A method for fermenting kudzu by microorganisms

Abstract

The application discloses a method for fermenting bletilla striata by microorganisms, which comprises the following steps: inoculating lactobacillus rhamnosus into bletilla striata powder, and fermenting the bletilla striata by the lactobacillus rhamnosus. The method can effectively improve the content of total flavonoids, soluble polysaccharides and main isoflavones including 3-OH puerarin, daidzein and genistein in the bletilla striata. The prepared bletilla striata product has higher biological activity, is more beneficial to human body absorption and utilization, and can more effectively improve the symptoms of type 2 diabetes. Meanwhile, the preparation method is simple and easy to operate, and can save cost, so that the method has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation processing, in particular to a method for fermenting pueraria lobata by microorganisms. BACKGROUND

[0002] Pueraria lobata is the dried tuber of Pueraria lobata (Willd.) Ohwi var. lobata (Willd.) Benth. Pueraria thomsonii It has the characteristics of unique nutrition, medicine and food. Pueraria lobata has many pharmacological effects such as anti-myocardial ischemia, hypoglycemic, hypolipidemic, antipyretic, and antiviral. The main components that play a role in pueraria lobata are starch, polysaccharide and isoflavone components. 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. Isoflavone components 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, which can improve insulin sensitivity by increasing AMPK phosphorylation and glucose transporter 4 (GLUT4) expression, and promoting glucose uptake. Genistein in pueraria lobata also has good estrogenic effects and antioxidant capacity, and it is also a potential anti-fat and lipid-lowering agent. Studies have shown that 3'-hydroxy puerarin 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 polysaccharide cannot be directly degraded and absorbed by enzymes encoded by the human genome, and isoflavone components such as daidzein glycosides in the body cannot be directly absorbed by the small intestine wall, and must be converted to free glycosides to be absorbed.

[0003] In order to further improve the bioavailability of pueraria lobata, researchers at home and abroad have carried out a large number of researches on starch and polysaccharide components in pueraria lobata. Through enzymolysis, gelatinization, saccharification and other methods, the utilization rate of starch and polysaccharide is improved, but there are problems such as complex operation process and large energy loss.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a method for fermenting pueraria lobata by microorganisms, which can effectively improve the content of total flavonoids, soluble polysaccharides and main isoflavones in pueraria lobata.

[0006] The present application is realized as follows:

[0007] In a first aspect, the present application provides a method for fermenting pueraria lobata by microorganisms, which comprises: adding lactobacillus rhamnosus liquid to pueraria lobata fermentation base material, mixing and fermenting to obtain liquid microbial fermentation product.

[0008] In some embodiments, the Lactobacillus rhamnosus is Lactobacillus rhamnosus HCS01-013; the preservation number is CGMCC No. 19510.

[0009] In some embodiments, the inoculation amount of the Lactobacillus rhamnosus bacterial solution is 6 wt%-12 wt%.

[0010] In some embodiments, the concentration of the Lactobacillus rhamnosus bacterial solution is 6.0×10 6 CFU / mL-1.2×10 7 CFU / mL.

[0011] In some embodiments, the preparation method of the Pueraria ferment includes: mixing the crushed and sieved Pueraria powder with water, sterilizing the Pueraria powder after fully absorbing water, and cooling to obtain the Pueraria ferment.

[0012] In some embodiments, the mesh number of the sieved Pueraria powder is 80-100 mesh.

[0013] In some embodiments, the ratio of the Pueraria powder to water is 1:20-30 (g:mL).

[0014] In some embodiments, the high-temperature sterilization condition is 120℃-122℃ for 20 min-30 min.

[0015] In some embodiments, the fermentation condition is: temperature 32℃-37℃, and fermentation time 24 h-60 h.

[0016] In the second aspect, the application further provides a Pueraria ferment prepared by the above method.

[0017] In the third aspect, the application further provides an application of the above Pueraria ferment in preparing a product for improving the symptoms of type 2 diabetes.

[0018] The application has the following beneficial effects:

[0019] The application inoculates Lactobacillus rhamnosus in Pueraria powder, and ferments the Pueraria powder by using the Lactobacillus rhamnosus, so as to effectively increase the total flavonoids, soluble polysaccharides, and main isoflavones including 3-OH puerarin, daidzein, and genistein in the Pueraria powder. The preparation method of the application can obtain a Pueraria product with higher biological activity, which is more beneficial to human body absorption and utilization, and can more effectively improve the symptoms of type 2 diabetes; at the same time, the preparation method of the application is simple and easy to operate, and can save costs, so the method of the application has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation to the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0021] Figure 1 Animal experiment technical roadmap for Example 2;

[0022] Figure 2 Phenotype index detection results in Example 2;

[0023] Figure 3 Influence of fermented Puerariae radix and Puerariae radix on biochemical values of db / db mice in Example 2 (compared with the model group, P <0.05;** P <0.01);

[0024] Figure 4 HE staining diagram of pathological liver tissue of db / db mice in Example 2;

[0025] Figure 5 HE staining diagram of pathological pancreas tissue of db / db mice in Example 2. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the following will clearly and completely describe the technical solutions in the embodiments of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by market purchase.

[0027] The polysaccharide, isoflavone and other components in Puerariae radix are the main components for exerting the pharmacological effects, but these components cannot be directly absorbed and utilized by the human body. In order to improve this problem, the glycoside components in Puerariae radix are converted into aglycone substances and the polysaccharide content is improved through the microbial fermentation technology, and then the polysaccharide is decomposed into secondary metabolites through the activity of the enzymes of the microorganisms, so as to improve the bioavailability of Puerariae radix.

[0028] In the present application, the microbial fermentation technology is used to ferment Puerariae radix by probiotic lactic acid bacteria, so as to improve the flavor and taste of Puerariae radix and improve the bioavailability of Puerariae radix. Specifically, the method for fermenting Puerariae radix by microorganisms in the present application is as follows:

[0029] S1. Preparation of Puerariae radix fermentation base material

[0030] Select the powder of no spot, no insect, crush and sieve, then mix the powder and water, make the raw material fully absorb water, high temperature sterilization, until the powder of fermented base cooling.

[0031] In the present application, the powder of B. japonica is crushed and sieved to 80-100 mesh. If the mesh size is too high, it may lead to: (1) low production efficiency, increased resistance during screening, affecting production efficiency, longer screening time, or even greater power required to drive the screening equipment; (2) screen clogging, small pore size, and fine powder particles or impurities more easily clogging the screen, resulting in reduced screening efficiency, and even frequent screen cleaning, further reducing production efficiency; (3) too fine powder, high mesh size may make the powder too fine, which is beneficial for some applications (such as pharmaceuticals, food fine processing, etc.), but in some applications that require a certain particle size (such as feed, some food raw materials, etc.), the powder may not be suitable.

[0032] Low mesh size may lead to: (1) high impurity content in the powder, affecting product quality; (2) uneven particle size of the medicinal material, and when the mesh size is too low, the particle size range of the sieved powder is large, which is not conducive to the uniformity and stability of the product; (3) unable to meet specific requirements, in some applications that have strict requirements on the particle size of the powder (such as cosmetics, fine chemicals, etc.), low mesh size may not meet the requirements, resulting in the product not meeting the expected effect.

[0033] In the present application, the powder of B. japonica is mixed with water to fully absorb water, which aims to: by fully mixing with water, a heterogeneous colloid can be formed, improving the solubility and utilization of the powder in water; during the water absorption and gelatinization process, the nutrients in the powder (such as starch, plant proteins, etc.) are more easily surrounded and dissolved by water molecules, thereby improving their bioavailability.

[0034] The mass-volume ratio of the powder of B. japonica to water is 1:20~1:30 (g:mL), and a high ratio may lead to: (1) high concentration of the powder, resulting in a too thick paste with poor flowability, which is not conducive to subsequent processing and uniform mixing; (2) incomplete dissolution: due to insufficient water, some of the powder may not be completely dissolved or gelatinized.

[0035] A low mass-volume ratio of the powder of B. japonica to water may lead to: (1) the final fermented product may not achieve the expected effect, such as not forming a stable structure or not providing sufficient mouthfeel and flavor; (2) too much water, reducing the content of nutrients per unit volume or unit mass, which may not meet the specific nutritional requirements.

[0036] Further, the powder of B. japonica is fully water-absorbed and then subjected to high-temperature sterilization at 120℃~122℃ for 20 min~30 min.

[0037] S2. Fermentation of kudzu vine fermentation base

[0038] Lactobacillus rhamnosus liquid is inoculated into kudzu vine fermentation base material, and the mixture is mixed and fermented to obtain liquid microbial fermentation product.

[0039] The probiotic selected by the present invention is Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), which mostly exists in the intestines of humans and animals. In bacterial taxonomy, it belongs to the genus Lactobacillus, subspecies rhamnosus. It is an anaerobic, acid-resistant, non-spore-forming Gram-positive probiotic. The main function of this bacterium is to regulate intestinal flora, prevent and treat diarrhea, excrete toxins and enhance the body's immunity. At present, there is no research on the application of Lactobacillus rhamnosus in fermenting kudzu.

[0040] This invention is the first to ferment kudzu root using Lactobacillus rhamnosus. The selected Lactobacillus rhamnosus is Lactobacillus rhamnosus HCS01-013, with a deposit number of CGMCC No. 19510. This strain is currently available and was deposited with the General Microbiology Center of the China Culture Collection Administration on March 25, 2020, and is disclosed in CN112608865A.

[0041] Specifically, the concentration of the Lactobacillus rhamnosus bacterial solution was 6.0×10 6 CFU / mL~1.2×10 7 CFU / mL, and the inoculation amount of the Lactobacillus rhamnosus bacterial solution in the present invention is 6wt%~12wt%.

[0042] When the above strains are used for Pueraria lobata fermentation, if the inoculation amount is too high, it may lead to (1) excessive consumption of nutrients: high concentrations of lactic acid bacteria will quickly consume nutrients in the culture medium, resulting in significant changes in the nutrient composition in a short period of time, which may affect the stability of the subsequent fermentation process and the quality of the product; (2) mycelial entanglement and reduced ventilation: high concentrations of bacteria may cause mycelia to entangle together, which not only increases the mutual shielding between bacteria, but also reduces the ventilation in the fermentation liquid, thereby affecting the fermentation efficiency; (3) product inhibition and changes in metabolic pathways: in some cases, high concentrations of lactic acid bacteria will produce a large amount of metabolites, which may inhibit the growth of lactic acid bacteria themselves and the synthesis of products; in addition, high concentrations may also cause changes in the metabolic pathways of lactic acid bacteria, affecting the type and yield of the final product.

[0043] Too low inoculation amount may lead to: (1) prolonged fermentation time: low concentration of lactic acid bacteria needs longer time to reach sufficient bacterial concentration during fermentation, thus prolonging the fermentation period. This not only increases production cost, but also may reduce fermentation efficiency; (2) insufficient utilization of effective components: too low bacterial concentration means that effective components in the culture medium cannot be fully utilized, leading to waste of resources and reduction of fermentation product yield; (3) easy to be contaminated: low concentration of lactic acid bacteria may not be able to effectively compete for nutrients and space in the culture medium, thus being more susceptible to contamination by miscellaneous bacteria. The presence of miscellaneous bacteria further affects the stability of the fermentation process and the quality of the product.

[0044] After inoculating the Lactobacillus rhamnosus bacterial solution, the fermented base of pueraria is fermented, and the fermentation conditions are: micro-aerobic fermentation, temperature 32℃~37℃, fermentation time 24 h~60 h.

[0045] By the above preparation method, a pueraria fermented product can be obtained. By comparing the contents of main pharmacodynamic and nutritional components before and after fermentation, it is found that the fermentation of pueraria by the above steps can improve the flavor and taste of pueraria, improve the bioavailability of pueraria, and more effectively improve the symptoms of type 2 diabetes, such as reducing body weight, reducing food intake and water intake, inhibiting blood glucose rise, reducing insulin resistance and chronic inflammation, and improving liver and kidney protection. In practical application, the pueraria fermented product obtained by the present application can be used for preparing pueraria products, which helps to expand the development of pueraria resources and improve the economic value of pueraria.

[0046] The features and performance of the present application are further described in detail below in conjunction with examples.

[0047] Example 1

[0048] This example is a method for microbial fermentation of pueraria, and the specific steps are as follows:

[0049] (1) Select pueraria decoction pieces without spots and insect damage, crush through an 80-mesh sieve, and accurately weigh pueraria powder and water mixed at a ratio of 1:25 (g:mL);

[0050] (2) After the raw materials are fully water-absorbed, sterilize at 120℃~122℃ for 20 min~30 min until the pueraria fermentation base is cooled;

[0051] (3) Take the bacterial solution of Lactobacillus rhamnosus HCS01-013 (concentration 1.0×10 7 CFU / mL) with a total inoculation amount of 10% (mass fraction) to inoculate the pueraria fermentation base, mix uniformly, ferment, and the fermentation conditions are: micro-aerobic fermentation, temperature 37℃, fermentation time 36 h, to obtain a liquid microbial fermented product of pueraria;

[0052] (4) Take out the fermentation broth 8000 r / min centrifugal 2 times, each time 10 min, take the supernatant 4℃ refrigeration preservation for testing.

[0053] Take the above step of the powder of the fermented product, determine its total flavonoids, major isoflavones, soluble polysaccharide, reducing sugar, total phenol content, test method and results as follows:

[0054] 1.1 Total flavonoids content determination before and after the fermentation of the powder

[0055] 1.1.1 Preparation of standard solution and drawing of standard curve

[0056] 1 mg of the reference substance of puerarin was precisely weighed and placed in a 10 mL volumetric flask, and dissolved with 30 % ethanol solution to constant volume. With this as the mother liquor, 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 were precisely pipetted therefrom, and diluted to 10 mL with 30 % ethanol, to prepare a series of reference substance solutions with gradient concentrations. The above reference substance solutions were measured for absorbance values at 250 nm with 30 % ethanol as the reference solution, to prepare a standard curve. The vertical coordinate was the absorbance value, and the horizontal coordinate was the concentration of the reference substance of puerarin (μg / mL), to draw the standard curve. The regression equation was Y = 91.571X + 0.01, and R² = 0.9997. The results showed that the content of puerarin had a good linear relationship within 3-9 μg / mL.

[0057] 1.1.2 Determination of samples

[0058] 0.1 mL of the fermentation broth was taken, diluted to 5 mL with 30 % ethanol, and 1 mL was taken and diluted to 5 mL with 30 % ethanol, and shaken well, and the content was determined by ultraviolet. The absorbance value was measured at 250 nm wavelength with 30 % ethanol solution as the reference solution, and the content of total flavonoids was calculated.

[0059] 1.2 Determination of isoflavone content before and after the fermentation of the powder

[0060] 1.2.1 Chromatographic conditions

[0061] 5C18-MS-II C18 column (250 mm x 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 1.0 mL / min; column temperature 30℃; injection volume 10 μL; detection wavelength 250 nm.

[0062] 1.2.2 Preparation of standard solution and standard curve

[0063] An appropriate amount of 3'-hydroxymorin, morin, morin apioside, daidzin, genistin, daidzein, genistein reference substance was weighed, accurately measured, and dissolved in methanol to make a stock solution of 10 mL. An appropriate amount of 3'-hydroxymorin, morin, morin apioside, daidzin, genistin, daidzein, and genistein stock solution was accurately pipetted into a 10 mL volumetric flask, and 50% methanol was added to the mark to obtain a reference solution with a mass concentration of 15.15, 251.50, 25.00, 40.00, 25.00, 15.06, and 25.05 μg / mL, respectively. 5 mL of the reference stock solution was accurately transferred, and 50% ethanol solution was added to dilute it by 2 times. The solution was filtered through a 0.22 μm microporous filter, and the filtrate was injected for chromatographic determination. The linear regression equation of each component was obtained with the concentration as the abscissa and the peak area as the ordinate, as shown in Table 1.

[0064] Table 1 Linear investigation results of each component

[0065]

[0066] 1.2.3 Preparation of test sample solution

[0067] 1 mL of the fermented powder of kudzu was accurately measured in a 10 mL volumetric flask, and 50% methanol was added to the mark. The solution was filtered through a 0.22 μm microporous filter to obtain the test sample solution.

[0068] 1.3 Determination of soluble polysaccharide content before and after fermentation of powder of kudzu

[0069] 1.3.1 Preparation of standard solution and standard curve

[0070] According to the phenol-sulfuric acid method, 50 mg of anhydrous glucose was accurately weighed in a 50 mL capacity, and distilled water was used to make up the volume to obtain a 1 mg / mL glucose solution. 10 g of phenol was added to 190 mL of water to obtain a 5% phenol solution in a brown bottle. 10 mL of the prepared glucose solution was diluted, and the volume was made up to 50 mL to obtain a 0.2 mg / mL glucose standard solution. 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, and 1.3 mL of the glucose standard solution were taken in 20 mL stoppered test tubes, respectively, and distilled water was added to each test tube to make the volume of the solution 2 mL. Then, 1 mL of 5% phenol solution was added to each test tube, and the mixture was mixed thoroughly. After 5 minutes, the mixture was heated in a boiling water bath for 15 minutes, then cooled to room temperature. The absorbance of the solution was measured at 490 nm, and the glucose concentration was taken as the horizontal coordinate and the absorbance as the vertical coordinate to draw a standard curve. The regression equation was Y = 14.98x + 0.0179, R² = 0.9996, and the results showed that the glucose content had a good linear relationship in the range of 0-0.13 mg.

[0071] 1.3.2 Determination of Test Solution

[0072] 2 mL of the diluted powder kudzu fermentation broth was taken in a test tube, and 2 mL of deionized water was used as a blank control. The rest of the operations were the same as above. The soluble polysaccharide content in the diluted fermentation broth was calculated according to the regression equation of the glucose solution curve.

[0073] 1.4 Determination of Reducing Sugar Content Before and After Fermentation of Powder Kudzu

[0074] 1.4.1 Preparation of Standard Solution and Drawing of Standard Curve

[0075] A 10 mg anhydrous glucose standard was accurately weighed in a 10 mL volumetric flask, and distilled water was used to prepare a 1 mg / mL anhydrous glucose standard. 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution were taken in stoppered tubes, respectively, and distilled water was added to 1 mL. Then, 2 mL of DNS reagent was added, and the mixture was mixed thoroughly. After 5 minutes in a boiling water bath, the mixture was cooled to room temperature, and 9.0 mL of distilled water was added. The absorbance was measured at 540 nm. The glucose concentration was taken as the horizontal coordinate, and the absorbance was taken as the vertical coordinate to draw a standard curve. The linear equation was Y = 0.9589x - 0.0168, R² = 0.9984.

[0076] 1.4.2 Determination of Test Solution

[0077] Take 1.0 mL of the diluted 1.0 mL of the Pueraria lobata fermented liquid and the blank solution in a test tube with a plug, and determine according to the above steps. Take the blank control tube as the reference, and determine the absorbance at a wavelength of 540 nm to calculate the reducing sugar content.

[0078] 1.5 Total phenol content determination before and after Pueraria lobata fermentation

[0079] 1.5.1 Preparation of standard solution and drawing of standard curve

[0080] Precisely take 24.90 mg of gallic acid reference substance and place it in a 25 mL volumetric flask. Dilute to the calibration mark with ultrapure water. Prepare the stock solution, and then precisely take 0.50 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, and 2 mL of the solution from the stock solution, respectively, and dilute to 5 mL with ultrapure water to prepare a series of concentration gradient reference substance solutions. Take the above reference substance solutions, take ultrapure water as the reference solution, mix 20 μL of the reference substance and 100 μL of 0.2 mol Folin phenol reagent in a 96-well enzyme-labeled plate, and stand for 5 min of light-avoiding reaction. Then add 80 μL of 10% Na2CO3, and after 30 min, determine the absorbance at a wavelength of 765 nm by full-wavelength scanning to draw a standard curve, with the vertical coordinate being the absorbance value and the horizontal coordinate being the gallic acid reference substance concentration (mg / mL). The regression equation Y=91.571X+0.01, R 2 =0.9997. The results show that the total phenol content has a good linear relationship in the concentration range of 0.0153~0.1071 mg / mL.

[0081] 1.5.2 Determination of test sample solution

[0082] Take 20 μL of the sample diluted by a certain multiple and 100 μL of 0.2 mol / L Folin phenol reagent, mix them in a 96-well enzyme-labeled plate, and stand for 5 min of light-avoiding reaction. Add 80 μL of 10% Na2CO3 in the 96-well enzyme-labeled plate, and after 30 min, determine the absorbance at a wavelength of 765 nm, and the result is expressed as mg GAE / mL of sample.

[0083] The detection results of the changes in the content of the index components before and after Pueraria lobata fermentation are shown in Table 2:

[0084] Table 2 Changes in the content of index components before and after Pueraria lobata fermentation

[0085]

[0086] Note: * P<0.05, ** P<0.01, *** P<0.001

[0087] According to the results of Table 2, it can be concluded that the total flavonoid content in fermented Pueraria lobata is increased to 36.54 mg / g, which is 1.109 times that of Pueraria lobata before fermentation, and the difference is significant (P<0.05).

[0088] The main isoflavone components in Pueraria lobata are 3-OH puerarin, puerarin, puerarin apioside, daidzin, genistin, daidzein and genistein. Compared with Pueraria lobata before fermentation, the contents of 3-OH puerarin, daidzein and genistein in fermented Pueraria lobata are increased, and the contents of daidzin and genistin are decreased. Therefore, it can be proved that the fermentation method of the present application can convert the glycoside components in Pueraria lobata into aglycone substances.

[0089] Compared with before fermentation, the soluble polysaccharide content in fermented Pueraria lobata is increased to 12.01 mg / mL, which is 1.093 times that of Pueraria lobata before fermentation, and the difference is significant (P<0.001). It may be because lactobacillus is a kind of probiotics with strong carbohydrate metabolism, which metabolizes free glucose or small molecule glucose polymers with starch as carbon source in the fermentation process, thereby increasing the content of soluble polysaccharide.

[0090] Compared with before fermentation, the total phenol content in fermented Pueraria lobata is increased, which may be because the microorganisms in the fermentation process deglycosylate the glycosylated phenolic compounds, thereby releasing the soluble and insoluble bound phenolic compounds from the plant cell wall, and increasing the total phenol content.

[0091] Example 2

[0092] This example is a verification of the effect of the fermented Pueraria lobata obtained in Example 1 on type 2 diabetes of C57BL / KsJ-db / db mice, which is as follows:

[0093] 1. Experimental animals

[0094] In this study, 60 male C57BL / KsJ-db / db mice (fasting blood glucose ≥11.1 mmol·L -1 ) of 8 weeks old and 10 db / m mice of SPF level were purchased from Changzhou Kaivens Co., Ltd. (License No.: SCXK (Su) 2021-0013). Before the formal experiment, the mice in each group were adaptively fed for 3 days to ensure stable experimental conditions. This experiment has obtained the approval of the Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine (Approval No.: JZLLSC20240554).

[0095] 2. Experimental method

[0096] 2.1 Preparation of fermented Pueraria lobata and Pueraria lobata solution

[0097] The fermented pueraria was prepared according to the embodiment 1, the fermented liquid was concentrated by rotary evaporation to a certain concentration, then the fermented liquid was poured out, pre-frozen for 12 hours at-20℃, and then the fermented product was freeze-dried. The yield was calculated as 56.95% for the fermented product and 54.28% for the unfermented product. When used, the fermented pueraria and the pueraria were dissolved in ultrapure water respectively, and stored in a 4℃ refrigerator.

[0098] 2.2 Experimental animal grouping and administration

[0099] The experimental groups and the corresponding treatment methods are shown in Table 1. Figure 1 During the feeding of the experimental animals, the day and night were alternated for 12 hours, the temperature was maintained at 25±1℃, the humidity was maintained at 55%-65%, and the air flow was maintained. After adaptive feeding for 3 days, the fasting blood glucose≥11.1mmol / L was detected as the T2DM modeling condition. The db / m mice were set as the normal group (N), and the db / db mice were randomly divided into 6 groups: model group (M), positive group (MET, 0.36 g / kg), low-dose pueraria group (PL, 1.23 g / kg), high-dose pueraria group (PH, 2.47 g / kg), low-dose fermented pueraria group (FPL, 1.30 g / kg), and high-dose fermented pueraria group (FPH, 2.59 g / kg). Each administration group was continuously administered by gavage for 5 weeks, and the normal group and the model group were administered with normal saline (10 mL / kg) by gavage.

[0100] 2.3 Detection of phenotypic indicators

[0101] On the first day of administration, the body weight of each group of mice was measured, and then the mice were administered by gavage according to the body weight. The body weight of the mice was measured regularly every week and recorded. From the next day of formal administration, the food intake and water intake of the mice were weighed and recorded at the same time every week. The fasting blood glucose was determined after the mice were fasted for 12 hours without water at the same time every week. The detection method was to prick the tail tip vein of the mice with a matched blood sampling needle, and slightly squeeze out a small drop of blood for absorption by blood sampling paper. The blood glucose value was displayed by a blood glucose meter.

[0102] 2.4 Sample collection

[0103] 2.4.1 Collection of serum samples

[0104] After the experimental animals were treated by 12-hour fasting without water, the next day, the animals were sacrificed and dissected. Before sacrifice, the fasting blood glucose value was determined by a micro blood sampling instrument, and the whole blood sample was collected by orbital venous plexus puncture. The blood sample was collected in a 1.5 mL centrifuge tube, and the supernatant was obtained after standing at room temperature for 2 hours, centrifugation at 3500 rpm for 15 minutes at 4℃. The obtained serum sample was quickly frozen in liquid nitrogen and then transferred to a-80℃ ultra-low temperature freezer for long-term storage.

[0105] 2.4.2 Collection of tissue samples

[0106] 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 left lobe of 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, frozen in liquid nitrogen and then transferred to a -80°C ultra-low temperature freezer for long-term storage.

[0107] 2.5 Determination of biochemical indicators and organ indices

[0108] 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 alpha (TNF-α) and interleukin-1 beta (IL-1β) in serum were detected. All operations followed the requirements of the kit operation specification. Liver index, pancreas index, kidney index and insulin resistance index (HOMA-IR) were calculated according to the formula: fasting insulin value x fasting blood glucose value / 22.5.

[0109] 2.6 Histopathology

[0110] Liver tissue and pancreas tissue were fixed in tissue fixative. After dehydration, paraffin embedding and sectioning were performed, with a section thickness of 4-5 mm. HE staining was performed, and electron microscopy scanning was performed to take photographs. Sample collection and analysis were performed using a Lecia Applaction Stiue image system, and histopathological changes were observed.

[0111] 3 Experimental results

[0112] 3.1 Analysis of phenotypic indicators

[0113] The results are shown in Figure 2 , where Figure 2 A represents the effect of fermented pueraria and pueraria on the body weight of db / db mice; Figure 2 B represents the effect of fermented pueraria and pueraria on the food intake of db / db mice; Figure 2 C represents the effect of fermented pueraria and pueraria on the water intake of db / db mice; Figure 2 D represents the effect of fermented pueraria and pueraria on the blood glucose of db / db mice; (*P<0.05; **P<0.01 compared with the model group).

[0114] As shown in Figure 2 A, the model group of diabetic mice showed symptoms of weight loss in the later stage, and the administration of MET, PL, PH and FPH significantly reversed the weight loss symptoms of diabetic mice.Figure 2 B in the figure represents the food intake of db / db mice. After drug intervention, the food intake of mice in the MET and FPH groups began to decline in the second week compared with the model group, and at the fifth week, the food intake of mice in the MET, FPL, and FPH groups was significantly lower than that in the model group ( P <0.01), the food intake of mice in the PL and PH groups was lower than that in the model group, but there was no significant difference. Figure 2 C represents the water intake of db / db mice. It can be seen that the water intake of mice in the normal group remained stable, while that of the model group was consistently the highest, consistent with the typical symptom of type 2 diabetes, polydipsia. MET, FPL, and FPH intervention significantly reduced water intake in mice, thereby alleviating symptoms. P <0.05).

[0115] like Figure 2 As shown in Figure D, fermented kudzu root and kudzu root had a certain effect on the fasting blood glucose levels of db / db mice. The experimental results showed that throughout the entire administration period, the blood glucose levels of the model group mice were always higher than those of the normal group, and as the feeding time prolonged, the blood glucose levels of the model group mice showed a continuous upward trend. By the 5th week, the blood glucose levels of the MET group mice were significantly lower than those of the model group ( P <0.01), and the blood glucose levels of mice in the PH group were significantly decreased ( P <0.01), and the blood glucose level of mice in the FPL group was slightly lower than that in the model group ( P <0.05), the blood glucose level of mice in the FPH group decreased most significantly ( P <0.01), while mice in the PL group showed a certain downward trend in blood glucose, but the difference was not statistically significant. These results suggest that Pueraria lobata fermentation products may inhibit the increase in blood glucose to some extent.

[0116] According to the above results, it can be seen that compared with kudzu vine and kudzu vine fermentation products, kudzu vine fermentation products have a greater impact on various phenotypes.

[0117] 3.2 Analysis of biochemical indicators

[0118] The results of biochemical indicators, inflammatory factors and organ indexes are shown in Figure 3, wherein A represents the detection result of INS, B represents the result of HOMA-IR, C represents the detection result of ALT, D represents the detection result of AST, E represents the detection result of ALP, F represents the detection result of TC, G represents the detection result of TG, H represents the detection result of LDL-C, I represents the detection result of TNF-α, J represents the detection result of IL-1B, 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; blood lipid indicators such as ALT, AST, ALP, TC, TG and LDL-C participate in the process of gluconeogenesis, and pro-inflammatory factors such as TNF-α and IL-1β interfere with insulin signals, leading to chronic inflammation, resulting in dysfunction, apoptosis and necrosis of beta cells, thereby affecting the secretion of insulin.

[0119] After the intervention treatment of fermented Pueraria and Pueraria, the indicators 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 different doses of fermented Pueraria and Pueraria have an improvement effect on insulin resistance and chronic inflammation of diabetic mice; compared with the model group, the insulin resistance index groups MET and FPH are significantly reduced P <0.01 or P <0.05), other administration groups showed a downward trend compared with the model group, and there was no significant difference; compared with the model group, the liver index of the FPL group decreased P <0.01), the kidney index of each administration group showed a downward trend P <0.05, P <0.01 or P <0.01), the pancreas index showed an upward trend, indicating that different doses of fermented Pueraria and Pueraria can improve the atrophy of the pancreas of type 2 diabetic mice and have a certain protective effect on the liver and kidney. From this, it can also be seen that fermented Pueraria has a better improvement effect on diabetic mice and a more obvious protective effect on the liver and kidney.

[0120] 3.3 Histopathological analysis of liver and pancreas

[0121] HE staining was used to determine the effect of fermented Pueraria and Pueraria administration on liver histological changes Figure 4 ), Figure 4 A in the above table is the HE staining result of liver tissue under × 20, Figure 4B is the HE staining result of liver tissue under x40. The overall structure of liver tissue of normal group mice is basically normal, the arrangement of tissue hepatocytes is regular, the structure of tissue hepatocytes is round and full, no obvious loose edema, fatty degeneration and necrosis of hepatocytes, the tissue hepatic sinusoids are arranged radially along the central vein, no obvious congestion and expansion of tissue hepatic sinusoids, and no obvious inflammatory cell infiltration. The arrangement of hepatocytes of the model group is irregular, the structure is loose, and a large number of hepatocytes are obviously edematous, part of which is edematous to vacuolar degeneration, the structure of tissue hepatic sinusoids is unclear, and a large number of inflammatory cells can be seen in the tissue. After metformin, different doses of fermented pueraria and pueraria intervention treatment, no obvious congestion and expansion of tissue hepatic sinusoids, reduction of hepatocyte vacuolar fat droplets, improvement of cell nucleus pyknosis and apoptosis to different degrees, no obvious inflammatory cell infiltration in the tissue, and the improvement effect of high dose group is better than that of low dose group, especially FPH group.

[0122] In addition, the effect of fermented pueraria and pueraria on pancreatic islet cells was also studied, and the staining results are shown in Figure 5 Figure 5 A is the HE staining result of pancreatic tissue under x20, Figure 5 B is the HE staining result of pancreatic tissue under x40. The overall structure of pancreatic tissue of normal group mice is normal, the arrangement of tissue islet cells is regular and clear, the number of islet cells is rich, the shape is full, the structure of acinar epithelial cells is full, the number of islet cells is rich, the shape is full, the structure of acinar epithelial cells is full, and no obvious loose edema, necrosis and other degeneration can be seen, and no obvious inflammatory cell infiltration can be seen. The arrangement of acinar of the model group mice is irregular, a large number of acinar epithelial cells are obviously necrotic, the cell nucleus is pyknotic and deeply stained, and no islet cells are basically seen. After drug treatment, the arrangement of islet cells is regular and clear, the number of islet cells is increased, the cell nucleus pyknosis and inflammatory cell infiltration are improved to a certain extent.

[0123] In summary, the fermented pueraria obtained by the fermentation method of the application has higher content of effective components than unfermented pueraria, higher biological activity, and better improvement effect on type 2 diabetes.

[0124] Experimental example 1

[0125] This experiment is a comparison of the fermentation effects of different strains, as follows:

[0126] (1) Select pueraria decoction pieces without spots and insect damage, crush through an 80 mesh sieve, and accurately weigh pueraria powder and water at a ratio of 1:25 (g:mL);

[0127] (2) After the raw materials are fully water-absorbed, sterilize at 120°C~122°C for 20min~30min until the pueraria fermentation base material cools down;

[0128] ​(3) Take six different strains, Roy's mucus lactobacillus HCS02-001 (CGMCC No. 19746), paracasein lactobacillus HCS17-040 (CGMCC No. 19747), rhamnose lactobacillus HCS01-013 (CGMCC No. 19510), fermented mucus lactobacillus HCS08-005 (CGMCC No. 16259), plant lactobacillus HCS03-001 (CGMCC No. 16258), and plant lactobacillus P9 (CGMCC No. 16662) with a bacterial liquid concentration of 8.0 x 10 6 CFU / mL, inoculate into the powder ginger fermentation base at a total inoculation amount of 8% (by mass fraction), mix uniformly, ferment, and ferment under the following conditions: micro-aerobic fermentation, temperature 37°C, fermentation time 60 h, to obtain different strain powder ginger fermented liquid microbial fermentation products;

[0129] (4) Centrifuge the fermentation liquid at 8000 r / min for 2 times, 10 min each time, and store the supernatant at 4°C for preservation before testing.

[0130] Take the powder ginger fermentation product prepared in the above step, determine the viable count, pH, total acid, total flavonoids, and main isoflavone content, perform principal component analysis (PCA) on the above indexes, and according to the related characteristics of the basic physicochemical indexes and functional activity indexes of different lactobacillus fermentation, and comprehensively consider the growth of the bacteria and the conversion degree of the functional factors, finally optimize the fermentation dominant strain, and the test method and results are as follows:

[0131] 1 Lactobacillus viable count

[0132] The total viable count (TVC) in the sample liquid was detected by the dilution plate counting method. The sample liquid was gradiently diluted in a clean bench. 1 mL of 10 -5 , 10 -6 , and 10 -7 sample dilution liquid was taken, and the medium was poured. The plate medium was inverted and placed in a constant temperature and humidity box, and cultured at 37°C for 48 h, and counted.

[0133] 2 pH determination

[0134] Reference national standard GB 5009.237-2016 "Determination of pH value of food".

[0135] 3 Total acid (TA) content determination

[0136] Reference national standard GB 12456-2021 "Determination of total acid in food".

[0137] Diluted NaOH was added to the burette, rinsed once to remove bubbles, and then diluted to the 0 mark. 2-4 drops of phenolphthalein were added to the diluted sample. The 0.01 mol·L -1 NaOH was titrated to a faint pink color for 30 s without fading, and the volume of NaOH consumed V1 was recorded (read to one decimal place). The above steps were repeated three times. A blank control was performed using the same volume of CO2-free water instead of the sample solution, and the volume of NaOH consumed V2 was recorded.

[0138] X = ([C x (V1-V2)] x K x N) / M x 1000 (g / Kg or g / L)

[0139] C: concentration of the NaOH standard titration solution;

[0140] K: lactic acid is equal to 0.090;

[0141] N: dilution multiple of the sample solution;

[0142] M: mass (g) or volume (mL) of the sample;

[0143] V1, V2: volume of NaOH consumed by the sample solution and the blank control;

[0144] 4 Determination of total flavonoid content in powder kudzu before and after fermentation

[0145] 4.1 Preparation of the standard solution and drawing of the standard curve

[0146] 1 mg of the reference substance of puerarin was accurately weighed into a 10 mL volumetric flask, dissolved with 30 % ethanol solution, and diluted to the mark. This was used as the mother liquor, and 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 were accurately pipetted from the mother liquor into 10 mL volumetric flasks, respectively, and diluted to the mark with 30 % ethanol to prepare a series of reference substance solutions with different concentrations. The absorbance value was measured at 250 nm with 30 % ethanol as the reference solution to draw the standard curve. The vertical coordinate was the absorbance value, and the horizontal coordinate was the concentration of the reference substance of puerarin (μg / mL) to draw the standard curve. The regression equation was Y = 91.571X + 0.01, and R² = 0.9997. The results showed that the content of puerarin had a good linear relationship in the range of 3-9 μg / mL.

[0147] 4.2 Determination of the sample

[0148] 0.1 mL of the fermentation broth was taken, diluted to 5 mL with 30 % ethanol, and then 1 mL was taken and diluted to 5 mL with 30 % ethanol, shaken well, and subjected to ultraviolet determination of the content. The absorbance value was measured at 250 nm with 30 % ethanol solution as the reference solution, and the total flavonoid content was calculated.

[0149] 5 Determination of isoflavone content in powder of Pueraria montana var. lobata before and after fermentation

[0150] 5.1 Chromatographic conditions

[0151] 5 C18-MS-II C18 column (250 mm x 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; sample injection volume 10 μL; detection wavelength 250 nm.

[0152] 5.2 Preparation of standard solution and drawing of standard curve

[0153] An appropriate amount of 3'-hydroxy puerarin, puerarin, puerarin apioside, daidzin, genistin, daidzein, and genistein reference substances was weighed, accurately measured, and dissolved in methanol to make a mother liquor. An appropriate amount of 3'-hydroxy puerarin, puerarin, puerarin apioside, daidzin, genistin, daidzein, and genistein mother liquor was accurately pipetted into a 10 mL volumetric flask, and 50% methanol was added to the mark to obtain reference substance solutions with concentrations of 15.15, 251.50, 25.00, 40.00, 25.00, 15.06, and 25.05 μg / mL, respectively. 5 mL of the reference substance mother liquor was accurately transferred, and 50% ethanol solution was added to dilute it by 2 times. The solution was filtered through a 0.22 μm microporous filter, and the filtrate was injected for chromatographic determination. The concentration was taken as the abscissa, and the peak area was taken as the ordinate to obtain the linear regression equations of each component, as shown in Table 1.

[0154] 5.3 Preparation of test sample solution

[0155] 1 mL of powder of Pueraria montana var. lobata fermentation liquor was accurately measured in a 10 mL volumetric flask, and 50% methanol was added to the mark. The solution was filtered through a 0.22 μm microporous filter to obtain the test sample solution.

[0156] The detection results were as follows:

[0157] Table 3 Characteristics of powder of Pueraria montana var. lobata fermented by different lactobacilli (X ± S)

[0158]

[0159] Note: Duncan's multiple comparison was used. The same column marked with different capital letters means that there is a significant difference between groups (P < 0.01); marked with different lowercase letters means that there is a significant difference between groups (P < 0.05); marked with the same lowercase letter means that there is no significant difference between groups (P > 0.05).

[0160] As can be seen from Table 3, the viable cell count of the pueraria after 60 h of fermentation reached 8.0 lg (CFU·mL -1 , among which the viable cell count of the pueraria fermented by lactobacillus rhamnosus reached 8.50 lg (CFU·mL-1). The pH value of the pueraria fermentation base was 6.36, and after fermentation by lactobacillus, some acid substances increased, and the pH value of each pueraria fermentation group decreased. The total acid content also increased due to the action of microorganisms, and the total acid content of lactobacillus rhamnosus was 1.03 g·L -1 , which was 2.5 times the content of the pueraria fermentation group. Lactobacillus improved the total flavonoid content during fermentation, and the initial total flavonoid content of pueraria was 20.29 mg·g -1 , and after 60 h of fermentation, the total flavonoid content of the lactobacillus rhamnosus fermentation group was 22.07 mg·g -1 , which was about 8.8% higher than before fermentation. The total flavonoid content of other groups also increased. The flavonoids in pueraria mainly included 3'-hydroxy puerarin, puerarin, daidzein, daidzin, genistein and genistin. It was found through determination that the content of puerarin in the pueraria fermentation liquid was the highest, reaching 8.10 mg·g -1 , while that in the pueraria base before fermentation was 7.9 mg·g -1 , and the content of puerarin increased after fermentation. There was no significant difference between the lactobacillus fermentation group and the pueraria base group, and the content of puerarin in lactobacillus reuteri was the lowest, reaching 7.88 mg·g -1 . The content of daidzin in the pueraria fermentation liquid was also lower than that in the pueraria base. Compared with the control group, the content of daidzein and genistein in the pueraria fermentation liquid of many groups increased significantly after fermentation, among which the content of daidzein and genistein in the lactobacillus rhamnosus fermentation group was 2.93 mg·g -1 and 0.13 mg·g -1 , respectively.

[0161] Table 4 Comprehensive score and ranking of pueraria root fermentation enzyme fermented by different strains

[0162]

[0163] According to the principal component score (Y1-Y3), the variance contribution rate corresponding to each principal component is taken as the weight, and a comprehensive evaluation model is constructed: Y=0.56Y1+0.28Y2+0.16Y3. According to the model, the comprehensive total score of each group is calculated, see table 4. The first is lactobacillus rhamnosus, and the comprehensive quality of lactobacillus rhamnosus fermented with powder of Gertu is better.

[0164] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for microbial fermentation of kudzu vine, characterized in that: include: Adding Lactobacillus rhamnosus culture liquid to the kudzu fermentation base, mixing and fermenting to obtain a liquid microbial fermentation product; The Lactobacillus rhamnosus is Lactobacillus rhamnosus HCS01-013; Accession number: CGMCC No.19510, deposited in the General Microbiology Center of China Culture Collection Administration on March 25, 2020; The preparation method of the kudzu vine fermentation base comprises: mixing pulverized and sifted kudzu vine powder with water, sterilizing the kudzu vine powder at high temperature after fully absorbing water, and cooling the kudzu vine powder to obtain the kudzu vine fermentation base.

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

3. 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.

4. The method according to claim 1, wherein The kudzu powder is sieved through a mesh number of 80-100 mesh; the ratio of the kudzu powder to water is 1:20 g / mL to 1:30 g / mL.

5. The method according to claim 4, characterized in that The high temperature sterilization condition is 120° C. to 122° C. for 20 min to 30 min.

6. 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.

7. The kudzu vine fermentation product prepared by the method according to any one of claims 1 to 6.

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