria flavonoids in the second fermentation solution (18.39 µg / mL) further increased by 17.44% compared with the concentration of pueraria flavonoids in the first fermentation solution (15.66 µg / mL). This indicates that the concentration of pueraria flavonoids in the two fermentation broths gradually increased. On the one hand, the reason may be that the microorganisms secrete a variety of extracellular enzymes during the fermentation process, which can destroy the structure of the cell wall in the pueraria juice and promote the dissolution of pueraria flavonoids in the pueraria juice; on the other hand, it may be that the microorganisms themselves synthesize, secrete and release some pueraria flavonoids, causing the increase in the concentration of pueraria flavonoids in the first fermentation broth. It may also be that the microbial fermentation produces some metabolites, such as organic acids, which change the pH conditions of the fermentation environment and improve the stability and solubility of flavonoids.

[0074] Table 10 shows the analysis results of physical and chemical indicators before and after Pueraria enzyme fermentation.

[0075] Table 10 Analysis results of physicochemical indicators of Pueraria enzyme before and after fermentation

[0076] As can be seen from Table 10, the pH value gradually decreased and the total acid content continued to rise. The reason is that the microorganisms produced and consumed metabolites during their fermentation process, producing a variety of organic acids; the reducing sugar content first decreased and then increased, while the reducing sugar content in the primary fermentation liquid was 0. Since it was during the primary fermentation process, the microorganisms consumed the carbon source in the Pueraria juice during their fermentation process, resulting in a decrease in the reducing sugar content in the primary fermentation liquid. However, 15% brown sugar was added in the secondary fermentation to increase the carbon source and promote the fermentation of microorganisms, which increased the reducing sugar content in the secondary fermentation liquid; the lactic acid bacteria content showed a trend of increasing first and then decreasing. The reason may be that during the first fermentation, the fermentation liquid has sufficient nutrients and oxygen content, and Lactobacillus plantarum proliferates rapidly, which increases the lactic acid bacteria content in the first fermentation liquid. As the secondary fermentation proceeds, the mixed strains are inoculated and nutrients are added again, which leads to a decrease in the oxygen content in the secondary fermentation liquid, resulting in a decrease in the lactic acid bacteria content in the secondary fermentation liquid. Only vitamin B2 and vitamin B6 were detected in the secondary fermentation liquid. On the one hand, this is because they are produced by the fermentation of yeast and Lactobacillus plantarum during the secondary fermentation process. On the other hand, the addition of lemon slices and brown sugar during the secondary fermentation leads to the production of vitamins in the secondary fermentation liquid.

[0077] Table 11 shows the changes in amino acid types during Pueraria enzyme fermentation as the number of fermentations increases.

[0078] Table 11 The changes of amino acid types during Pueraria enzyme fermentation with the number of fermentation times

[0079] As shown in Table 11, the fermentation of microorganisms increases the amino acid content in Pueraria juice, indicating that in addition to having nutritional functions and promoting digestion, the amino acids in the secondary fermentation liquid also produce certain flavors. For example, valine and threonine are converted into characteristic flavor substances such as acetaldehyde through microbial conversion, making the flavor of the secondary fermentation liquid better.

[0080] Figure 8 The following are the test results of the effects of different stabilizers on the stability of pueraria enzyme. Figure 8 a, 8b, 8c are the effects of different addition amounts of xanthan gum, sodium alginate, and pectin on the stability of pueraria enzyme. Figure 8 It can be seen that xanthan gum and sodium alginate have a significant effect on the stability of pueraria enzyme. However, the effect of pectin on the stability of pueraria enzyme is not ideal. With the increase of pectin addition, the precipitation centrifugation of pueraria enzyme first decreases and then increases. When the addition amount is 0.15%, the precipitation centrifugation rate is greater than the initial state, and when the pectin addition amount is 0.05%, the centrifugal precipitation rate is the lowest, but it is also almost the same as the initial state. It is not as good as xanthan gum and sodium alginate on the stability of pueraria enzyme. Therefore, sodium alginate is selected as a stabilizer.

[0081] Sensory evaluation of pueraria enzyme: 10 students were randomly selected to evaluate 9 samples of different formulations based on color, aroma, taste, and tissue morphology. The reference literature for the sensory evaluation index assessment standard of pueraria enzyme is shown in Table 12.

[0082] Table 12 Sensory index evaluation standards of Pueraria enzyme

[0083] Table 13 shows the sensory evaluation scores of pueraria enzyme under different conditions.

[0084] Table 13 Sensory evaluation scores of pueraria enzyme under different conditions

[0085] As shown in Table 13, the best combination of Pueraria enzyme flavor preparation is pH 5.0, the amount of white sugar added is 6%, and the amount of stabilizer added is 0.05%.

[0086] Alcohol detoxification test Several healthy ICR male mice were selected and adaptively fed for 3 days. Then, they were randomly divided into model group and different drug administration groups (Kuang root liquid, once fermented Kudzu root liquid under the best combination conditions, and twice fermented Kudzu root liquid under the best combination conditions) according to the experimental conditions. After fasting but not water for 12 hours, the drug administration groups were gavaged with corresponding drugs (7.0 mL / kg), and the model group was gavaged with the same dose of pure water. 30 minutes later, each group was gavaged with 54° liquor (12.5 mL / kg), and the intoxication latency (time from alcohol infusion to loss of righting reflex) and sobering time (time from loss of righting reflex to recovery of righting reflex) of each group of mice were observed and recorded.

[0087] Table 14 Experimental results of different kudzu root liquids for alcohol sobering and preventing drunkenness (n=8)

[0088] Note: Compared with the model group, indicates P < 0.05, P < 0.01 The hangover-removing and anti-drunkenness effects and sobering-up time were: secondary fermented Pueraria root liquid > primary fermented Pueraria root liquid > Pueraria root liquid > model group.

[0089] Compared with the traditional single fermentation of pueraria enzyme, the present application combines the single fermentation with the secondary fermentation to significantly increase the acetaldehyde dehydrogenase activity, pueraria flavonoids and physical and chemical components of pueraria enzyme and produce a variety of essential amino acids for the human body, making the alcohol-detoxifying pueraria enzyme more nutritious and flavorful. The pueraria enzyme is fermented twice by mixed bacteria of rice wine yeast and plant lactobacillus Z38 to prepare a new alcohol-detoxifying health drink-enzyme, which improves the comprehensive development and utilization of pueraria, a plant with medicinal and edible homology, and provides an important reference for the subsequent development and research of pueraria enzyme.

[0090] The above are only preferred specific implementations of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. An ultrasound-assisted production process of pueraria enzyme, characterized in that: The following steps are involved: S1. Pueraria root juice is obtained by crushing, beating, homogenizing, centrifuging and steaming Pueraria root diced as raw material; S2. Adjusting the pH value of the Pueraria juice, inoculating Lactobacillus plantarum and rice wine yeast Z38, and then sonicating and fermenting once to obtain a fermentation broth; S3. Adding a carbon source to the primary fermentation broth, adjusting the pH, and then inoculating Lactobacillus plantarum and rice wine yeast Z38, and then performing a secondary fermentation to obtain a secondary fermentation broth; S4. Add stabilizers and flavoring agents to the secondary fermentation broth, and adjust the pH value to obtain the ultrasound-assisted pueraria enzyme.

2. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S2, the time of the primary fermentation is 1-7 days, and the temperature of the primary fermentation is 28-36°C.

3. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S2, the ultrasonic time is 30-120 minutes, and the ultrasonic frequency is 20KHz - 50KHz.

4. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S2, the pH value of the Pueraria juice is adjusted to 4.5-6.

5.

5. The ultrasound-assisted puerarin production process according to claim 1, characterized in that: In steps S2 and S3, the inoculation amount of Lactobacillus plantarum Z38 and rice wine yeast is 3%, and the inoculation ratio of Lactobacillus plantarum Z38 and rice wine yeast is 1-3:1-3; the preservation number of Lactobacillus plantarum Z38 is CCTCC M 20242392, which was deposited in the China Center for Type Culture Collection on October 31, 2024.

6. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S3, the carbon source includes one or more of brown sugar and lemon slices.

7. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S3, the pH value is adjusted to 4.5-5.

5.

8. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S4, the stabilizer includes one or more of xanthan gum, sodium alginate, and pectin, and the flavoring agent includes white sugar.

9. The ultrasound-assisted pueraria enzyme production process according to claim 1, characterized in that: In step S4, the pH value is adjusted to 4.0-5.

0.

10. An ultrasound-assisted pueraria enzyme prepared by the production process according to any one of claims 1 to 9.

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