Method for microbial fermentation of pachyrhizua angulatus

Abstract

The invention discloses a method for microbial fermentation of pachyrhizua angulatus, which comprises the following steps: inoculating lactobacillus rhamnosus into pachyrhizua angulatus powder, and fermenting the pachyrhizua angulatus by using the lactobacillus rhamnosus, so that the contents of total flavonoids, soluble polysaccharides and main isoflavones including 3-OH puerarin, daidzein and genistein in the pachyrhizua angulatus can be effectively increased. By adopting the preparation method disclosed by the invention, a pueraria thomsonii product with higher biological activity can be obtained, the pueraria thomsonii product can be more easily absorbed and utilized by a human body, and symptoms of type 2 diabetes mellitus can be more effectively improved; meanwhile, the preparation method disclosed by the invention is simple and easy to implement, and the cost can be saved, so that the method disclosed by the invention has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation processing, and in particular to a method for fermenting kudzu vine root with microorganisms. Background Art

[0002] Pueraria lobata Pueraria thomsonii The dried tubers of Pueraria lobata have unique nutrition and are both good for medicine and food. Pueraria lobata has multiple pharmacological effects such as anti-myocardial ischemia, hypoglycemia, hypolipidemic, antipyretic, and antiviral. The main components that exert their medicinal effects are starch, polysaccharides, and isoflavones. As a prebiotic substance, Pueraria lobata polysaccharide can regulate the structure of intestinal flora, improve alcoholic fatty liver and lipid peroxidation, and maintain the health of the body. Isoflavones in Pueraria lobata, such as puerarin, daidzein, and genistein, have good pharmacological effects. Studies have shown that daidzein has good estrogenic effects and antioxidant capacity, and also has good hypoglycemic effects. It can increase insulin sensitivity by increasing AMPK phosphorylation and glucose transporter 4 (GLUT4) expression, as well as promoting glucose uptake. Genistein in Pueraria lobata also has good estrogenic effects and antioxidant capacity, and it is also a highly potential anti-fat increase agent and lipid-lowering agent. Other studies have shown that 3′-hydroxypuerarin can promote glucose utilization in insulin-resistant adipocytes and inhibit the production of free fatty acids, thereby improving insulin resistance. However, there are still some problems in the development and utilization of Pueraria lobata, such as poor taste and low bioavailability. Pueraria lobata polysaccharides cannot be directly degraded and absorbed by enzymes encoded by the human genome, and its isoflavone components, such as soy isoflavones in the form of glycosides, cannot be directly absorbed by the small intestinal wall in the body and must be converted into free aglycones before they can be absorbed.

[0003] In order to further improve the bioavailability of Pueraria lobata, researchers at home and abroad have conducted a lot of research on the starch and polysaccharide components in Pueraria lobata, and improved the utilization rate of starch and polysaccharides through enzymatic hydrolysis, gelatinization, saccharification and other methods. However, there are problems such as complicated operating procedures and large energy consumption losses.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a method for microbial fermentation of kudzu vine, by which the content of total flavonoids, soluble polysaccharides and main isoflavones in kudzu vine can be effectively increased.

[0006] The present invention is achieved in that: In a first aspect, the present invention provides a method for microbial fermentation of kudzu vine, comprising: adding Lactobacillus rhamnosus culture liquid to a kudzu vine fermentation base material, mixing and fermenting to obtain a liquid microbial fermentation product.

[0007] In some embodiments, Lactobacillus rhamnosus is Lactobacillus rhamnosus HCS01-013; the preservation number is: CGMCC No. 19510.

[0008] In some embodiments, the inoculation amount of the Lactobacillus rhamnosus bacterial liquid is 6 wt%-12 wt%.

[0009] In some embodiments, the concentration of the Lactobacillus rhamnosus bacterial liquid is 6.0×10 6 CFU / mL - 1.2×10 7 CFU / mL.

[0010] In some embodiments, the preparation method of the kudzu root fermentation substrate includes: mixing the pulverized and sieved kudzu root powder with water, and after the kudzu root powder fully absorbs water, performing high-temperature sterilization, and then cooling to obtain the kudzu root fermentation substrate.

[0011] In some embodiments, the mesh number of the sieved kudzu root powder is 80-100 meshes.

[0012] In some embodiments, the ratio of kudzu root powder to water is 1:20~30 (g:mL).

[0013] In some embodiments, the conditions for high-temperature sterilization are sterilization at 120°C~122°C for 20 min~30 min.

[0014] In some embodiments, the fermentation conditions are: temperature 32°C~37°C, fermentation time 24 h~60 h.

[0015] In a second aspect, the present invention also provides the kudzu root ferment obtained by the above method.

[0016] In a third aspect, the present invention also provides the application of the above kudzu root ferment in the preparation of a product for improving the symptoms of type 2 diabetes.

[0017] The present invention has the following beneficial effects: By inoculating Lactobacillus rhamnosus in kudzu root powder and fermenting kudzu root with Lactobacillus rhamnosus, the present invention can effectively increase the contents of total flavonoids, soluble polysaccharides and main isoflavones including 3-OH puerarin, daidzein and genistein in kudzu root. The preparation method of the present invention can obtain a kudzu root product with higher biological activity, which is more beneficial to human absorption and utilization, and can more effectively improve the symptoms of type 2 diabetes; at the same time, the preparation method of the present invention is simple and easy to implement, and can save costs. Therefore, the method of the present invention has good application prospects. Description of the Drawings

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0019] Figure 1 It is the technical roadmap of the animal experiment for Example 2; Figure 2 It is the detection result of the phenotypic index in Example 2; Figure 3 It is the influence of fermented kudzu root and kudzu root on the biochemical values of db / db mice in Example 2 (compared with the model group, * P <0.05; ** P <0.01); Figure 4 It is the HE staining diagram of the pathological liver tissue of db / db mice in Example 2; Figure 5 It is the HE staining diagram of the pathological pancreatic tissue of db / db mice in Example 2. Detailed implementation manners

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0021] Components such as polysaccharides and isoflavones in kudzu root are the main components for its medicinal effects, but these components cannot be directly absorbed and utilized by the human body. To improve this problem, the present invention converts the glycoside components in kudzu root into aglycone substances through microbial fermentation technology and increases the polysaccharide content, and then through the action of the active enzymes of microorganisms, decomposes the polysaccharides into secondary metabolites to improve the bioavailability of kudzu root.

[0022] In the present invention, the microbial fermentation technology adopted is to ferment kudzu root with probiotic lactic acid bacteria to improve the flavor and taste of kudzu root and its bioavailability. Specifically, the method for microbial fermentation of kudzu root in the present invention is as follows: S1. Preparation of kudzu root fermentation base material Select kudzu root slices without spots and insect pests, crush and sieve them, then mix the kudzu root powder and water. After the raw materials are fully hydrated, sterilize them at high temperature and wait for the kudzu root fermentation base material to cool.

[0023] Among them, the mesh number of the screened kudzu powder after pulverization is 80 - 100 meshes. If the mesh number is too high, it will lead to: (1) Low production efficiency. The resistance increases during the screening process, thus affecting the production efficiency. The screening process may take a longer time and may even require greater power to drive the screening equipment; (2) The sieve holes are easily blocked. With a small aperture, fine kudzu powder particles or impurities are more likely to block the sieve mesh, resulting in a decline in the screening effect. Even frequent cleaning of the sieve mesh is required, further reducing the production efficiency; (3) The powder quality is too fine. An excessively high mesh number will make the kudzu powder too delicate. Although it is beneficial for certain applications (such as pharmaceuticals, fine food processing, etc.), in some applications that require a certain particle size (such as feed, some food raw materials, etc.), the overly fine kudzu powder may not be applicable.

[0024] A too low mesh number may lead to: (1) A relatively high impurity content in the kudzu powder, affecting the product quality; (2) Uneven particle size of the medicinal materials. Secondly, when the mesh number is too low, the particle size range of the screened kudzu powder is relatively large, which is not conducive to the uniformity and stability of the product. (3) Inability to meet specific requirements. In some applications with strict requirements for the particle size of kudzu powder (such as cosmetics, fine chemicals, etc.), a too low mesh number may not be able to meet the requirements, resulting in the product not achieving the expected effect.

[0025] In the present invention, the purpose of mixing the kudzu powder with water to allow the raw materials to fully absorb water is: By fully mixing with water, a heterogeneous colloid can be formed, improving the solubility and utilization rate of the kudzu powder in water; during the processes of water absorption swelling and gelatinization, nutrients in the kudzu powder (such as starch, plant protein, etc.) are more easily surrounded and dissolved by water molecules, thereby improving its biological utilization rate.

[0026] The mass - volume ratio of the kudzu powder to water is 1:20 - 1:30 (g:mL). A too high ratio may lead to: (1) An overly high concentration of the kudzu powder will cause the paste to be too viscous and have poor fluidity, which is not conducive to subsequent processing and uniform mixing; (2) Incomplete dissolution: Due to insufficient water, some of the kudzu powder may not be completely dissolved or gelatinized.

[0027] A too low ratio of the mass - volume ratio of the kudzu powder to water may lead to: (1) The final fermented product cannot achieve the expected effect, such as being unable to form a stable structure or unable to provide sufficient taste and flavor; (2) Excessive water, resulting in a reduced content of nutritional components per unit volume or unit mass, and may not be able to meet specific nutritional requirements.

[0028] Furthermore, after the kudzu powder fully absorbs water, it is subjected to high - temperature sterilization. The sterilization conditions are: sterilization at 120°C - 122°C for 20 min - 30 min.

[0029] S2. Fermentation of the kudzu fermentation base Inoculate the Pueraria thomsonii fermentation base material with Lactobacillus rhamnosus bacterial liquid, mix well and ferment to obtain a liquid microbial fermentate.

[0030] The probiotic selected in the present invention is Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), which is mostly present in the intestines of humans and animals. Taxonomically, it belongs to the genus Lactobacillus, the rhamnosus subspecies. It is an anaerobic and acid-tolerant, spore-free Gram-positive probiotic. The main functions of this bacterium are to regulate the intestinal flora, prevent and treat diarrhea, expel toxins and enhance the body's immunity. At present, there is no research on the application of Lactobacillus rhamnosus in fermenting Pueraria thomsonii.

[0031] The present invention first uses Lactobacillus rhamnosus to ferment Pueraria thomsonii. The selected Lactobacillus rhamnosus is Lactobacillus rhamnosus HCS01-013; the deposit number is: CGMCC No. 19510. This strain is an existing strain, which was deposited in the China General Microbiological Culture Collection Center on March 25, 2020 and has been disclosed in CN112608865A.

[0032] Specifically, the concentration of the Lactobacillus rhamnosus bacterial liquid is 6.0×10 6 CFU / mL~1.2×10 7 CFU / mL, and the inoculation amount of the Lactobacillus rhamnosus bacterial liquid in the present invention is 6wt%~12wt%.

[0033] When the above-mentioned strain is fermenting Pueraria thomsonii, if the inoculation amount is too high, it may lead to: (1) rapid consumption of nutrients: high-concentration lactic acid bacteria will quickly consume the nutrients in the culture medium, resulting in obvious changes in the nutrient components in a short time, which may affect the stability of the subsequent fermentation process and the quality of the product; (2) hyphal entanglement and reduction of aeration volume: high-concentration bacterial cells may cause the hyphae to entangle together, which not only increases the mutual shielding between the bacterial cells, but also reduces the aeration volume in the fermentation broth, thus affecting the fermentation efficiency; (3) product inhibition and change of metabolic pathway: in some cases, high-concentration lactic acid bacteria will produce a large amount of metabolic products, and these products may have an inhibitory effect on the growth of lactic acid bacteria themselves and the synthesis of products; in addition, high concentration may also cause changes in the metabolic pathway of lactic acid bacteria, affecting the types and yields of the final products.

[0034] Too low inoculum may lead to: (1) Prolonged fermentation time: Low concentrations of lactic acid bacteria require more time to reach sufficient cell concentrations during fermentation, thus extending the fermentation cycle. This not only increases production costs but may also reduce fermentation efficiency; (2) Insufficient utilization of active ingredients: Too low cell concentration means that the active ingredients in the culture medium cannot be fully utilized, resulting in waste of resources and reduced yields of fermentation products; (3) Susceptibility to contamination: Low concentrations of lactic acid bacteria may not be able to effectively compete for nutrients and space in the culture medium, making them more susceptible to contamination by miscellaneous bacteria. The presence of miscellaneous bacteria will further affect the stability of the fermentation process and the quality of the products.

[0035] After inoculating the Lactobacillus rhamnosus bacterial solution, the fermented kudzu base material is fermented under the following conditions: microaerobic fermentation, temperature 32°C - 37°C, fermentation time 24 h - 60 h.

[0036] Through the above preparation method, a fermented kudzu product can be obtained. By comparing the contents of the main medicinal effects and nutritional components before and after fermentation, it is found that fermenting kudzu using the above steps can improve the flavor and taste of kudzu, increase the bioavailability of kudzu, and can more effectively improve type 2 diabetes symptoms, such as reversing weight loss, reducing food and water intake, inhibiting blood sugar elevation, reducing insulin resistance and chronic inflammation, and enhancing the protection of the liver and kidneys. In practical applications, the fermented kudzu product obtained in the present invention can be used to prepare kudzu products, which helps to expand the development of kudzu resources and increase the economic value of kudzu.

[0037] The characteristics and properties of the present invention are further described in detail below in conjunction with the embodiments.

[0038] Example 1 This example is a method for microbial fermentation of kudzu, and the specific steps are as follows: (1) Select kudzu slices without spots or insect damage, crush them through an 80-mesh sieve, and accurately weigh and mix kudzu powder and water at a ratio of 1:25 (g:mL); (2) After allowing the raw materials to fully absorb water, sterilize them at 120°C - 122°C for 20 min - 30 min until the fermented kudzu base material cools; (3) Take the bacterial solution of Lactobacillus rhamnosus HCS01-013 (concentration 1.0×10 7 CFU / mL) and inoculate it into the fermented kudzu base material at a total inoculum of 10% (by mass fraction), mix evenly, and ferment. The fermentation conditions are: microaerobic fermentation, temperature 37°C, fermentation time 36 h, to obtain a liquid microbial fermented product of fermented kudzu; (4) Take out the fermentation broth and centrifuge it at 8000 r / min for 2 times, 10 min each time, and take the supernatant and store it at 4°C for future testing.

[0039] Take the fermented product of Pueraria thomsonii prepared in the above steps, and determine the contents of total flavonoids, main isoflavones, soluble polysaccharides, reducing sugars, total phenols, etc. The test methods and results are as follows: 1.1 Determination of total flavonoid content before and after fermentation of Pueraria thomsonii 1.1.1 Preparation of standard solution and drawing of standard curve Precisely weigh 1 mg of puerarin reference substance and place it in a 10 mL volumetric flask. Dissolve and make up the volume with 30% ethanol solution. Taking this as the stock solution, respectively and precisely pipette 0.3 mL, 0.40 mL, 0.50 mL, 0.60 mL, 0.70 mL, 0.80 mL, 0.90 mL of the solution from it, and make up the volume to 10 mL with 30% ethanol in 10 mL volumetric flasks respectively to prepare a series of reference substance solutions with concentration gradients. Using 30% ethanol as the reference solution for the above reference substance solutions, measure the absorbance value at 250 nm to draw the standard curve. The ordinate is the absorbance value, and the abscissa is the concentration of puerarin reference substance (μg / mL) to draw the standard curve. The regression equation is Y = 91.571X + 0.01, R² = 0.9997. The results show that there is a good linear relationship for the puerarin content in the range of 3 - 9 μg / mL.

[0040] 1.1.2 Determination of samples Take 0.1 mL of the fermentation broth, add 30% ethanol to make up the volume to 5 mL, then take 1 mL and add 30% ethanol to make up the volume to 5 mL, shake well, and measure the content by ultraviolet determination. Using 30% ethanol solution as the reference solution, measure the absorbance value at a wavelength of 250 nm, and calculate the total flavonoid content.

[0041] 1.2 Determination of isoflavone components content before and after fermentation of Pueraria thomsonii 1.2.1 Chromatographic conditions 5C18-MS-II C18 chromatographic column (250 mm×4.6 mm, 5 μm), the mobile phase is acetonitrile - 0.1% phosphoric acid aqueous solution, gradient elution: 0 - 10 min, 8% - 18% acetonitrile; 10 - 20 min, 18% - 30% acetonitrile; 20 - 25 min, 30% - 50% acetonitrile; 25 - 28 min, 50% - 55% acetonitrile; 28 - 30 min, 55% - 8%; the volume flow rate of acetonitrile is 1.0 mL / min; the column temperature is 30°C; the injection volume is 10 μL; the detection wavelength is 250 nm.

[0042] 1.2.2 Preparation of standard solution and drawing of standard curve Weigh appropriate amounts of 3′-hydroxy puerarin, puerarin, puerarin apioglucoside, daidzin, genistin, daidzein, and genistein reference standards accurately. Dissolve them in methanol and make up to 10 mL to prepare the stock solution. Then, accurately pipette appropriate amounts of the stock solutions of 3′-hydroxy puerarin, puerarin, puerarin apioglucoside, daidzin, genistin, daidzein, and genistein into a 10 mL volumetric flask, and dilute to the mark with 50% methanol to obtain reference standard solutions with mass concentrations of 15.15, 251.50, 25.00, 40.00, 25.00, 15.06, and 25.05 μg / mL respectively. Precisely transfer 5 mL of the reference standard stock solution, dilute it successively by 2-fold with 50% ethanol solution, filter through a 0.22 μm microporous filter membrane, and inject the subsequent filtrate for determination under the chromatographic conditions. Using the concentration as the abscissa and the peak area as the ordinate, the linear regression equations of each component are shown in Table 1.

[0043] Table 1 Results of linear investigation of each component

[0044] 1.2.3 Preparation of test solution Precisely measure 1 mL of the fermented kudzu root liquid into a 10 mL volumetric flask, dilute to the mark with 50% methanol, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0045] 1.3 Determination of soluble polysaccharide content before and after fermentation of kudzu root 1.3.1 Preparation of standard solution and drawing of standard curve Determine according to the phenol-sulfuric acid method. Accurately weigh 50 mg of anhydrous glucose into a 50 mL volumetric flask, dissolve it with distilled water and make up to the mark to obtain a 1 mg / mL glucose solution. Dissolve 10 g of phenol in 190 mL of water to obtain a 5% phenol solution in a brown bottle. Take 10 mL of the above-prepared glucose solution, dilute it, and make up to 50 mL to obtain a 0.2 mg / mL glucose standard solution. Respectively take 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, and 1.3 mL of the above glucose standard solution into 20 mL stoppered test tubes, then add distilled water to each test tube until the volume of the solution in each test tube reaches 2 mL. Then add 1 mL of 5% phenol solution to each test tube, mix well, quickly add 5 mL of concentrated sulfuric acid solution, mix gently, let stand for 5 min, then place it in a water bath and heat in a boiling water bath for 15 min. Take it out and cool to room temperature. Measure the absorbance of the above solutions at 490 nm. Using the glucose concentration as the abscissa and the absorbance as the ordinate, draw the standard curve. The regression equation is Y = 14.98x + 0.0179, R² = 0.9996. The results show that there is a good linear relationship between the glucose content and 0 - 0.13 mg.

[0046] 1.3.2 Determination of the test solution Take 2 mL of the fermented kudzu root solution diluted by a certain multiple in a test tube, use 2 mL of deionized water as the blank control, and perform the remaining operations as above. Calculate the content of soluble polysaccharides in the diluted fermented solution according to the regression equation of the above glucose solution curve.

[0047] 1.4 Determination of the reducing sugar content before and after the fermentation of kudzu root 1.4.1 Preparation of the standard solution and drawing of the standard curve Using the DNS colorimetric method, accurately weigh 10 mg of anhydrous glucose standard sample, place it in a 10 mL volumetric flask, and prepare an anhydrous glucose standard solution with a concentration of 1 mg / mL using distilled water. Respectively pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the glucose standard solution into stoppered tubes, add distilled water to 1 mL, then add 2 mL of DNS reagent, mix well, place in a boiling water bath for 5 min, cool to room temperature, add 9.0 mL of distilled water, and measure the absorbance at 540 nm. Draw a standard curve with the glucose concentration as the abscissa and the absorbance value as the ordinate, and obtain the linear equation Y = 0.9589x - 0.0168, R² = 0.9984.

[0048] 1.4.2 Determination of the test solution Take 1.0 mL of the fermented kudzu root solution and the blank solution diluted to a certain concentration in a stoppered test tube, and perform the determination according to the above steps. Using the blank control tube as the reference, measure the absorbance value at a wavelength of 540 nm, and calculate the reducing sugar content.

[0049] 1.5 Determination of the total phenol content before and after the fermentation of kudzu root 1.5.1 Preparation of the standard solution and drawing of the standard curve Accurately weigh 24.90 mg of gallic acid reference substance and place it in a 25 mL volumetric flask, and make up to the scale line with ultrapure water. Prepare the mother liquor, and then accurately pipette 0.50 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2 mL of the solution from it and make up to 5 mL to prepare a series of concentration gradient reference substance solutions. For the above reference substance solutions, using ultrapure water as the reference solution, mix 20 μL of the reference substance and 100 μL of 0.2 mol Folin-Ciocalteu reagent in a 96-well microplate, let stand in the dark for 5 min, then add 80 μL of 10% Na 2 CO 3 ₃, after 30 min, perform a full wavelength scan to measure the absorbance value at a wavelength of 765 nm, draw a standard curve, with the ordinate being the absorbance value and the abscissa being the concentration of the gallic acid reference substance (mg / mL). The regression equation is Y = 91.571X + 0.01, R 2= 0.9997. The results showed that there was a good linear relationship in the total phenol content in the concentration range of 0.0153 - 0.1071 mg / mL.

[0050] 1.5.2 Determination of the test solution Take 20 μL of the sample diluted by a certain multiple and mix it with 100 μL of 0.2 mol / L Folin-Ciocalteu reagent in a 96-well microplate, and let it stand in the dark for 5 min. Add 80 μL of 10% Na 2 CO 3 to the 96-well microplate. After 30 min, measure the absorbance at a wavelength of 765 nm, and the result is expressed as mg GAE / mL of the sample.

[0051] The detection results of the changes in the contents of the target components before and after the fermentation of Pueraria thomsonii are shown in Table 2: Table 2 Changes in the contents of the target components before and after the fermentation of Pueraria thomsonii

[0052] Note: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 According to the results in Table 2, it can be concluded that compared with Pueraria thomsonii before fermentation, the total flavonoid content in Pueraria thomsonii after fermentation increased to 36.54 mg / g, which was 1.109 times that of Pueraria thomsonii before fermentation, and the difference was significant (P < 0.05).

[0053] The main isoflavone components in Pueraria thomsonii are 3-OH puerarin, puerarin, puerarin apioglucoside, daidzin, genistin, daidzein, genistein. Compared with Pueraria thomsonii before fermentation, the contents of 3-OH puerarin, daidzein, and genistein in Pueraria thomsonii after fermentation increased, while the contents of daidzin and genistin decreased. Therefore, it can be proved that the fermentation method of the present invention can convert the glycoside components in Pueraria thomsonii into aglycone substances.

[0054] Compared with before fermentation, the soluble polysaccharide content in Pueraria thomsonii after fermentation increased to 12.01 mg / mL, which was 1.093 times that of Pueraria thomsonii before fermentation, and the difference was significant (P < 0.001). It may be because Lactobacillus is a kind of probiotic with strong carbohydrate metabolism. During the fermentation process, it uses starch as a carbon source and metabolizes to produce free glucose or small molecular glucose polymers, increasing the content of soluble polysaccharides.

[0055] Compared with before fermentation, the total phenol content in Pueraria thomsonii after fermentation increased. This may be because microorganisms de-glycosylated the glycosylated phenolic compounds during the fermentation process, thus releasing the soluble conjugated phenolic compounds and insoluble bound phenolic compounds from the plant cell wall, increasing the total phenol content.

[0056] Example 2 This example is to verify the effect of the kudzu root ferment obtained in Example 1 on type 2 diabetes in C57BL / KsJ-db / db mice, which is specifically as follows: 1. Experimental animals In this study, 60 male C57BL / KsJ-db / db mice at 8 weeks of age and specific pathogen-free (SPF) level (fasting blood glucose ≥ 11.1 mmol·L -1 ) and 10 db / m mice were purchased from Changzhou Cavens Co., Ltd. (license number: SCXK (Su) 2021-0013). Before the formal experiment, each group of mice was fed adaptively for 3 days to ensure stable experimental conditions. This experiment has been approved by the Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine (approval number: JZLLSC20240554).

[0057] 2. Experimental methods 2.1 Preparation of fermented kudzu root and kudzu root solution According to Example 1, the kudzu root ferment was prepared. After the fermentation broth was rotary evaporated and concentrated to a certain concentration, the fermentation broth was poured out, pre-frozen at -20°C for 12 h, and then the ferment was freeze-dried. The yield of the ferment was calculated to be 56.95%, and the yield of the unfermented material was 54.28%. When used, the fermented kudzu root and kudzu root were respectively dissolved in ultrapure water and stored in a 4°C refrigerator.

[0058] 2.2 Grouping and administration of experimental animals The experimental groups and the corresponding treatment methods are as Figure 1 shown. During the feeding of experimental animals, the day and night alternate for 12 h, the temperature is maintained at 25 ± 1°C, the humidity is maintained at 55% - 65%, and the air is kept flowing. After 3 days of adaptive feeding, the fasting blood glucose ≥ 11.1 mmol / L was detected as the molding condition of T2DM. The db / m mice were set as the normal group (N), and the db / db mice were randomly divided into 6 groups: the model group (M), the positive group (MET, 0.36 g / kg), the low-dose kudzu root group (PL, 1.23 g / kg), the high-dose kudzu root group (PH, 2.47 g / kg), the low-dose fermented kudzu root group (FPL, 1.30 g / kg), and the high-dose fermented kudzu root group (FPH, 2.59 g / kg). Each administration group was continuously intragastrically administered for 5 weeks according to the dose, and the normal group and the model group were intragastrically administered with normal saline (10 mL / kg).

[0059] 2.3 Detection of phenotypic indicators On the first day of drug administration, the body weights of mice in each group were measured, and then intragastric administration was performed according to the body weight. The changes in the body weights of mice were regularly measured and recorded every week; starting from the day after the official drug administration, the food intake and water intake of mice were weighed and recorded at the same time period every week; fasting blood glucose was measured 12 h after fasting but not water deprivation at the same time every week. The detection method was to puncture the tail vein of the mouse with a matching blood collection needle, and gently squeeze out a small blood drop for the blood collection test strip to inhale, and the blood glucose meter showed the blood glucose value.

[0060] 2.4 Sample collection 2.4.1 Collection of serum samples After the experimental animals were treated with fasting but not water deprivation for 12 hours, they were sacrificed and dissected on the second day. Before sacrifice, the fasting blood glucose value was measured using a micro blood collection instrument, and whole blood samples were collected by puncturing the orbital venous plexus. The blood samples were collected in 1.5 mL centrifuge tubes, left to stand at room temperature for 2 h, centrifuged at 3500 rpm at 4 °C for 15 min, and the supernatant was taken. The obtained serum samples were quickly frozen in liquid nitrogen and then transferred to an ultra-low temperature refrigerator at -80 °C for long-term storage.

[0061] 2.4.2 Collection of tissue samples The pancreas, liver, kidney, colon, and cecal contents were obtained by dissection. The liver, kidney, and pancreas were weighed, and the wet weight data of each organ were recorded. The pancreas and the left lobe of the liver were immediately immersed in paraformaldehyde tissue fixative for subsequent histopathological examination; the remaining liver tissue, colon tissue, and cecal contents were aliquoted into cryotubes, quickly frozen in liquid nitrogen, and then transferred to an ultra-low temperature refrigerator at -80 °C for long-term storage.

[0062] 2.5 Determination of biochemical indicators and organ indices Insulin (INS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), alkaline phosphatase (ALP), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the serum were detected. All operations followed the requirements of the kit operation specifications. At the same time, the liver index, pancreas index, and kidney index were calculated. The insulin resistance index (HOMA-IR) was calculated according to the fasting insulin value × fasting blood glucose value / 22.5.

[0063] 2.6 Histopathology The liver tissue and pancreas tissue were fixed in the tissue fixative. After dehydration, paraffin embedding and sectioning were performed. The section thickness was 4 - 5 mm, HE staining was performed, electron microscope scanning and photographing were carried out, and sample collection and analysis were performed using the Lecia Applaction Stiue image system to observe the histopathological changes.

[0064] 3 Experimental Results 3.1 Analysis of Phenotypic Indexes The results are as Figure 2 shown, where Figure 2 A in it is the effect of fermented kudzu root and kudzu root on the body weight of db / db mice; Figure 2 B in it is the effect of fermented kudzu root and kudzu root on the food intake of db / db mice; Figure 2 C in it is the effect of fermented kudzu root and kudzu root on the water intake of db / db mice; Figure 2 D in it is the effect of fermented kudzu root and kudzu root on the blood glucose of db / db mice; (compared with the model group, *P<0.05; **P<0.01).

[0065] As Figure 2 shown by A in it, the diabetic mice in the model group showed symptoms of weight loss in the later stage. After MET, PL, PH, and FPH administration interventions, the symptoms of weight loss in diabetic mice could be significantly reversed. Figure 2 B in it is the food intake of db / db mice. After administration interventions, compared with the model group, the food intake of mice in the MET and FPH groups began to show a downward trend in the second week, and at the fifth week, the food intake of mice in the MET, FPL, and FPH groups was significantly lower than that of the model group ( P <0.01), and the food intake of mice in the PL and PH groups was lower than that of the model group, but there was no significant difference. Figure 2 C in it is the water intake of db / db mice. It can be seen that the water intake of mice in the normal group has always remained stable, and the water intake of the model group has always been the highest, which is in line with the typical symptom of type 2 diabetes, "polydipsia". After MET, FPL, and FPH administration interventions, the water intake of mice can be significantly reduced to relieve the symptoms ( P <0.05).

[0066] As Figure 2 shown by D in it, fermented kudzu root and kudzu root both had a certain effect on the fasting blood glucose level of db / db mice. The experimental results showed that throughout the administration period, the blood glucose level of mice in the model group was always higher than that of the normal group, and with the extension of the feeding time, the blood glucose value of mice in the model group showed a continuous upward trend. By the fifth week, compared with the model group, the blood glucose level of mice in the MET group was significantly decreased ( P <0.01), the blood glucose value of mice in the PH group was significantly decreased ( P <0.01), the blood glucose level of mice in the FPL group was slightly lower than that of the model group ( P <0.05), the blood glucose of mice in the FPH group decreased most significantly ( P <0.01), while although the mice in the PL group showed a certain downward trend in blood glucose, the difference was not statistically significant. These results indicate that the fermentation products of kudzu root may inhibit the increase of blood glucose to a certain extent.

[0067] It can also be seen from the above results that compared with Pueraria thomsonii, the fermented product of Pueraria thomsonii has a greater impact on each phenotype.

[0068] 3.2 Analysis of Biochemical Index Results The results of biochemical indexes, inflammatory factors and organ indexes are shown in Figure 3 , where A represents the test result of INS, B represents the result of HOMA-IR, C represents the test result of ALT, D represents the test result of AST, E represents the test result of ALP, F represents the test result of TC, G represents the test result of TG, H represents the test result of LDL-C, I represents the test result of TNF-α, J represents the test result of IL-1Β, K represents the result of liver coefficient, L represents the result of pancreas coefficient, and M represents the result of kidney coefficient. INS can promote glucose utilization, inhibit gluconeogenesis and inhibit the production of liver glucose; These blood lipid indexes such as ALT, AST, ALP, TC, TG, and LDL-C participate in the process of gluconeogenesis, and these pro-inflammatory factors such as TNF-α and IL-1β will interfere with insulin signaling, leading to chronic inflammation, resulting in β-cell dysfunction, apoptosis and necrosis, thus affecting insulin secretion.

[0069] After the intervention treatment with fermented Pueraria thomsonii and Pueraria thomsonii, the indexes such as ALT, AST, ALP, TC, TG, LDL-C, TNF-α and IL-1β showed a downward trend compared with the model group ( P <0.05; P <0.01; P <0.01; P <0.01), indicating that fermented Pueraria thomsonii and Pueraria thomsonii at different doses have an improving effect on insulin resistance and chronic inflammation in diabetic mice; Compared with the model group, the insulin resistance index groups of MET and FPH were significantly reduced ( P <0.01 or P <0.05), and the other drug administration groups showed a downward trend compared with the model group, without significant difference; Compared with the model group, the liver index of the FPL group decreased ( P <0.01), and the kidney indexes of each drug administration group showed a downward trend ( P <0.05, P <0.01 or P <0.01), and the pancreas index showed an upward trend, indicating that fermented Pueraria thomsonii and Pueraria thomsonii at different doses can improve pancreatic atrophy in type 2 diabetic mice and have a certain protective effect on the liver and kidneys. It can also be seen from this that the fermented Pueraria thomsonii has a better improvement effect on diabetic mice and a more obvious protective effect on the liver and kidneys.

[0070] 3.3 Histopathological Analysis of Liver and Pancreas The effects of fermented kudzu root and kudzu root administration on liver histological changes were determined using HE staining ( Figure 4 ), Figure 4 where A in Figure 4 is the HE staining result of liver tissue at ×20 magnification, and B in

[0071] is the HE staining result of liver tissue at ×40 magnification. The overall structure of the liver tissue in the normal group of mice was basically normal, with regular arrangement of hepatocytes, round and full-structured hepatocytes, no obvious loosening, edema, fatty degeneration, necrosis or other degeneration of hepatocytes, the hepatic sinusoids arranged radially along the central vein, no obvious congestion or dilation of the hepatic sinusoids, and no obvious infiltration of inflammatory cells. In the model group, the arrangement of hepatocytes was irregular and the structure was loose. Many hepatocytes showed obvious edema, and some were edematous to vacuolar degeneration. The structure of the hepatic sinusoids was unclear, and a large number of inflammatory cells were infiltrated. After intervention with metformin, fermented kudzu root and kudzu root at different doses, there was no obvious congestion or dilation of the hepatic sinusoids, the vacuolar lipid droplets in hepatocytes decreased, and the nuclear pyknosis and apoptosis were improved to varying degrees. There was no obvious infiltration of inflammatory cells. The improvement effect in the high-dose group was better than that in the low-dose group, especially in the FPH group. Figure 5 shown, Figure 5 where A in Figure 5 is the HE staining result of pancreatic tissue at ×20 magnification, and B in

[0072] In addition, the effects of fermented kudzu root and kudzu root on pancreatic islet cells were also studied. The staining results are as

[0073] Experimental Example 1 This experiment was a comparison of the fermentation effects of different strains, as follows: (1) Select kudzu root slices without spots or insect damage, crush them through an 80-mesh sieve, and accurately weigh the kudzu root powder and mix it with water at a ratio of 1:25 (g:mL); (2) After the raw materials are fully hydrated, sterilize them at 120°C - 122°C for 20 - 30 minutes until the kudzu root fermentation substrate cools; (3) Six different strains were taken, Lactobacillus mucosae HCS02-001 (CGMCC No. 19746), Lactobacillus casei paracasei HCS17-040 (CGMCC No. 19747), Lactobacillus rhamnosus HCS01-013 (CGMCC No. 19510), Lactobacillus mucosae fermentum HCS08-005 (CGMCC No. 16259), Lactiplantibacillus plantarum HCS03-001 (CGMCC No. 16258), Lactobacillus plantarum P9 (CGMCC No. 16662), with the bacterial liquid concentration of 8.0×10 6 CFU / mL, inoculated into the kudzu root fermentation substrate at a total inoculation amount of 8% (by mass fraction), mixed evenly, and fermented. The fermentation conditions were: microaerobic fermentation, temperature 37 °C, fermentation time 60 h, to obtain the liquid microbial fermentates of kudzu root fermented by different strains; (4) The fermented liquid was centrifuged twice at 8000 r / min for 10 min each time, and the supernatant was taken and stored at 4 °C for testing.

[0074] Take the kudzu root fermentation product prepared in the above steps, measure its viable bacteria count, pH, total acid, total flavonoids, main isoflavones and other contents, conduct principal component analysis (Principal component analysis, PCA) on the above indicators, and finally optimize the dominant fermentation strains according to the relevant characteristics such as the basic physical and chemical indicators and functional activity indicators of different lactic acid bacteria fermentation, and comprehensively consider the bacterial growth situation and the conversion degree of functional factors. The test methods and results are as follows: 1 Viable count of Lactobacillus The total viable count (Total viable count, TVC) in the sample solution was detected by the dilution plate counting method. The sample solution was gradient diluted in a laminar flow hood. 1 mL of 10 -5 , 10 -6 and 10 -7 sample solution dilutions were taken, and the culture medium was added and poured. The plate culture medium was inverted and placed in a constant temperature and humidity box, cultured at 37 °C for 48 h, and counted.

[0075] 2 pH determination Refer to the national standard GB 5009.237-2016 "Determination of pH value of foods".

[0076] 3 Determination of total acid (TA) content Refer to the national standard GB 12456-2021 "Determination of total acid in foods".

[0077] The diluted NAOH was added to the burette, rinsed once to remove air bubbles, and fixed at the 0 scale. 2-4 drops of phenolphthalein were added to the diluted sample. Using 0.01 mol·L-1 Titrate with NaOH until it turns slightly red and does not fade for 30 s, and record the volume of NaOH consumed, V1 (read to one decimal place). Repeat the above steps 3 times. For the blank control, use the same volume of CO-free 2 water to replace the sample solution for the blank experiment, and record the volume of NaOH consumed, V2.

[0078] X = ([C×(V1 - V2)]×K×N) / M×1000 (g / Kg or g / L) C: Concentration of the standard NaOH titration solution; K: Lactic acid equals 0.090; N: Dilution factor of the sample solution; M: Mass (g) or volume (mL) of the test sample; V1, V2: Volumes of NaOH consumed by the sample solution and the blank control; 4 Determination of the total flavonoid content before and after the fermentation of Pueraria thomsonii 4.1 Preparation of the standard solution and drawing of the standard curve Precisely weigh 1 mg of puerarin reference substance and place it in a 10 mL volumetric flask. Dissolve and make up the volume with 30% ethanol solution. Using this as the stock solution, accurately pipette 0.3 mL, 0.40 mL, 0.50 mL, 0.60 mL, 0.70 mL, 0.80 mL, and 0.90 mL of the solution from it respectively, and make up the volume to 10 mL in 10 mL volumetric flasks with 30% ethanol to prepare a series of reference substance solutions with different concentration gradients. For the above reference substance solutions, using 30% ethanol as the reference solution, measure the absorbance value at 250 nm to draw the standard curve. The ordinate is the absorbance value, and the abscissa is the concentration of the puerarin reference substance (μg / mL) to draw the standard curve. The regression equation is Y = 91.571X + 0.01, R² = 0.9997. The results show that there is a good linear relationship for the puerarin content in the range of 3 - 9 μg / mL.

[0079] 4.2 Determination of the sample Take 0.1 mL of the fermentation broth, add 30% ethanol to make up the volume to 5 mL, then take 1 mL and add 30% ethanol to make up the volume to 5 mL, shake well, and measure the content by ultraviolet method. Using 30% ethanol solution as the reference solution, measure the absorbance value at a wavelength of 250 nm, and calculate the total flavonoid content.

[0080] 5 Determination of the content of isoflavone components before and after the fermentation of Pueraria thomsonii 5.1 Chromatographic conditions 5C18-MS-IIC18 chromatographic column (250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile - 0.1% phosphoric acid aqueous solution, gradient elution: 0 - 10 min, 8% - 18% acetonitrile; 10 - 20 min, 18% - 30% acetonitrile; 20 - 25 min, 30% - 50% acetonitrile; 25 - 28 min, 50% - 55% acetonitrile; 28 - 30 min, 55% - 8%; acetonitrile volume flow rate 1.0 mL / min; column temperature 30 °C; injection volume 10 μL; detection wavelength 250 nm.

[0081] 5.2 Preparation of standard solution and drawing of standard curve Weigh appropriate amounts of reference substances of 3'-hydroxy puerarin, puerarin, puerarin apiin, daidzin, genistin, daidzein, and genistein, accurately weigh them, dissolve and dilute to 10 mL with methanol to prepare a stock solution. Then, accurately pipette appropriate amounts of the stock solutions of 3'-hydroxy puerarin, puerarin, puerarin apiin, daidzin, genistin, daidzein, and genistein into 10 mL volumetric flasks, and dilute to the mark with 50% methanol to obtain reference substance solutions with mass concentrations of 15.15, 251.50, 25.00, 40.00, 25.00, 15.06, and 25.05 μg / mL respectively. Accurately transfer 5 mL of the reference substance stock solution, dilute it successively by 2 times with 50% ethanol solution, filter through a 0.22 μm microporous filter membrane, and inject the subsequent filtrate under the chromatographic conditions for determination. Taking the concentration as the abscissa and the peak area as the ordinate, the linear regression equations of each component are shown in Table 1.

[0082] 5.3 Preparation of test solution Accurately pipette 1 mL of the fermented kudzu root liquid into a 10 mL volumetric flask, dilute to the mark with 50% methanol, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0083] The test results are as follows: Table 3 Characteristics of different Lactobacillus-fermented kudzu roots (X ± S)

[0084] Note: Multiple comparisons were performed by the Duncan method. Different capital letters in the same column indicate extremely significant differences between groups (P < 0.01); different lowercase letters indicate significant differences between groups (P < 0.05); the same lowercase letters indicate no significant differences between groups (P > 0.05).

[0085] As can be seen from Table 3, the viable count in the kudzu root after 60 h of fermentation reached 8.0 lg (CFU·mL -1), in which the viable count of kudzu fermented by Lactobacillus rhamnosus can reach up to 8.50 lg (CFU·mL-1) at most. The pH value of the kudzu fermentation substrate is 6.36. After fermentation by Lactobacillus, some acidic substances increase, and the pH values of each kudzu fermentation group decrease. The total acid content also increases due to the action of microorganisms. The total acid content of Lactobacillus rhamnosus is 1.03 g·L -1 , which is 2.5 times that of the kudzu fermentation group. Lactobacillus improves the total flavonoid content during the fermentation process. The initial total flavonoid content of kudzu is 20.29 mg·g -1 . After 60 h of fermentation, the total flavonoid content of the Lactobacillus rhamnosus fermentation group is 22.07 mg·g -1 , which is about 8.8% higher than that before fermentation. The total flavonoid content of other groups also increases. The main flavonoid compounds in kudzu are 3'-hydroxy puerarin, puerarin, daidzein, daidzin, genistein, and genistin. It is found by determination that the content of puerarin in the kudzu fermentation broth is the highest, which is 8.10 mg·g -1 , while it is 7.9 mg·g -1 in the kudzu substrate before fermentation. The content of puerarin increases after fermentation. There is no significant difference between the Lactobacillus fermentum group and the kudzu substrate group. The content of puerarin in the Lactobacillus reuteri group is the lowest, which is 7.88 mg·g -1 . The content of daidzin in the kudzu fermentation broth is also lower than that in the kudzu substrate. Compared with the control group, the contents of daidzein and genistein in the fermentation broth of multiple groups of kudzu increase significantly after fermentation. The contents of daidzein and genistein in the Lactobacillus rhamnosus fermentation group are 2.93 mg·g -1 and 0.13 mg·g -1 .

[0086] Table 4 Comprehensive scores and rankings of kudzu enzymes fermented by different strains

[0087] According to the scores of each principal component (Y1 - Y3), with the variance contribution rate corresponding to each principal component as the weight, a comprehensive evaluation model is constructed: Y = 0.56Y1 + 0.28Y2 + 0.16Y3. According to this model, the comprehensive total scores of each group are calculated, as shown in Table 4. The Lactobacillus rhamnosus ranks first in the comprehensive total score. Combining with the PCA score, it can be seen that the comprehensive quality of kudzu fermented by Lactobacillus rhamnosus is better.

[0088] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for microbial fermentation of kudzu vine, characterized in that: include: Lactobacillus rhamnosus bacterial liquid is added to the kudzu vine fermentation base material, mixed and fermented to obtain liquid microbial fermentation product.

2. The method according to claim 1, characterized in that The Lactobacillus rhamnosus is Lactobacillus rhamnosus HCS01-013; its preservation number is CGMCC No.19510.

3. The method according to claim 1, characterized in that The inoculation amount of the Lactobacillus rhamnosus bacterial liquid is 6 wt% to 12 wt%.

4. The method according to claim 1, characterized in that: The concentration of the Lactobacillus rhamnosus bacterial solution is 6.0×10 6 CFU / mL~1.2×10 7 CFU / mL.

5. The method according to claim 1, characterized in that The preparation method of the kudzu vine fermentation base material comprises: mixing pulverized and sieved kudzu vine powder with water, sterilizing the kudzu vine powder at high temperature after fully absorbing water, and obtaining the kudzu vine fermentation base material after cooling.

6. The method according to claim 5, characterized in that The kudzu powder is sieved through 80-100 meshes; the ratio of the kudzu powder to water is 1:20 g / mL to 1:30 g / mL.

7. The method according to claim 5, characterized in that The high temperature sterilization condition is sterilization at 120°C to 122°C for 20 min to 30 min.

8. The method according to claim 1, characterized in that The fermentation conditions are: temperature 32°C to 37°C, and fermentation time 24 h to 60 h.

9. The kudzu vine fermented product prepared by the method according to any one of claims 1 to 8.

10. Use of the kudzu vine fermented product according to claim 9 in preparing a product for improving symptoms of type 2 diabetes.

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