A composite microbial agent and application thereof in preparation of an antioxidative and blood vessel protecting and liver protecting product

Abstract

This invention provides a compound microbial agent and its application in the preparation of antioxidant, blood vessel-protecting, and liver-protecting products, belonging to the field of microbial fermentation technology. The compound microbial agent of this invention includes fermenting lactic acid bacteria 30818 and Delbufoss yeast Y56. This invention uses a compound microbial agent prepared from fermenting lactic acid bacteria 30818 and Delbufoss yeast Y56 to conduct liquid fermentation of kudzu root. This not only effectively protects and extracts the antioxidant active ingredients in kudzu root, but also further enhances the antioxidant capacity of the extract through biotransformation during fermentation. Simultaneously, the preparation method of the kudzu root extract in this invention ensures the safety and naturalness of the extract.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a compound microbial agent and its application in the preparation of antioxidant, blood vessel-protecting and liver-protecting products. Background Technology

[0002] Kudzu root is a plant used both as food and medicine, rich in isoflavones, puerarin, saponins, kudzu polysaccharides, alkaloids, and other active components. In traditional Chinese medicine, kudzu root is often used as an adjunct treatment for fever, diarrhea, and angina pectoris. Modern pharmacological studies have confirmed that kudzu root possesses various biological activities. For example, isoflavone compounds such as puerarin and daidzein in kudzu root have been shown to have extremely strong antioxidant activity, effectively protecting venous endothelial cells from cell death caused by reactive oxygen species (ROS) and damage to mitochondria.

[0003] However, the bioavailability of these glycosides is relatively low, attributed to their limited intestinal absorption. To overcome this limitation, microbial fermentation has emerged as a promising strategy to enhance the bioactivity and biotransformation efficiency of herbal compounds. Probiotic strains such as *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Lactococcus lactis* have been successfully used to ferment various herbal substrates, such as red ginseng, white-leaf squash, and ellagitannins, thereby enhancing their therapeutic potential against inflammation, oxidative stress, and metabolic disorders.

[0004] Currently, people are paying increasing attention to the health benefits of natural ingredients. Kudzu root, as a natural product rich in nutrients and medicinal value, has shown potential health benefits in areas such as anti-oxidation, vascular protection, and blood sugar reduction, leading to its increasing application in health products and biomedicine. However, the antioxidant active ingredients in kudzu root suffer from low bioavailability. Therefore, how to increase the content of antioxidant active ingredients in kudzu root extract is a problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a compound microbial agent and its application in the preparation of antioxidant, vascular-protective, and liver-protective products. This invention utilizes a compound microbial agent prepared from fermenting lactic acid bacteria 30818 and *Saccharomyces cerevisiae* Y56 for liquid fermentation of kudzu root. This not only effectively protects and extracts the antioxidant active ingredients from kudzu root but also further enhances the antioxidant capacity of the extract through biotransformation during fermentation. Simultaneously, the preparation method of the compound fermented extract (kudzu root extract) of this invention ensures the safety and naturalness of the extract.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a compound microbial agent, which includes fermenting lactic acid bacteria 30818 and Delbufoss spore-forming yeast Y56; wherein the mass ratio of fermenting lactic acid bacteria 30818 to Delbufoss spore-forming yeast Y56 is 3~0.5:0.5~3; The fermenting lactic acid bacteria 30818 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38478; The aforementioned *Delbus spore-forming yeast* Y56 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38477.

[0007] This invention provides a method for preparing the aforementioned composite microbial agent, which involves mixing fermenting lactic acid bacteria 30818 and Delbufoss yeast Y56 at a mass ratio of 3~0.5:0.5~3; The preparation method of the fermented lactic acid bacteria 30818 is as follows: the fermented lactic acid bacteria 30818 strain is inoculated into nutrient broth and cultured for 46-50 hours to obtain a viable count of 8×10⁻⁶. 6 ~3×10 7 Fermenting lactic acid bacteria 30818 at CFU / mL; The method for preparing the *Delbus sporozoans* Y56 is as follows: *Delbus sporozoans* Y56 is inoculated into a nutrient broth and cultured for 46-50 hours to obtain a viable count of 8 × 10⁻⁶ cells / year. 6 ~3×10 7 CFU / mL of Delbufoss Y56.

[0008] This invention provides the application of the aforementioned compound microbial agent or the compound microbial agent obtained according to the preparation method in the preparation of antioxidant, vascular-protective, and liver-protective products, wherein the product is kudzu root extract.

[0009] This invention provides a kudzu root extract, which is obtained by fermentation using the aforementioned compound microbial agent.

[0010] The present invention also provides a method for preparing the kudzu root extract, comprising the following steps: (1) Mix kudzu root powder with water to obtain kudzu root base liquid; (2) Saccharify the kudzu root base liquid to obtain a saccharified base liquid; (3) Cool the saccharified base solution and add enzyme to inactivate the enzyme and obtain the enzymatic hydrolysate; (4) Add the compound microbial agent to the enzymatic hydrolysate and ferment for 3-7 days to obtain the fermentation broth; (5) Centrifuge and filter the fermentation broth, take the supernatant, sterilize it, and obtain kudzu root extract.

[0011] Preferably, the mass ratio of kudzu root powder to water is 1:5~25.

[0012] Preferably, the saccharification method is as follows: heating the kudzu root base liquid to 90~110℃ and boiling for 50~70 minutes.

[0013] Preferably, the cooling and enzyme treatment is performed by two cooling processes: first, cooling to 70°C, adding α-amylase, and hydrolyzing for 50-70 minutes; second, cooling to 60°C, adding saccharifying enzyme, and hydrolyzing for 50-70 minutes. The enzyme activity of α-amylase is 5-15 U / mg, and the enzyme activity of saccharifying enzyme is 80-120 U / mg.

[0014] Preferably, the fermentation temperature is 28~36℃.

[0015] The present invention also provides an antioxidant, blood vessel-protecting, and liver-protecting fermented kudzu root liquor, wherein the liquor uses the kudzu root extract or the kudzu root extract obtained according to the preparation method as the main active ingredient or the only active ingredient.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts compound fermented kudzu root extract, which improves the extraction efficiency and stability of antioxidant active ingredients in kudzu root and reduces the damage to active ingredients that may be caused by traditional high temperature extraction methods.

[0017] This invention promotes the conversion and enhancement of antioxidant active ingredients in the compound fermentation extract through the synergistic effect of Delbufoss Y56 and fermenting lactic acid bacteria 30818.

[0018] This invention is applied to kudzu root liqueur products, breaking through the simple soaking process and adopting a unique natural fermentation technique. Through biological fermentation technology, kudzu root is transformed into a kudzu root fermentation liquid rich in prebiotics and active substances. Then, taking advantage of the fact that these active ingredients are easily soluble in ethanol, these active ingredients are better extracted in the wine, further enhancing the conversion efficiency and variety of antioxidant active ingredients in the fermentation liquid. Attached Figure Description

[0019] Figure 1 The effect of strain ratio on the puerarin content of kudzu root fermentation broth; Figure 2 The effect of the material-to-liquid ratio on the puerarin content of kudzu root fermentation broth; Figure 3 The effect of fermentation temperature on the puerarin content of kudzu root fermentation broth; Figure 4 The effect of fermentation time on the puerarin content of kudzu root fermentation broth.

[0020] Biological Preservation Instructions

[0021] Fermenting lactic acid bacteria 30818, Latin name Lactobacillus fermentum, is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 29, 2026, with accession number CGMCC No. 38478.

[0022] Torulaspora delbrueckii Y56 is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 29, 2026, with accession number CGMCC No. 38477. Detailed Implementation

[0023] This invention provides a compound microbial agent, which includes fermenting lactic acid bacteria 30818 and Delbufoss spore-forming yeast Y56; wherein the mass ratio of fermenting lactic acid bacteria 30818 to Delbufoss spore-forming yeast Y56 is 3~0.5:0.5~3, preferably 2~0.8:0.8~2, and more preferably 1:1; The fermenting lactic acid bacteria 30818 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38478; The aforementioned *Delbus spore-forming yeast* Y56 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38477.

[0024] The present invention provides a method for preparing the aforementioned composite microbial agent, which is prepared by mixing fermenting lactic acid bacteria 30818 and Delbufoss spore-forming yeast Y56 in a mass ratio of 3~0.5:0.5~3. The preferred mass ratio of fermenting lactic acid bacteria 30818 to Delbufoss spore-forming yeast Y56 is 2~0.8:0.8~2, and more preferably 1:1. The preparation method of the fermented lactic acid bacteria 30818 is as follows: the fermented lactic acid bacteria 30818 strain is inoculated into nutrient broth and cultured for 46-50 hours to obtain a viable count of 8×10⁻⁶. 6 ~3×10 7 The fermentation bacteria 30818 (CFU / mL) are used; the culture temperature is 35-40℃, preferably 36-38℃, more preferably 37℃; the culture time is preferably 47-49h, more preferably 48h; the shaking frequency during culture is 100-200r / min, preferably 130-170r / min; the viable count of the fermentation bacteria 30818 is preferably 9×10⁻⁶. 6 ~2×107 CFU / mL, further preferably 1×10⁻⁶ 7 CFU / mL; The method for preparing the *Delbus sporozoans* Y56 is as follows: *Delbus sporozoans* Y56 is inoculated into a nutrient broth and cultured for 46-50 hours to obtain a viable count of 8 × 10⁻⁶ cells / year. 6 ~3×10 7 The *Saccharomyces delbue* Y56 at CFU / mL is cultured at a temperature of 30–34°C, preferably 31–33°C, more preferably 32°C, for a culture time of 47–49 h, more preferably 48 h, with an oscillation frequency of 100–200 r / min, preferably 130–170 r / min; the viable count of the *Saccharomyces delbue* Y56 is preferably 9 × 10⁻⁶. 6 ~2×10 7 CFU / mL, further preferably 1×10⁻⁶ 7 CFU / mL.

[0025] In this invention, by optimizing the preparation conditions of fermenting lactic acid bacteria 30818 and Delbrio spore yeast Y56, the activity and purity of the seed liquid were ensured, providing a better fermentation environment for the subsequent fermentation process, thereby guaranteeing the stability and effectiveness of the compound fermentation extract.

[0026] This invention provides the application of the aforementioned compound microbial agent or the compound microbial agent obtained according to the preparation method in the preparation of antioxidant, vascular-protective, and liver-protective products, wherein the product is kudzu root extract.

[0027] This invention provides a kudzu root extract, which is obtained by fermentation using the aforementioned compound microbial agent.

[0028] The present invention also provides a method for preparing the kudzu root extract, comprising the following steps: (1) Mix kudzu root powder with water to obtain kudzu root base liquid; (2) Saccharify the kudzu root base liquid to obtain a saccharified base liquid; (3) Cool the saccharified base solution and add enzyme to inactivate the enzyme and obtain the enzymatic hydrolysate; (4) Add the compound microbial agent to the enzymatic hydrolysate and ferment for 3-7 days to obtain the fermentation broth; (5) Centrifuge and filter the fermentation broth, take the supernatant, sterilize it, and obtain kudzu root extract.

[0029] In this invention, kudzu root powder is mixed with water to obtain kudzu root base liquid; kudzu root powder and water are mixed at a mass ratio of 1:5 to 25 to obtain kudzu root base liquid, wherein the mass ratio of kudzu root powder to water is preferably 1:8 to 15, and more preferably 1:10.

[0030] In this invention, the kudzu root base liquid is saccharified to obtain a saccharified base liquid; preferably, the kudzu root base liquid is heated to 90~110℃ and boiled for 50~70min to obtain a saccharified base liquid, wherein the heating temperature is preferably 95~105℃, more preferably 100℃, and the boiling time is preferably 55~65min, more preferably 60min.

[0031] In this invention, the saccharified base solution is subjected to cooling and enzyme addition treatment to inactivate the enzyme and obtain an enzymatic hydrolysate. Preferably, the saccharified base solution is cooled to 70°C, and 0.3%~0.7% (by mass) of kudzu root powder α-amylase is added. After enzymatic hydrolysis for 50~70 min, the solution is further cooled to 60°C, and 0.3%~0.7% (by mass) of kudzu root powder saccharifying enzyme is added. After enzymatic hydrolysis for 50~70 min, the enzyme is inactivated to obtain the enzymatic hydrolysate. The amount of α-amylase added is preferably 0.4%~0.6% (by mass) of kudzu root powder, more preferably 0.5%. The α-amylase has an enzyme activity of 5-15 U / mg, preferably 8-12 U / mg, and more preferably 10 U / mg; the amount of saccharifying enzyme added is preferably 0.4%-0.6% of the mass fraction of kudzu root powder, more preferably 0.5%, and the enzyme activity of the saccharifying enzyme is 80-120 U / mg, preferably 90-110 U / mg, and more preferably 100 U / mg; the enzymatic hydrolysis time of both α-amylase and saccharifying enzyme is preferably 55-65 min, more preferably 60 min.

[0032] In this invention, the enzymatic hydrolysis of α-amylase and saccharifying enzyme promotes the more effective release of active ingredients in kudzu root, providing abundant substrates for subsequent fermentation extraction. The fermentation extraction process using lactic acid bacteria and yeast not only effectively preserves the antioxidant active ingredients in kudzu root and avoids the potential damage to active ingredients caused by traditional high-temperature extraction methods, but also reduces the use of chemical solvents, ensuring the safety and naturalness of the extract. Furthermore, the synergistic effect of microbial fermentation further transforms and enhances the antioxidant active ingredients in kudzu root, significantly improving the antioxidant activity and stability of the extract. This invention further optimizes the enzyme activity range of α-amylase and saccharifying enzyme, enabling more effective decomposition of kudzu root powder and more effective release of active ingredients, thereby improving enzymatic hydrolysis efficiency and the quality of the extract.

[0033] In this invention, the aforementioned compound microbial agent is added to the enzymatic hydrolysate, and fermentation is carried out for 3-7 days to obtain a fermentation broth. Preferably, 3%-7% of the volume of the enzymatic hydrolysate compound microbial agent is added to the enzymatic hydrolysate, and fermentation is carried out at 28-36℃ for 3-7 days to obtain a fermentation broth. The amount of compound microbial agent added is preferably 4%-6% of the volume of the enzymatic hydrolysate, more preferably 5%. The compound microbial agent is prepared by mixing fermenting lactic acid bacteria 30818 and Delbufoss yeast Y56 at a mass ratio of 3-0.5:0.5-3, wherein the mass ratio of fermenting lactic acid bacteria 30818 to Delbufoss yeast Y56 is preferably 2-0.8:0.8-2, more preferably 1:1. The fermenting lactic acid bacteria 30818 is prepared by inoculating the fermenting lactic acid bacteria 30818 strain into a nutrient broth and culturing for 46-50 hours to obtain a viable count of 8 × 10⁻⁶. 6 ~3×10 7 The fermentation bacteria 30818 (CFU / mL) are used; the culture temperature is 35-40℃, preferably 36-38℃, more preferably 37℃; the culture time is preferably 47-49h, more preferably 48h; the shaking frequency during culture is 100-200r / min, preferably 130-170r / min; the viable count of the fermentation bacteria 30818 is preferably 9×10⁻⁶. 6 ~2×10 7 CFU / mL, further preferably 1×10⁻⁶ 7 The preparation method of *Saccharomyces delbue* Y56 is as follows: *Saccharomyces delbue* Y56 is inoculated into nutrient broth and cultured for 46-50 hours to obtain a viable count of 8 × 10⁻⁶. 6 ~3×10 7 The *Saccharomyces delbue* Y56 at CFU / mL is cultured at a temperature of 30–34°C, preferably 31–33°C, more preferably 32°C, for a culture time of 47–49 h, more preferably 48 h, with an oscillation frequency of 100–200 r / min, preferably 130–170 r / min; the viable count of the *Saccharomyces delbue* Y56 is preferably 9 × 10⁻⁶. 6 ~2×10 7 CFU / mL, further preferably 1×10⁻⁶ 7 CFU / mL.

[0034] In this invention, by employing mixed fermentation of two strains, the synergistic effect between the two strains is utilized to enhance the conversion efficiency and variety of antioxidant active ingredients in the fermentation broth, thereby improving the antioxidant performance of the extract. This invention further optimizes the fermentation conditions to ensure the biological activity of fermenting lactic acid bacteria 30818 and Delbrio spore yeast Y56, which helps to rapidly produce metabolites that are beneficial to the release and conversion of antioxidant active ingredients, further enhancing the antioxidant capacity of the extract.

[0035] In this invention, the fermentation broth is centrifuged and filtered, the supernatant is collected and sterilized to obtain kudzu root extract; preferably, the fermentation broth is centrifuged at 8000~12000 r / min for 8~12 min, the supernatant is collected and sterilized to obtain kudzu root extract; the centrifugation speed is preferably 9000~11000 r / min, more preferably 10000 r / min, and the centrifugation time is preferably 9~11 min, more preferably 10 min.

[0036] In this invention, a compound fermented kudzu root extract with high antioxidant activity and puerarin content was successfully prepared using the method of this invention. This extract not only retains the natural active ingredients in kudzu root, but also significantly enhances its antioxidant capacity and puerarin content through biotransformation during fermentation. Applying such a compound fermented extract to the fields of health products or biomedicine can provide consumers with a new source of natural antioxidants.

[0037] The present invention also provides an antioxidant, blood vessel-protecting, and liver-protecting fermented kudzu root liquor, wherein the liquor uses the kudzu root extract or the kudzu root extract obtained according to the preparation method as the main active ingredient or the only active ingredient.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1: Preparation of Compound Fermentation Agent

[0040] A compound bacterial agent was prepared using fermenting lactic acid bacteria strain 30818 preserved in the Zhongyuan Food Laboratory and yeast strain Y56 of Delbufos.

[0041] Take 1 mL of Lactobacillus 30818 bacterial culture and inoculate it into 100 mL of nutrient broth. Incubate at 37°C on a shaker for 48 hours with a shaking frequency of 150 r / min to obtain Lactobacillus 30818 seed culture. The viable count of the seed culture is 1 × 10⁻⁶. 7 CFU / mL.

[0042] Take 1 mL of *Saccharomyces delbue* Y56 bacterial culture and inoculate it into 100 mL of nutrient broth. Incubate at 32°C on a shaker for 48 h with a shaking frequency of 150 r / min to obtain *Saccharomyces delbue* Y56 seed culture. The viable count of the seed culture is 1 × 10⁻⁶. 7 CFU / mL.

[0043] The seed culture of fermenting lactic acid bacteria 30818 and the seed culture of Delbufos sporogenes Y56 were mixed at different mass ratios to obtain compound fermentation agents with different strain ratios, which were used for subsequent experiments.

[0044] Example 2: Preparation of Kudzu Root Extract

[0045] (1) Take 100g of kudzu root powder (purchased from Beijing Tongrentang (Sichuan) Health Pharmaceutical Co., Ltd.), add 1.5kg of water, mix well, and obtain kudzu root base liquid; (2) Heat the kudzu root base liquid obtained in step (1) to 100℃, boil and saccharify for 1 hour, then cool down to 70℃ and add 0.5g α-amylase (enzyme activity of 10U / mg), stir evenly and enzymatically hydrolyze for 60 minutes, then cool down to 60℃ and add 0.5g saccharifying enzyme (enzyme activity of 100U / mg), stir evenly and enzymatically hydrolyze for 60 minutes, inactivate the enzyme, and obtain the enzymatic hydrolysate; (3) Inoculate the enzyme hydrolysate with 5% of the volume of the compound fermentation agent (obtained by the method of Example 1, which is a mixture of fermentation lactic acid bacteria 30818 seed liquid and Delbu spore yeast Y56 seed liquid in a 1:1 mass ratio), and ferment at 32°C for 5 days to obtain the fermentation liquid.

[0046] (4) Centrifuge the fermentation broth at 10000r / min for 10min, filter it, take the supernatant, sterilize it, and obtain kudzu root extract.

[0047] Example 3

[0048] The kudzu root extract was prepared using the same method as in Example 2, except that the mass ratio of the fermenting lactic acid bacteria 30818 seed liquid to the Delbrio spore yeast Y56 seed liquid in the compound fermentation agent was adjusted from 1:1 to 3:1, 2:1, 1:2 and 1:3.

[0049] Example 4

[0050] The kudzu root extract was prepared using the same method as in Example 2, except that the mass ratio of kudzu root powder to water was adjusted from 1:10 to 1:5, 1:15, 1:20, and 1:25.

[0051] Example 5

[0052] The kudzu root extract was prepared using the same method as in Example 2, except that the fermentation temperature was adjusted from 32°C to 28°C, 30°C, 34°C, and 36°C.

[0053] Example 6

[0054] The kudzu root extract was prepared using the same method as in Example 2, except that the fermentation time was adjusted from 4 days to 3 days, 5 days, 6 days, and 7 days.

[0055] Comparative Example

[0056] Pueraria extract was prepared using the same method as in Example 2, except that fermentation was not performed. The obtained puerarin content was 1.69 mg / L.

[0057] Example of effect 1

[0058] Using puerarin content as the evaluation index, the quality of kudzu fermentation broth obtained in Examples 2-6 and the comparative example (blank) was judged, and the results are shown in […]. Figures 1-4 .

[0059] Determination of puerarin content: Place the pueraria fermentation broth in a high-speed centrifuge and centrifuge at 10000 r / min for 10 min. Take 1 mL of the supernatant and place it in a graduated tube. Add 8 mL of 50% (volume fraction) methanol solution, mix thoroughly, and extract by sonication for 20 min. Cool to room temperature, transfer to a 10 mL volumetric flask, and then dilute to volume with 50% (volume fraction) methanol solution. Mix well, filter through a filter membrane, and test.

[0060] Preparation of the puerarin standard curve

[0061] Puerarin standard was prepared into a 2 mg / mL puerarin standard stock solution using 70% ethanol. Subsequently, it was diluted with 70% ethanol at concentrations of 1, 5, 10, 50, 100, and 200 μg / mL to prepare a series of working standard solutions. These working standard solutions were injected into a high-performance liquid chromatograph (HPLC), and the peak area of ​​each component was measured. A standard curve was plotted with the mass concentration of the corresponding working standard solution on the x-axis and the peak area on the y-axis.

[0062] Chromatographic conditions: C18 column (250mm×4.6mm, 5μm); injection volume 10μL; column temperature 30℃; detection wavelength 247nm; mobile phase acetonitrile-0.1% (volume fraction) phosphoric acid solution; liquid chromatography elution program according to national standard GB / T22261-2024.

[0063] Qualitative and quantitative analysis: qualitative analysis was performed using retention time, and quantitative analysis was performed using the external standard method.

[0064] Puerarin enhancement rate (%) = (puerarin content in fermentation broth - puerarin content in blank broth) ÷ puerarin content in blank broth × 100%.

[0065] Figure 1 It can be seen that as the inoculation ratio of fermenting lactic acid bacteria 30818 and Delbrio spore-forming yeast Y56 decreased, the increase in puerarin in the kudzu root fermentation broth first leveled off and then rose rapidly, reaching its maximum of 47% when the inoculum ratio was 1:1. As the inoculum ratio continued to decrease, the increase rate of puerarin continued to decline. It's possible that when the proportion of lactic acid bacteria in the inoculation ratio is too high (3:1, 2:1), the accumulation of metabolites and competitive inhibition prevent the synergistic conversion ability from being fully utilized. When the inoculation ratio is 1:1, the two strains reach an optimal synergistic state in metabolism and enzyme systems. The acid produced by the fermenting lactic acid bacteria 30818 lowers the pH of the system, creating a suitable growth environment for *Saccharomyces cerevisiae* Y56. The growth and metabolism of *Saccharomyces cerevisiae* Y56 may in turn promote the activity of fermenting lactic acid bacteria 30818. The enzyme systems secreted by both strains (such as β-glucosidase) complement each other, thus significantly improving the bioconversion efficiency of puerarin. However, when the proportion of *Saccharomyces cerevisiae* Y56 is too high (1:2, 1:3), the intensified competition for carbon sources and disruption of metabolic balance may significantly reduce the puerarin enhancement rate. In conclusion, the puerarin fermentation broth has the best quality when the strain ratio is 1:1.

[0066] Depend on Figure 2 It can be seen that as the material-to-liquid ratio decreases, the enhancement rate of puerarin in the kudzu fermentation broth shows a trend of first increasing and then decreasing, reaching a maximum of 61% at a material-to-liquid ratio of 1:10. The reason for this may be related to the influence of substrate concentration on microbial metabolism in the fermentation system. Appropriate kudzu powder addition can provide sufficient substrate for microbial growth and enzymatic reactions, promoting the conversion and accumulation of puerarin. When the material-to-liquid ratio is too high, the water content in the system is relatively reduced, which may affect the fluidity, metabolic activity, and mass transfer efficiency of the microorganisms, thus affecting the enhancement rate of puerarin. Conversely, when the material-to-liquid ratio is too low, the substrate concentration in the fermentation system is insufficient, and microbial growth and metabolism lack sufficient material basis, resulting in a relatively low bioconversion of puerarin and ultimately a low enhancement rate. Therefore, in this fermentation system, a material-to-liquid ratio of 1:10 may be a more suitable addition condition.

[0067] Depend on Figure 3It was observed that the increase in puerarin concentration in the kudzu fermentation broth gradually increased with rising fermentation temperature, reaching a maximum of 60% at 34℃. As the fermentation temperature continued to rise, the increase in puerarin concentration decreased. This may be closely related to the optimal growth temperature of the microorganisms used in the fermentation. The metabolic activity and product synthesis efficiency of microorganisms often reach their highest levels within a specific temperature range. Temperatures that are too low will slow down microbial growth and enzymatic reaction rates, while temperatures that are too high may lead to reduced enzyme activity or inhibited cell metabolism, thus affecting the biotransformation efficiency of puerarin. Therefore, in this fermentation system, 34℃ may be the most suitable temperature condition for promoting puerarin accumulation.

[0068] Depend on Figure 4 It was observed that the increase in puerarin in the kudzu fermentation broth initially rose with increasing fermentation time, reaching a maximum of 49% after 4 days. As fermentation time continued to increase, the increase in puerarin in the kudzu fermentation broth initially leveled off and then decreased sharply, reaching only 0.5% by day 6. This may be because in the early stages of fermentation, the active metabolism of microorganisms and the secreted enzymes (such as β-glucosidase) effectively promoted the conversion and accumulation of puerarin precursors; however, as fermentation progressed, substrate consumption, metabolic product accumulation, and decreased microbial activity led to a reduction in conversion efficiency. Therefore, the dynamic changes in enzyme activity and substrate availability during microbial fermentation are key factors affecting puerarin accumulation.

[0069] Example 2

[0070] Using puerarin as an indicator, L9(3) 4 An orthogonal experiment was conducted (Table 1, specific method is described in Example 2) to determine the optimal formula for kudzu root fermentation broth (where the ratio of bacterial strains is fermenting lactic acid bacteria 30818: Delbufoss Y56), and the results are shown in Tables 2 and 3.

[0071] Table 1 Factor Level Table

[0072] Table 2. Results and Analysis of Orthogonal Experiments

[0073] Table 3. Analysis of Variance of Orthogonal Experiment Results

[0074] Table 2 shows that the order of influence of each factor on the puerarin enhancement rate is A>D>B>C, i.e., inoculum ratio > substrate-to-liquid ratio > fermentation time > fermentation temperature. The optimal fermentation process is A2B2C1D2, which means an inoculum ratio of 1:1, a fermentation time of 4 days, a fermentation temperature of 32℃, and a substrate-to-liquid ratio of 1:10. Table 3 shows that factors A (inoculum ratio) and D (substrate-to-liquid ratio) have a significant impact on the puerarin enhancement rate (P<0.05), which is consistent with the intuitive analysis results. The experimental results were verified under optimal conditions, and the results showed that the puerarin enhancement rate in the kudzu fermentation broth was 63%. Therefore, the optimal fermentation process combination was determined to be A2B2C1D2.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent includes fermenting lactic acid bacteria 30818 and Delbufoss spore-forming yeast Y56; wherein the mass ratio of fermenting lactic acid bacteria 30818 to Delbufoss spore-forming yeast Y56 is 3~0.5:0.5~3. The fermenting lactic acid bacteria 30818 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38478; The aforementioned *Delbus spore-forming yeast* Y56 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38477.

2. The method for preparing the composite microbial agent according to claim 1, characterized in that, It is prepared by mixing fermenting lactic acid bacteria 30818 and Delbufoss spore-forming yeast Y56 at a mass ratio of 3~0.5:0.5~3; The preparation method of the fermented lactic acid bacteria 30818 is as follows: the fermented lactic acid bacteria 30818 strain is inoculated into nutrient broth and cultured for 46-50 hours to obtain a viable count of 8×10⁻⁶. 6 ~3×10 7 Fermenting lactic acid bacteria 30818 at CFU / mL; The method for preparing the *Delbus sporozoans* Y56 is as follows: *Delbus sporozoans* Y56 is inoculated into a nutrient broth and cultured for 46-50 hours to obtain a viable count of 8 × 10⁻⁶ cells / year. 6 ~3×10 7 CFU / mL of Delbufoss Y56.

3. The application of the composite microbial agent according to claim 1 or the composite microbial agent obtained by the preparation method according to claim 2 in the preparation of antioxidant, blood vessel protective, and liver protective products, characterized in that, The product is kudzu root extract.

4. A kudzu root extract, characterized in that, The kudzu root extract was obtained by fermentation using the compound microbial agent described in claim 1.

5. The method for preparing the kudzu root extract according to claim 4, characterized in that, Includes the following steps: (1) Mix kudzu root powder with water to obtain kudzu root base liquid; (2) Saccharify the kudzu root base liquid to obtain a saccharified base liquid; (3) Cool the saccharified base solution and add enzyme to inactivate the enzyme and obtain the enzymatic hydrolysate; (4) Add the compound microbial agent of claim 1 to the enzymatic hydrolysate and ferment for 3-7 days to obtain the fermentation broth; (5) Centrifuge and filter the fermentation broth, take the supernatant, sterilize it, and obtain kudzu root extract.

6. The preparation method according to claim 5, characterized in that, The mass ratio of kudzu root powder to water is 1:5~25.

7. The preparation method according to claim 5, characterized in that, The saccharification method is as follows: heat the kudzu root base liquid to 90~110℃ and boil for 50~70 minutes.

8. The preparation method according to claim 5, characterized in that, The cooling and enzyme addition treatment is performed by two cooling processes. The first cooling is performed at 70°C, and α-amylase is added and enzymatic hydrolysis is carried out for 50-70 minutes. The second cooling is performed at 60°C, and saccharifying enzyme is added and enzymatic hydrolysis is carried out for 50-70 minutes. The enzyme activity of α-amylase is 5-15 U / mg, and the enzyme activity of saccharifying enzyme is 80-120 U / mg.

9. The preparation method according to claim 5, characterized in that, The fermentation temperature is 28~36℃.

10. A fermented kudzu root liquor with antioxidant, blood vessel-protecting, and liver-protecting properties, characterized in that... The liqueur uses the kudzu root extract as described in claim 4 or the kudzu root extract obtained by any one of the preparation methods described in claims 5 to 9 as the main active ingredient or the only active ingredient.

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