A synergistically compounded microbial starter and its application in targeted improvement of flavor of fermented gengen beverage

Abstract

The application discloses a kind of composite microorganism fermenting agent with significant synergistic effect and its application in the directional promotion of fermented flavour of gordon root beverage.The application provides a deep processing scheme with gordon root as raw material for the technical problems of existing gordon root beverage, such as heavy earthy smell, rough taste and single function activity.The fermenting agent is compounded by specific proportion of fermented Lactobacillus mucus and cheese lactobacillus.The application of the fermenting agent in gordon root beverage fermentation, with the metabolic effect of lactic acid bacteria, not only effectively converts or covers the original bad flavor of gordon root, generates pleasant fermentation aromatic components, significantly improves the taste and flavor of the product, effectively guarantees the stability and antibacterial ability of fermentation process, and greatly improves the number of live bacteria and the content of organic acid in the beverage, giving the product good intestinal probiotic function.

Description

Technical Field

[0001] This invention belongs to the field of food microbial fermentation technology, specifically involving a synergistic compound microbial fermentation agent and its application in the targeted enhancement of the flavor of fermented kudzu root beverages. Background Technology

[0002] Kudzu root, a plant belonging to the genus *Pueraria* of the legume family (Leguminosae), is used medicinally for its underground rhizome and is an important ingredient listed in my country's list of food and medicine homologous ingredients. Kudzu root is rich in nutrients, mainly containing abundant starch, dietary fiber, isoflavones, triterpenoids, coumarins, alkaloids, and amino acids. Starch accounts for approximately 20%–30% of the fresh weight of kudzu root, of which amylose accounts for about 25%.

[0003] The main active substances in kudzu root are flavonoids and isoflavones, such as puerarin, daidzein, genistein, and gentiopicrin, which have pharmacological and health-promoting effects, including improving cardiovascular circulation, reducing myocardial oxygen consumption, lowering blood sugar, preventing hypertension and arteriosclerosis, enhancing immunity, and possessing antibacterial and antiviral properties. However, the development of kudzu root is mostly limited to primary processing, and its high-value-added transformation pathways, especially the development of specialty flavor products, are severely lagging behind.

[0004] Existing lactic acid bacteria fermentation technologies for plant-based beverages suffer from problems such as blind selection of strains and uncontrollable flavor development. Direct application to kudzu often results in products with mediocre flavor and poor stability. Therefore, how to create kudzu fermented beverages with rich flavor, stable quality, and functional properties through innovative strain combinations and targeted regulation of the fermentation process has become a key technological bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a synergistic compound microbial fermentation agent and its application in the targeted enhancement of the flavor of fermented kudzu root beverages.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a composite microbial fermentation agent based on synergistic effect, characterized in that: the composite microbial fermentation agent is obtained by combining fermenting Lactobacillus mucilaginosus and Lactobacillus casei; The compound microbial fermentation agent can increase the total amount of volatile flavor substances and inhibit miscellaneous bacteria in the kudzu fermentation system.

[0009] As a preferred embodiment of the composite microbial fermentation agent of the present invention, the total viable bacteria concentration of the composite microbial fermentation agent is 10. 6 ~10 8 CFU / mL.

[0010] As a preferred embodiment of the composite microbial fermentation agent of the present invention, the ratio of viable bacteria of the fermenting *Lactobacillus mucinus* and *Lactobacillus casei* is 1:1.

[0011] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a compound microbial fermentation agent in the targeted enhancement of the flavor of fermented kudzu root beverages.

[0012] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for enhancing the flavor of fermented kudzu root beverage, characterized in that: the above-mentioned compound microbial fermentation agent is added to the kudzu root enzymatic hydrolysate to be fermented for fermentation.

[0013] In a preferred embodiment of the method described in this invention, the inoculum amount of the composite microbial fermentation agent is 2.0% v / w.

[0014] In a preferred embodiment of the method described in this invention, the fermentation time is 24 to 72 hours.

[0015] In a preferred embodiment of the method described in this invention, the fermentation temperature is 37°C.

[0016] In a preferred embodiment of the method described in this invention, the total amount of volatile flavor compounds in the product obtained by fermentation is significantly increased compared to the unfermented kudzu root hydrolysate.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide a fermented kudzu root beverage, characterized in that it is prepared by the above-described method, and the content of volatile flavor substances of acids and alcohols in the beverage is significantly increased.

[0018] Beneficial effects of this invention: (1) Innovative strain combination: For the first time, it was discovered and verified that fermenting Lactobacillus mucinus and Lactobacillus casei in a 1:1 ratio have a unique synergistic effect in the kudzu root fermentation system.

[0019] (2) Flavor-oriented enhancement: It can target and significantly increase the total amount of volatile flavor substances (20 to 40 times, up to 95 times), especially the content of acids (such as glacial acetic acid) that give beverages a refreshing taste and alcohols (such as n-hexanol) that give beverages a rich aroma.

[0020] (3) Stable and controllable fermentation process: The compound fermentation agent has a strong competitive advantage, can effectively inhibit miscellaneous bacteria, ensure stable fermentation process, and high product consistency.

[0021] (4) Balancing function and flavor: While optimizing the flavor, attention was paid to the changes of flavonoids, the core functional component of kudzu root, during the fermentation process, which provided a technical basis for developing products that combine "flavor and health".

[0022] (5) The compound fermentation agent prepared by the present invention can increase the total amount of volatile flavor substances in the product to up to 95 times that of the unfermented group (792.05 vs 8.33 μg / mL). The content of key flavor substances such as acids and alcohols is significantly increased, which solves the technical problems of single flavor and unstable quality of traditional kudzu fermented products. The final product has the dual nutritional and health value of kudzu and lactic acid bacteria. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The pH change curves of six lactic acid bacteria in kudzu root enzymatic hydrolysate are shown; where Lb: Lactobacillus delbrueckii subsp. bulgaricus; Lc: Lactobacillus casei; Lp: Lactobacillus plantarum; Pp: Pediococcus pentosaceus; La: Lactobacillus acidophilus; Lf: Lactobacillus fermentum. Figure 2 The graph shows the total acidity variation of six lactic acid bacteria in kudzu root enzymatic hydrolysate; where Lb: Lactobacillus delbrueckii subsp. bulgaricus; Lc: Lactobacillus casei; Lp: Lactobacillus plantarum; Pp: Pediococcus pentosaceus; La: Lactobacillus acidophilus; Lf: Lactobacillus fermentum. Figure 3 The graph shows the changes in reducing sugars in the enzymatic hydrolysate of kudzu root of six lactic acid bacteria; where Lb: Lactobacillus delbrueckii subsp. bulgaricus; Lc: Lactobacillus casei; Lp: Lactobacillus plantarum; Pp: Pediococcus pentosacchari; La: Lactobacillus acidophilus; and Lf: Lactobacillus fermentum. Figure 4The graph shows the total flavonoid content of six lactic acid bacteria in kudzu root hydrolysate on the second day; where Lb: Lactobacillus delbrueckii subsp. bulgaricus; Lc: Lactobacillus casei; Lp: Lactobacillus plantarum; Pp: Pediococcus pentosaceus; La: Lactobacillus acidophilus; Lf: Lactobacillus fermentum. Figure 5 The graphs show the changes in viable cell counts of *Lactobacillus casei* and *Lactobacillus fermentatus* during individual and combined fermentation in kudzu root enzymatic hydrolysate; where Lc: *Lactobacillus casei*; Lf: *Lactobacillus fermentatus*; Lc+Lf: *Lactobacillus casei* + *Lactobacillus fermentatus*. Figure 6 The pH change curves are shown for Lactobacillus casei and Lactobacillus fermentation in kudzu root enzymatic hydrolysate during individual and combined fermentation; where Lc: Lactobacillus casei; Lf: Lactobacillus fermentation; Lc+Lf: Lactobacillus casei + Lactobacillus fermentation; Figure 7 The graphs show the changes in total acidity during the individual and combined fermentation of *Lactobacillus casei* and *Lactobacillus fermentatus* in kudzu root hydrolysate; where Lc: *Lactobacillus casei*; Lf: *Lactobacillus fermentatus*; Lc+Lf: *Lactobacillus casei* + *Lactobacillus fermentatus*. Figure 8 The graph shows the changes in reducing sugar content during the individual and combined fermentation of *Lactobacillus casei* and *Lactobacillus fermentatus* in kudzu root enzymatic hydrolysate; where Lc: *Lactobacillus casei*; Lf: *Lactobacillus fermentatus*; Lc+Lf: *Lactobacillus casei* + *Lactobacillus fermentatus*. Figure 9 The graph shows the changes in total flavonoid content during the individual and combined fermentation of *Lactobacillus casei* and *Lactobacillus fermentatus* in kudzu root enzymatic hydrolysate; where Lc: *Lactobacillus casei*; Lf: *Lactobacillus fermentatus*; Lc+Lf: *Lactobacillus casei* + *Lactobacillus fermentatus*.

[0024] Figure 10 This is a heatmap showing the changes in volatile flavor compounds during the individual and combined fermentation of *Lactobacillus casei* and *Lactobacillus fermentatus* in kudzu root hydrolysate.

[0025] Figure 11 Bar charts showing the sensory evaluation of Lactobacillus casei and Lactobacillus fermentation in kudzu root hydrolysate after two days of individual and combined fermentation. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0029] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available.

[0030] MRS liquid culture medium was purchased from Qingdao Haibo Technology Biotechnology Co., Ltd.

[0031] All strains involved in the examples can be purchased from the corresponding preservation institutions.

[0032] The preparation method of kudzu enzymatic hydrolysate in this embodiment of the invention is as follows: Fresh kudzu root is washed and cut into pieces, and water equivalent to 1.5 times the weight of kudzu root is added to extract the juice. Then, it is gelatinized at 95°C for 30 min, cooled to 60°C, 6 U / g (based on the weight of fresh kudzu root) of α-amylase is added and liquefied for 2.5 h. 300 U / g (based on the weight of fresh kudzu root) of glucoamylase is added and saccharified for 1.5 h. After filtration through a 200-mesh filter, the filtrate is centrifuged at 6000 rpm for 10 min, and the supernatant is collected. The supernatant is sterilized at 115°C for 20 min. The resulting sterilized kudzu enzymatic hydrolysate is the kudzu enzymatic hydrolysate used in the experiment.

[0033] Detection method in this embodiment of the invention: Viable bacteria count: Refer to GB 4789.2-2022.

[0034] pH determination: Refer to GB 5009.237-2016.

[0035] Determination of total acid: Refer to the pH meter potentiometric titration method in GB 123456-2021.

[0036] Determination of reducing sugar content: Preparation of standard curve: Prepare standard solutions with reducing sugar concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL. Using deionized water as a blank, add 0.5 mL of DNS solution to 0.5 mL of the standard solution, mix well, and heat in a boiling water bath for 5 min to ensure complete reaction. After cooling, add 2 mL of deionized water, mix well, and measure the absorbance at a wavelength of 540 nm. Plot the absorbance on the ordinate and the reducing sugar content on the abscissa to construct a standard curve. The standard curve is: y = 1.2576x - 0.0204, R0 2 =0.9987. Determination of reducing sugar content in samples: Dilute the sample to a reducing sugar content of 0-1.0 mg / mL, and conduct the reaction according to the method for preparing the standard curve. Calculate the reducing sugar content using the standard curve.

[0037] Determination of total flavonoid content: Preparation of the total flavonoid standard curve: A 0.2 mg / ml puerarin standard solution was prepared using anhydrous ethanol, and then diluted to 0.002, 0.004, 0.006, 0.008, 0.01, 0.012, 0.014, 0.016, and 0.018 mg / ml standard solutions, respectively. Using anhydrous ethanol as a blank, the absorbance at 250 nm was measured. The standard curve was plotted with absorbance on the ordinate and puerarin mass concentration on the abscissa. The standard curve was: y = 96.1514x - 6.8727, R0 2 =0.9998. Determination of total flavonoid content in the sample: The absorbance was measured according to the standard curve preparation method, and the total flavonoid content was calculated through the standard curve.

[0038] Volatile flavor compounds: 5.000±0.001 ml of sample was placed in a 20.00 ml gas chromatograph, and 0.5 ml of 100 mg / L 2,4,6-trimethylpyridine (TMP) was added as an internal standard for semi-quantitative analysis. The extraction head was exposed in the headspace of the vial, approximately 1 cm above the sample surface. The sample was shaken at 60 °C for 30 s, and then desorbed into the injection port at 250 °C within 3 min. GC-MS conditions: A TG-WAXA column (60 m × 0.25 mm × 0.25 μm) was used. The column temperature program was: 40 °C initial temperature (hold for 1 min), increased to 180 °C at 3 °C / min, and then increased to 230 °C at 20 °C / min (hold for 15 min). Mass spectrometry conditions: EI ion source (emission current 80 μA, ionization energy 70 eV), mass scan range 30 m / z ~ 500 m / z, interface and ion source temperatures maintained at 250℃ and 260℃ respectively, detection voltage 1000 V. Accurate qualitative analysis of volatile components was performed based on retention indices established using external standards for C7 ~ C26 n-alkanes.

[0039] Sensory evaluation: Ten sensory evaluators with a background in food science were selected to evaluate the fermented products. The scoring was based on a 100-point scale, and the final average was taken. Specific scoring criteria are shown in Table 1.

[0040] Table 1

[0041] Example 1 This embodiment provides a method for screening strains based on synergistic compound microbial fermentation agents, specifically as follows: The six activated lactic acid bacteria strains were added to an equal volume of kudzu root enzymatic hydrolysate at a total inoculum of 2% (v / w), mixed thoroughly, and fermented at 37℃ for 4 days. Samples were taken daily from the start of fermentation (3 parallel samples per group) to detect the microbial growth, pH and total acid, reducing sugar content, and flavonoid content on the second day of fermentation.

[0042] (1) Stability of kudzu root enzymatic hydrolysate at 37℃ To eliminate the interference of non-biochemical reactions occurring during storage of the kudzu enzymatic hydrolysate at 37°C, the sterilized kudzu enzymatic hydrolysate was placed in a 37°C incubator for 4 days. Samples were taken on days 0, 1, 2, 3, and 4 to determine the viable cell count, pH, reducing sugar, and flavonoid content. The results are shown in Table 2.

[0043] As can be seen from the table above, when the sterilized kudzu enzymatic hydrolysate is cultured at 37°C, no microorganisms will grow during the culture process. Furthermore, the total acid and reducing sugar content does not change significantly during the culture process, while the pH and total flavonoids show a slight decreasing trend, but the overall changes are small.

[0044] (2) Activation of the strain The six lactic acid bacteria listed in Table 3—Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei, Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus acidophilus, and Lactobacillus fermentum—were inoculated into MRS liquid medium and activated for two generations. The first generation was incubated at 37°C for 48 hours, and the second generation was incubated at 37°C for 24 hours.

[0045] After incubation, centrifuge at 10000 rpm for 10 min at 4℃, discard the supernatant, wash twice with an equal volume of sterile 0.9% physiological saline, and then resuspend in sterile 0.9% physiological saline. Adjust the final colony count of each activated bacterium to 10-1. 8 CFU / mL, for later use.

[0046] GDMCC is the Guangdong Provincial Microbial Culture Collection Center; CICC is the China Industrial Microbial Culture Collection Center.

[0047] (3) The growth of microorganisms during the fermentation process was monitored and recorded at regular intervals every day from the start of fermentation. The results are shown in Table 4.

[0048] From the perspective of different fermentation days within the same group, the growth trends of microorganisms in each group in Table 4 are generally similar. They multiply rapidly on the first and second days, and tend to stabilize or even decrease in number on the third and fourth days.

[0049] Overall, all six different lactic acid bacteria strains showed good growth in kudzu root enzymatic hydrolysate, and the viable cell counts did not differ significantly during fermentation. This indicates that all six strains can grow in kudzu root enzymatic hydrolysate and exert their fermentation characteristics.

[0050] (4) The pH changes of each group during fermentation are as follows: Figure 1 As shown, lactic acid bacteria continuously ferment and produce acid during the fermentation process to lower the pH of the system. The pH of the system before fermentation is around 6.20.

[0051] On the first and second days of fermentation, the pH values ​​decreased significantly. On the first day, the pH values ​​for each group were 4.02, 4.20, 3.96, 4.03, 4.08, and 4.43, respectively. On the second day, the pH values ​​were 3.82, 3.88, 3.76, 3.85, 3.88, and 4.03, respectively. Towards the later stages of fermentation, on the third and fourth days, the pH values ​​for each group stabilized, showing no significant downward trend and remaining below 4.0.

[0052] (5) Changes in total acidity in each group during fermentation are as follows: Figure 2 As shown, the total acid content of the systems before fermentation was 1.38 mg / mL, 1.73 mg / mL, 1.72 mg / mL, 1.61 mg / mL, 1.42 mg / mL, and 1.34 mg / mL, respectively. After four days of fermentation, the total acid content of the systems was 9.31 mg / mL, 9.95 mg / mL, 9.74 mg / mL, 9.01 mg / mL, 11.12 mg / mL, and 10.34 mg / mL, respectively.

[0053] The changes in total acid content in the figure show that acid accumulation mainly occurs in the first three days of fermentation. The total acid content remains basically stable on the fourth day of fermentation. Furthermore, the Lc, La, and Lf groups produce more acid during fermentation. The formation of acidic substances has a positive effect on the formation of flavor compounds.

[0054] (6) In the production of fermented foods, the consumption of reducing sugars is directly related to the growth of microorganisms and the formation of metabolites, and is one of the key indicators for evaluating the dynamics of the fermentation process. The changes in reducing sugar content in each group during fermentation are shown in the figure below. Figure 3 As shown, the reducing sugar content in each group decreased rapidly in the early stage of fermentation, and the change slowed down on the fourth day of fermentation.

[0055] Before fermentation, the reducing sugar contents of each group were 57.12 mg / mL, 61.52 mg / mL, 55.21 mg / mL, 59.88 mg / mL, 58.90 mg / mL, and 58.42 mg / mL, respectively. On the fourth day of fermentation, the reducing sugar contents of each group were 45.65 mg / mL, 52.70 mg / mL, 47.61 mg / mL, 47.40 mg / mL, 38.12 mg / mL, and 43.98 mg / mL, respectively. In the early stage of fermentation, lactic acid bacteria use sugars for metabolism, accelerate growth, and produce acidic substances.

[0056] (7) Flavonoids are an important reference indicator for the quality of kudzu-related products and a significant indicator for evaluating the fermentation performance of lactic acid bacteria in kudzu enzymatic hydrolysate. The flavonoid content of each group on the second day of fermentation is as follows: Figure 4 As shown, the flavonoid content in the control group and each fermentation group was 0.96 mg / mL, 0.91 mg / mL, 0.97 mg / mL, 0.94 mg / mL, 0.94 mg / mL, 0.93 mg / mL, and 0.96 mg / mL, respectively. The flavonoid content in the Lb group, Lp group, Pp group, and La group was significantly lower than that in the control group, while the Lc group was significantly higher than that in the control group. There was no significant difference between the Lf group and the control group.

[0057] Example 2 This embodiment involves combining *Lactobacillus casei* and *Lactobacillus fermentum* selected in Example 1 for mixed fermentation, and comparing this with single fermentation of *Lactobacillus casei* and *Lactobacillus fermentum*, resulting in three groups: Lc group, Lf group, and Lc+Lf group, specifically: Lactobacillus casei and Lactobacillus fermentum were inoculated into MRS liquid medium and activated for two generations. The first generation was cultured at 37°C for 48 hours, and the second generation was cultured at 37°C for 24 hours.

[0058] After incubation, centrifuge at 10000 rpm for 10 min at 4℃, discard the supernatant, wash twice with an equal volume of sterile 0.9% physiological saline, and then resuspend in sterile 0.9% physiological saline. Adjust the final colony count of each activated bacterium to 10-1. 8 CFU / mL, for later use. The Lc+Lf group consists of a 1:1 mixture of *Lactobacillus casei* and *Lactobacillus fermentum*.

[0059] Add 2% (v / w) of the total inoculum to an equal volume of kudzu root enzymatic hydrolysate, mix well, and incubate at 37°C for 4 days. Take samples daily from the start of fermentation (3 parallel samples per group) to detect microbial growth, pH and total acid, reducing sugar content, and flavonoid content during fermentation.

[0060] (1) The growth of microorganisms during the fermentation process was monitored and recorded at regular intervals every day from the start of fermentation. The results are shown in the table below. Figure 5 The viable cell count in the Lc group significantly increased on the first day of fermentation, and showed a slight increasing trend in the later stages of fermentation, but the change was slow. Both the Lf and Lc+Lf groups reached their highest viable cell counts on the first day of fermentation, with the Lf group showing a gradual decrease in viable cell count from the second to the fourth day. The viable cell count in the Lc+Lf group began to decline on the second day, with no significant decreasing trend on the third and fourth days. Throughout the fermentation process, the viable cell count in all groups remained at 10. 8 above.

[0061] (2) The pH changes of each group during fermentation are as follows: Figure 6 As shown, the initial pH of each group before fermentation was 6.42-6.43, with no significant difference. On the first day of fermentation, the pH of the system decreased significantly; from the second to the fourth day of fermentation, it tended to stabilize but still showed a slight downward trend. On the fourth day of fermentation, the pH of the system was below 3.9 for all groups: Lc group: 3.83; Lf group: 3.90; Lc+Lf group: 3.86. The pH of the Lf group was significantly higher than that of the other two groups.

[0062] (3) Changes in total acidity in each group during fermentation are as follows: Figure 7 As shown, the total acid content of the systems before fermentation was 0.94 mg / mL, 1.04 mg / mL, and 1.02 mg / mL, respectively. After four days of fermentation, the total acid content of each group was 9.02 mg / mL, 9.84 mg / mL, and 10.18 mg / mL, respectively. The changes in total acid content in the graph show that acid accumulation mainly occurs in the early stage of fermentation, while the rate of acid formation slows down in the later stage. Furthermore, the total acid content of the mixed fermentation group (Lc+Lf group) was significantly higher than that of the other two groups on the fourth day. This indicates that Lc+Lf mixed fermentation produces more acid during fermentation, and the formation of acidic substances has a positive effect on the formation of flavor compounds.

[0063] (4) In the production of fermented foods, the consumption of reducing sugars is directly related to the growth of microorganisms and the formation of metabolites, and is one of the key indicators for evaluating the dynamics of the fermentation process. The changes in reducing sugar content in each group during fermentation are shown in the figure below. Figure 8As shown, the reducing sugar consumption rate of the Lf group and the Lc+Lf group was significantly higher than that of the Lc group, but the overall trend was basically the same. In the early stage of fermentation, sugars were rapidly used for growth, reproduction and acid production, while in the later stage of fermentation, the reducing sugar consumption rate decreased.

[0064] (5) Changes in flavonoid content in each group during fermentation, as shown in the figure. Figure 9 As shown, the total flavonoid content of each group before fermentation was 0.832 mg / mL, 0.843 mg / mL, and 0.835 mg / mL, respectively. The total flavonoid content of the Lc and Lf groups reached its maximum on the first day of fermentation, at 0.871 mg / mL and 0.867 mg / mL, respectively. The total flavonoid content decreased slightly from the second to the fourth day of fermentation. The total flavonoid content of the Lc+Lf group showed a slight upward trend from the first to the third day of fermentation, reaching its maximum on the third day at 0.867 mg / mL. The total flavonoid content decreased significantly on the fourth day of fermentation.

[0065] Example 3 This example demonstrates the effectiveness of combining different strains in improving the flavor of kudzu products. Specifically: (1) After the fermentation of each group in Example 2 was completed, the content and distribution of volatile flavor compounds in the products of each group were detected by GC-MS combined with headspace solid-phase microextraction. The results are shown in Table 5 and Figure 10 .

[0066] Table 5 shows that there were significant differences in the content of volatile flavor compounds among the fermented kudzu hydrolysate samples. Before fermentation, the total volatile flavor compound content of each group was 8.33 μg / ml. The total volatile flavor compound content of the Lc group reached its maximum on the first day, at 287.68 μg / ml; the Lf group reached its maximum on the second day, at 642.97 μg / ml; and the Lc+Lf group reached its maximum on the third day, at 792.05 μg / ml. The volatile flavor compounds produced were mainly acids, alcohols, and phenols, with alcohols accounting for approximately 50% of the total volatile flavor compounds. Compared with single fermentation groups, mixed fermentation with *Lactobacillus casei* and *Lactobacillus fermentatus* significantly increased the content of volatile flavor compounds.

[0067] Figure 10This study illustrates the changes in the types and contents of volatile compounds in the three groups of samples during fermentation. The figure shows that the types of volatile compounds in the kudzu root hydrolysate changed significantly during fermentation, with the generation of large amounts of acids (such as glacial acetic acid), alcohols (such as n-hexanol), and phenolic compounds (such as 2,4-di-tert-butylphenol). Compared to single fermentation groups, the mixed fermentation of *Lactobacillus casei* and *Lactobacillus fermentatus* resulted in a richer variety of volatile flavor compounds in the kudzu root fermentation broth.

[0068] (2) To verify the flavor-enhancing effect of lactic acid bacteria fermentation on kudzu root hydrolysate, a sensory experiment was conducted, and the results are as follows: Figure 11 As shown in the figure, the unfermented kudzu root hydrolysate and the kudzu root hydrolysate after 48 hours of fermentation were evaluated in terms of color, texture, aroma, and taste. As can be seen from the figure, all three fermentation groups showed significant improvements in color, aroma, and taste compared to the unfermented group, and the compound fermentation group showed even greater improvements in color, aroma, and taste compared to the single fermentation group.

[0069] This indicates a positive metabolic interaction between the two bacteria, potentially through mutual provision of growth factors or optimization of substrate utilization pathways, jointly driving a more vigorous flavor compound synthesis metabolic network, ultimately achieving a targeted and enhanced flavor enhancement of kudzu root beverages. This predictable and reproducible flavor enhancement effect represents a significant technological advancement of this invention.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A composite microbial fermentation agent based on synergistic effect, characterized in that: The compound microbial fermentation agent is obtained by combining fermenting Lactobacillus mucilaginosus and Lactobacillus casei. The compound microbial fermentation agent can increase the total amount of volatile flavor substances and inhibit miscellaneous bacteria in the kudzu fermentation system.

2. The compound microbial fermentation agent as described in claim 1, characterized in that: The total viable bacteria concentration of the compound microbial fermentation agent is 10. 6 ~10 8 CFU / mL.

3. The preparation method according to claim 1, characterized in that: The ratio of viable bacteria of the fermenting *Lactobacillus mucinus* and *Lactobacillus casei* is 1:

1.

4. The application of the compound microbial fermentation agent as described in any one of claims 1 to 3 in the targeted enhancement of the flavor of fermented kudzu root beverages.

5. A method for enhancing the flavor of fermented kudzu root beverage, characterized in that: The compound microbial fermentation agent according to any one of claims 1 to 3 is added to the kudzu root hydrolysate to be fermented for fermentation.

6. The method as described in claim 5, characterized in that: The inoculum size of the compound microbial fermentation agent is 2.0% v / w.

7. The method as described in claim 5, characterized in that: The fermentation time is 24 to 72 hours.

8. The method as described in claim 5, characterized in that: The fermentation temperature was 37°C.

9. The method as described in claim 5, characterized in that: The total amount of volatile flavor compounds in the product obtained from the fermentation was significantly increased compared to the unfermented kudzu hydrolysate.

10. A fermented kudzu root beverage, characterized in that: The beverage is prepared by the method according to any one of claims 5 to 9, and the content of volatile flavor compounds of acids and alcohols in the beverage is significantly increased.

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