A method for improving the bioavailability of daidzein prepared by transforming puerarin from kudzu root with Lactobacillus plantarum

Abstract

The invention discloses a method for improving the bioavailability of preparing daidzein by improving the conversion of puerarin in pueraria by lactobacillus plantarum, and belongs to the technical field of fermentation engineering. The fermentation process provided by the invention improves the activity of the bacterial cell by optimizing the selection and dosage of carbon and nitrogen sources during the fermentation process, the number of viable bacteria in the fermentation liquid is maintained at a high level in the whole fermentation process, and the yield of daidzein is greatly improved, reaching 603.27ppm, which is 44.72 times before optimization. The plant fermentation postbiotics prepared in this way can play more effects in the application of subsequent products. At the same time, daidzein with higher activity and more yield improves the absorption and utilization rate of human intestinal cells after taking it.

Description

Technical Field

[0001] The invention relates to a method for improving the bioavailability of daidzein prepared by transforming puerarin in kudzu root with lactobacillus plantarum, and belongs to the technical field of fermentation engineering. Background Art

[0002] Puerarin is the main functional active substance of the edible and medicinal plant Pueraria root, accounting for 70% of all Pueraria root isoflavones. It not only has therapeutic effects on diseases such as diabetes and cardiovascular diseases, but is also often used to treat liver damage caused by alcoholism because of its alcohol sobering and liver protection effects. However, due to its existence in the form of bound glycosides, puerarin cannot enter intestinal cells by free diffusion, thereby reducing its medicinal value of Pueraria root. As a free aglycone, daidzein is more easily absorbed by intestinal cells through the LPH transporter and SGLT1 transporter of the epithelial brush border. After absorption by the small intestine, the isoflavone compounds enter the blood and reach the liver through the portal vein for phase II metabolism, and the metabolites then enter the systemic circulation. In patent CN118086089 A, a strain of Lactobacillus plantarum CCFM1283 has been screened out, which can ferment puerarin to obtain high-concentration daidzein. However, the number of viable bacteria of Lactobacillus plantarum CCFM1283 in the fermentation broth synergistically fermented with puerarin is only 2×10 9 CFU / mL, which limits the degradation effect of the strain on puerarin. The yield of daidzein obviously still has a lot of room for improvement. Summary of the invention

[0003] The invention aims to improve the conversion rate of puerarin by plant lactobacillus CCFM1283, prepare high-concentration soybean aglycone fermentation postbiotics and improve the utilization rate of puerarin in human body.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] First, the composition of the fermentation medium of Lactobacillus plantarum CCFM1283 was optimized so that the number of viable bacteria in the fermentation liquid in the early stage of fermentation reached 7.12*10 9 CFU / mL, enhances the number of Lactobacillus plantarum CCFM1283 in unit volume of fermentation broth.

[0006] Second, use 700 mesh and above kudzu root powder for high temperature and high pressure extraction to obtain a saturated solution of puerarin content and increase the amount of conversion substrate.

[0007] Third, in the late fermentation stage, that is, after Lactobacillus plantarum CCFM1283 reaches the stable period, low-temperature slow fermentation is carried out to ensure the number of bacteria, delay the arrival of the bacteria's death period, increase its conversion time to puerarin, and ensure the high conversion of puerarin to daidzein.

[0008] The invention provides a fermentation medium for efficiently producing daidzein. The medium contains yeast powder, D-galactose, manganese sulfate monohydrate, K2HPO4, anhydrous sodium acetate, diammonium hydrogen citrate, MgSO4·7H2O, Tween 80, L-cysteine ​​hydrochloride and kudzu root powder.

[0009] In one embodiment, the culture medium comprises: yeast powder 20-30 g / L, D-galactose 35-40 g / L, manganese sulfate monohydrate 0.9-1.5 g / L, K2HPO4 2-4 g / L, anhydrous sodium acetate 2-4 g / L, diammonium hydrogen citrate 2-4 g / L, MgSO4·7H2O0.2-0.5 g / L, Tween 80 1-2 mL / L, L-cysteine ​​hydrochloride 1-2 g / L and kudzu root powder 5-6 g / L.

[0010] In one embodiment, the culture medium is suitable for Lactobacillus plantarum CCFM1283.

[0011] In one embodiment, the kudzu root powder has a mesh size of 700-1000.

[0012] The invention provides a method for producing daidzein. The method comprises the steps of inoculating plant lactobacillus CCFM1283 into the fermentation medium for fermentation.

[0013] In one embodiment, the fermentation is for at least 48 hours.

[0014] In one embodiment, the fermentation is a two-stage fermentation, wherein the first stage is at a temperature of 37-37.2°C for 12-13 hours; and the second stage is at a temperature of 21-22°C for at least 48 hours.

[0015] In one embodiment, during the fermentation process, 10 mol / L NaOH is used for titration to neutralize the acidic substances in the fermentation broth and control the pH of the fermentation broth to 5-6.

[0016] In one embodiment, the specific operating steps of the present invention are as follows:

[0017] (1) Activation of bacterial strains.

[0018] The plant lactobacillus CCFM1283 was spread on an MRS solid culture medium plate for culture to obtain a plate bacteria.

[0019] (2) Primary seed liquid culture.

[0020] The single colony on the MRS solid culture medium plate is picked up and cultured on the MRS shake flask seed culture medium to obtain the MRS shake flask seed bacteria.

[0021] (3) Secondary seed liquid culture.

[0022] The above-mentioned MRS shake flask seed bacteria were transferred to the shake flask seed culture medium for further expansion culture.

[0023] (4) Use the optimized high-density culture medium and superimpose 700-mesh Pueraria root base material for high-density culture.

[0024] First, the kudzu root base material is put into pure water for high temperature and high pressure extraction, and then the kudzu root fermentation raw materials are put into the fermentation medium for sterilization. The seed liquid is then transferred to the fermentation medium containing the kudzu root base material for cultivation to obtain the final product.

[0025] (5) Determination of the daidzein content of post-fermentation biomass.

[0026] Furthermore, in step (1), the fermentation medium is an MRS solid medium, comprising 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L ammonium citrate, 0.1 g / L magnesium sulfate, 1 mL / L Tween 80, 14 g / L agar powder, and the pH is adjusted to 6.5.

[0027] Furthermore, in steps (2) and (3), the MRS medium contains 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L ammonium citrate, 0.1 g / L magnesium sulfate, and 1 mL / L Tween 80, and the pH is adjusted to 6.5.

[0028] Furthermore, the components ( / L) of the Pueraria lobata base medium used for the expanded culture in step (4) are yeast powder (20-21 g), D-galactose (35-37 g), manganese sulfate monohydrate (0.9-1.1 g), K2HPO4 (2-5 g), anhydrous sodium acetate (2-5 g), diammonium hydrogen citrate (2-5 g), MgSO4·7H2O (2--5 g), Tween 80 (1-2 mL), L-cysteine ​​hydrochloride (1 g), and 700 mesh Pueraria lobata powder (5-6 g).

[0029] Furthermore, in step (4), 5-10 mol / L NaOH is used for titration to neutralize the acidic substances in the fermentation broth, and the pH is controlled during the entire fermentation process or not.

[0030] Furthermore, the sterilization conditions of the culture medium in all steps are 121-122° C., 0.10-0.11 MPa, 20-22 min.

[0031] Furthermore, the pre-fermentation culture conditions in step (4) are: temperature 37-37.2°C, time 12-13h.

[0032] Furthermore, the culture conditions in the late fermentation stage in step (4) are: temperature 21-22° C., time 13-48 h.

[0033] Furthermore, in step (4), the inoculation amount of the seed solution is 4-6% v / v, the temperature is 36.5-37.5° C., and the rotation speed is 50 rpm.

[0034] The invention also provides a method for culturing Lactobacillus plantarum CCFM1283 at a high density, wherein Lactobacillus plantarum CCFM1283 is inoculated into the culture medium for fermentation.

[0035] The invention also provides application of the culture medium in improving the conversion rate of puerarin by Lactobacillus plantarum CCFM1283.

[0036] The invention also provides application of the culture medium in improving the efficiency of producing daidzein by Lactobacillus plantarum CCFM1283.

[0037] The invention also provides application of the culture medium in improving the fermentation density of Lactobacillus plantarum CCFM1283.

[0038] Beneficial effects:

[0039] The fermentation process of the present invention improves the cell viability of Lactobacillus plantarum CCFM1283 by optimizing the selection and dosage of carbon and nitrogen sources, regulating the pH value in the fermentation process, and adjusting the temperature at different stages of the fermentation. The number of live bacteria in the fermentation liquid is ensured to be relatively high in the whole fermentation process, and the number of live bacteria in the fermentation liquid is always greater than 3.7*10 9 CFU / mL. The number of viable bacteria in the fermentation liquid of Lactobacillus plantarum CCFM1283 after fermentation at 37℃ for 48h in MRS medium containing kudzu root powder was 1.9*10 8 CFU / mL. Through optimization, the strain activity and conversion capacity of Lactobacillus plantarum CCFM1283 in the fermentation broth were guaranteed during the strong conversion period of puerarin to daidzein. Among them, the number of viable bacteria in the optimized fermentation broth at 48h was 19.47 times that of MRS culture.

[0040] The fermentation process provided by the present invention can improve the conversion efficiency of puerarin by Lactobacillus plantarum CCFM1283, so that the content of daidzein in the fermentation liquid reaches 603.27ppm, which is increased by 44.72 times, and can reach the effective amount for human consumption after being concentrated at a certain ratio. Compared with the MRS culture medium containing pueraria powder, the content of daidzein after fermentation increases by 8.755 times.

[0041] The soybean aglycone obtained by the fermentation process of the present invention comes from the food with medicinal and edible properties fermented by edible fungi, and while ensuring its edibility, the soybean aglycone content after fermentation is maximized. The soybean aglycone with higher activity and higher yield increases the absorption and utilization rate of human intestinal cells after taking it, so the plant fermentation postbiotics prepared by the present invention can play more functions in the application of subsequent products. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This study was conducted to explore the effects of different carbon sources on the number of viable bacteria and fermentation density in fermentation broth using carbon source screening culture medium.

[0043] Figure 2 This study was conducted to screen culture medium using nitrogen sources and explore the effects of different nitrogen sources on the number of viable bacteria and fermentation density in the fermentation broth.

[0044] Figure 3 The effect of different carbon-nitrogen ratios on the number of viable bacteria and fermentation density in the fermentation broth.

[0045] Figure 4 The effect of different total carbon and nitrogen contents on the number of viable bacteria and fermentation density in the fermentation broth.

[0046] Figure 5 To regulate the effect of temperature in the later stage of fermentation on the content of daidzein in the fermentation liquid.

[0047] Figure 6 The effect of 37℃ at each fermentation stage on the content of daidzein in the fermentation liquid. DETAILED DESCRIPTION

[0048] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0049] The culture medium involved in the following examples is as follows:

[0050] MRS liquid culture medium: yeast extract powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, pH 6.2-6.4.

[0051] MRS solid culture medium: yeast extract powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, agar powder 1.4 g / L, pH 6.2-6.4.

[0052] MRS fermentation medium containing kudzu root powder base: On the basis of the MRS medium components, 3-5g of kudzu root powder (from Wuxi Jiangyin Tianjiang Pharmaceutical) was added per liter.

[0053] The bacterial strains involved in the following examples are as follows:

[0054] Lactobacillus plantarum CCFM1283 has been disclosed in patent CN118086089A.

[0055] The preparation of the experimental samples involved in the following examples is as follows:

[0056] Preparation of Lactobacillus plantarum CCFM1283 seed solution: Take out the glycerol storage tube of Lactobacillus plantarum CCFM1283 from the -80℃ refrigerator and insert it into MRS liquid culture medium for activation. After the seed solution is passed to the second generation, it is expanded and cultured. After 12-13 hours, the number of viable bacteria in the final fermentation liquid is 1.5*10 9 CFU / mL (or OD value>7).

[0057] Determination method of daidzein: HPLC was used to determine the daidzein content in fermented post-biomass. The HPLC conditions were set as follows: the chromatographic detection system used Waters e2695; the chromatographic column was AtlantisTM T3 (5μm, 4.6×250mm); the flow rate was set to 1.0mL / min, and the detection wavelength was fixed at 280nm, and the injection volume was set to 5μL to ensure the accuracy of the analysis results. The mobile phase consisted of 0.1% acetic acid aqueous solution (A) and 100% methanol solution (B). In order to ensure the stability and separation effect of the chromatographic analysis, the column temperature was set to 35℃, and the elution gradient was carried out according to the following procedure: from 0 to 20min, the proportion of mobile phase B increased from 30% to 70%; from 20 to 21min, the proportion of mobile phase B decreased from 70% to 30%; from 21 to 25min, the proportion of mobile phase B was maintained at 30%. Preparation of standard curve: To establish the standard curve for quantitative analysis, this study first accurately weighed about 10 mg of puerarin, daidzein and daidzein reference substances into a 100 mL volumetric flask. Add methanol solution to the scale to prepare the mother solution. Then, take an appropriate amount of the mother solution to prepare standard solutions of different concentrations. Finally, prepare the standard curve under the mobile phase conditions used, and perform curve fitting to obtain the corresponding quantitative analysis results.

[0058] Example 1 Screening the optimal carbon source to increase the number of viable bacteria per unit volume of fermentation broth

[0059] Prepared a variety of common plant lactobacillus fermentation carbon sources, prepared different carbon source screening medium. Prepared seed liquid according to 3% (v / v) inoculation in the carbon source screening medium (Table 1), 37 ℃ overnight culture, the second day to measure the OD and pH value of different carbon source medium. Wherein the carbon source is respectively: glucose, trehalose, maltose, lactose, sucrose, starch, inulin, fructose, D-cellobiguanide, L-sorbose, D-mannitol, D-galactose. The final fermentation density of the fermented liquid is expressed by the absorbance of the fermented liquid at 600nm.

[0060] Table 1 Carbon source screening medium composition and content

[0061] Serial number Material Name g / L 1 Peptone 10 2 Yeast extract powder 5 3 Beef dip powder 10 4 <![CDATA[K2HPO4]]> 2 5 Anhydrous sodium acetate 5 6 Diammonium Hydrogen Citrate 2 7 <![CDATA[MgSO4·7H2O]]> 0.1 8 <![CDATA[MnSO4·H2O]]> 0.05 9 Twain 80 1 10 L-Cysteine ​​Hydrochloride 1 11 Carbon source to be screened 20g

[0062] Since the growth curve of Lactobacillus plantarum CCFM1283 has been determined in the early stage, the fermentation process can reach a stable period at 12 hours. After 12 hours of fermentation, the OD value of the fermentation liquid at 600nm is measured and recorded. Figure 1 The results showed that when D-galactose was used as the carbon source, the fermentation density of live bacteria in the fermentation broth was the highest.

[0063] Furthermore, the glucose in the MRS medium was replaced with D-galactose, and kudzu powder was added, and the culture was carried out for 12-13 hours with a seed liquid inoculation amount of 2-3%, and the daidzein content in the fermentation product before and after the replacement of the nitrogen source was detected. The results showed that the daidzein content of the MRS fermentation medium containing kudzu powder base material was 32.5ppm, and the plant lactobacillus only increased the yield of daidzein by 2.12 times to 68.9ppm after adding the MRS medium raw material for fermentation, but the use of D-galactose instead of the carbon source in the MRS fermentation medium containing kudzu powder base material increased the yield of daidzein to 5.90 times before fermentation, reaching 191.63ppm.

[0064] Example 2 Screening the optimal nitrogen source to increase the number of viable bacteria per unit volume of fermentation broth

[0065] A variety of common fermentation nitrogen sources of plant lactobacillus were prepared to prepare screening culture media with different nitrogen sources. The prepared seed solution was inoculated into the nitrogen source screening culture medium at an inoculum amount of 3% (v / v), cultured overnight at 37°C, and the OD and pH values ​​of the culture media with different nitrogen sources were measured the next day. The nitrogen sources were: soy peptone, yeast extract, yeast water extract, beef extract, casein peptone, bovine bone peptone, etc.

[0066] Prepare the culture medium for nitrogen source screening: 20g / L anhydrous glucose, 10g / L anhydrous dipotassium hydrogen phosphate, 10g / L anhydrous disodium hydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 0.2g / L manganese sulfate monohydrate, 1g / L Tween 80, 1g / L cysteine ​​hydrochloride, and 2g / L nitrogen source to be screened. The final fermentation density of the fermentation broth is expressed by the absorbance of the fermentation broth at 600nm. The specific results are shown in the attached figure. Figure 2 , the optimal nitrogen source was determined to be yeast extract powder, and the fermentation density was significantly higher than the nitrogen source used in MRS. The nitrogen source in the MRS medium containing kudzu root powder was completely replaced with yeast extract powder, and the culture was carried out for 12-13 hours with a seed liquid inoculation amount of 2-3%. The daidzein content in the fermentation product before and after the replacement of the nitrogen source was detected. The results showed that the daidzein yield increased to 142.2ppm, 4.55 times the 31.2ppm before fermentation, which was also significantly higher than the daidzein yield of fermentation using MRS raw materials.

[0067] Example 3: Optimizing the total amount of carbon and nitrogen sources and the carbon-nitrogen ratio to increase the yield of daidzein

[0068] In the fermentation base containing 3-5g / L kudzu root powder, 5% (v / v) seed liquid of Lactobacillus plantarum CCFM1283 was added, and the mixture was fermented at a constant temperature of 37°C for 12 hours. According to the optimal carbon source and the optimal nitrogen source selected in Example 1 and Example 2, their dosage in the fermentation medium and the ratio of carbon source dosage were further optimized. On the basis of MRS medium, the optimal carbon source D-galactose and the nitrogen source yeast extract powder were used to replace the carbon source and nitrogen source in the MRS medium to prepare the medium. Subsequently, the total amount of carbon and nitrogen and the carbon-nitrogen ratio in the MRS medium were changed to prepare the medium, and the inoculation amount was 5% (v / v) and the fermentation was carried out for 12 hours. According to the growth of Lactobacillus plantarum CCFM1283 cells, the appropriate total amount of carbon and nitrogen and the ratio were determined. When determining the total amount of carbon and nitrogen, the carbon-nitrogen ratio was fixed at 1:1, and when determining the carbon-nitrogen ratio, the total amount of carbon and nitrogen sources was fixed at 5%. Specific results are shown in the attached Figure 3 and Figure 4 , when the carbon-nitrogen ratio was 2:1 and the total carbon-nitrogen ratio was 5%, the fermentation liquid density was the highest. Subsequently, the optimal carbon-nitrogen source ratio of 2:1 (g / g) and the total carbon-nitrogen ratio (5%) were used to replace the carbon-nitrogen source ratio and total amount in the MRS basal medium, and kudzu root powder with a final concentration of 3-5g / ml was added. After fermentation for 12-13h with a 2-3% seed liquid inoculation amount, the soybean aglycone production increased to 8.38 times of 25.2ppm before fermentation, reaching 211.1ppm.

[0069] Example 4: Optimizing the temperature at each stage of fermentation to increase the yield of daidzein

[0070] Based on the determination of Examples 1, 2 and 3, the following culture medium was used for pilot culture (per L): yeast extract powder (20-21 g), D-galactose (25-32 g), manganese sulfate monohydrate 0.9-1.5 g / L, K2HPO4 2-4 g / L, anhydrous sodium acetate 2-4 g / L, diammonium hydrogen citrate 2-4 g / L, MgSO4·7H2O 0.2-0.5 g / L, Tween 80 1-2 mL / L, L-cysteine ​​hydrochloride 1-2 g / L, 700 mesh kudzu root powder (5-6 g), pH 5.0-6.5. During the whole fermentation process, the pH of the fermentation liquid was regulated to be stable between 5 and 6 by supplementing 20% ​​sodium hydroxide. By changing the culture temperature at different fermentation stages, a single factor experiment was designed to measure the cell density of the fermentation liquid at a wavelength of 600 nm. The results are shown in Table 1. Figure 5 The purpose of increasing the yield of soybean glycogen in the fermentation broth was achieved by adjusting the fermentation temperature at different stages. The results showed that when the temperature was 37-37.2℃ in the early stage of fermentation (12-13h) and 21-22℃ in the late stage of fermentation (13-48h), the number of live bacteria in the fermentation broth was greater than 3.71 billion, and the number of live bacteria reached 7.12*10 at 12-20h. 9CFU / mL. Only applicable to MRS medium cultured at 37℃ for 48h, the live bacteria in the fermentation liquid dropped to 1.9*10 8 CFU / mL. Through optimization, the strain activity and conversion ability of Lactobacillus plantarum CCFM1283 in the fermentation broth were guaranteed during the strong conversion period of puerarin to daidzein. Using the optimized culture medium, the number of viable bacteria in the fermentation broth was 19.47 times that of MRS culture medium. The daidzein content increased by 16.26 times after 48 hours, reaching 603.27ppm, and compared with the original MRS culture daidzein content of 37.12ppm, the daidzein content in the fermentation broth increased by 44.72 times. In addition, the daidzein yield was significantly increased by 8.755 times compared with the use of MRS fermentation raw materials.

[0071] Comparative Example 1: After adjusting the fermentation temperature, the yield of daidzein decreased

[0072] Specific implementation method Referring to Example 4, the difference is that the temperature during the fermentation process is adjusted so that the temperature is maintained at 37-37.2°C during the entire fermentation process. Figure 6 As shown in Figure 2, the fermentation density in the late fermentation period decreased to 2.21*10 9 CFU / mL, and the soybean aglycone production was only 379.28ppm.

[0073] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A fermentation medium for producing daidzein, characterized in that: The culture medium contains yeast extract powder, D-galactose, manganese sulfate monohydrate, phosphate, anhydrous sodium acetate, diammonium hydrogen citrate, magnesium salt, Tween 80, L-cysteine ​​hydrochloride, kudzu root powder and water.

2. The culture medium according to claim 1, characterized in that The culture medium comprises: 20-30 g / L yeast extract, 25-35 g / L D-galactose, 0.9-1.5 g / L manganese sulfate monohydrate, 2-4 g / L K2HPO4, 2-4 g / L anhydrous sodium acetate, 2-4 g / L diammonium hydrogen citrate, 0.2-0.5 g / L MgSO4·7H2O, 1-2 mL / L Tween 80, 1-2 g / L L-cysteine ​​hydrochloride and 5-6 g / L kudzu root powder.

3. The culture medium according to claim 1 or 2, characterized in that The culture medium is suitable for Lactobacillus plantarum CCFM1283.

4. A method for producing daidzein, characterized in that: The method comprises the steps of inoculating plant lactobacillus CCFM1283 into the fermentation medium for fermentation.

5. The method according to claim 4, characterized in that: Ferment for at least 48 hours.

6. The method according to claim 5, characterized in that The fermentation is a two-stage fermentation; the first stage is at a temperature of 37-37.2° C. for 12-13 hours; the second stage is at a temperature of 21-22° C. for at least 48 hours.

7. The method according to claim 6, characterized in that During the fermentation process, the pH of the fermentation liquid was controlled at 5-6.

8. A method for high-density cultivation of Lactobacillus plantarum CCFM1283, characterized in that: Inoculate Lactobacillus plantarum CCFM1283 into the culture medium according to any one of claims 1 to 3 for fermentation.

9. Use of the culture medium according to any one of claims 1 to 3 in improving the conversion rate of puerarin by Lactobacillus plantarum CCFM1283 or in improving the efficiency of producing daidzein by Lactobacillus plantarum CCFM1283.

10. Use of the culture medium according to any one of claims 1 to 3 in improving the fermentation density of Lactobacillus plantarum CCFM1283.

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