A compound lactic acid bacteria starter culture and its application, a method for producing Anoectochilus roxburghii fermentation broth and its products

Abstract

This invention relates to the field of microbial technology, and particularly to a compound lactic acid bacteria fermentation agent and its application, a method for producing *Anoectochilus roxburghii* fermentation broth, and related products. The compound lactic acid bacteria fermentation agent comprises *Lactobacillus plantarum* BXM2, *Lactobacillus fermentatus* J IAN, and *Lactobacillus paracasei* YYS-69. Fermenting *Anoectochilus roxburghii* broth using this compound fermentation agent results in stronger acid production, higher viable bacterial counts, and significantly improved antioxidant activity, total polyphenol and flavonoid content, and enhanced hypoglycemic activity. Sensory effects are also significantly improved. This compound lactic acid bacteria fermentation agent, when applied to the fermentation of traditional Chinese medicine, can enhance the antioxidant capacity and effective functional component content of the fermentation broth, thereby improving efficacy. It has great application prospects in the field of deep-processed *Anoectochilus roxburghii* products and even other fermented medicinal and edible products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound lactic acid bacteria fermentation agent and its application, a method for producing Anoectochilus roxburghii fermentation liquid and related products. Background Technology

[0002] *Anoectochilus roxburghii* (Wall.) Lindl., commonly known as Golden Thread Orchid, is an important traditional Chinese medicine in South China. In recent years, it has been widely used in medicine and cosmetics, with market demand increasing year by year. It has long been sought after by the market and is considered one of the most valuable medicinal plants. Golden Thread Orchid is rich in bioactive components such as alkaloids, polysaccharides, saponins, flavonoids, esters, and volatile oils. It possesses various medicinal properties, including clearing heat and cooling the blood, detoxifying and reducing swelling, dispelling wind and dampness, calming the nerves and liver, tonifying the kidneys and strengthening bones, promoting urination and relieving strangury, and moistening the lungs and relieving cough. It also plays a role in improving human immunity and in the recovery and treatment of chronic diseases of the liver, kidneys, and lungs. It has been used to prevent and treat diseases such as diabetes, hyperlipidemia, hepatitis, and tumors.

[0003] With societal development, people's demands for food are becoming increasingly diversified, with greater emphasis on its nutritional value and health benefits. Probiotic fermented products possess certain probiotic properties and are widely used in the prevention of physiological diseases. Research from the last century has demonstrated that probiotic fermented products can alleviate metabolic problems in the digestive tract and stomach. With the development of modern biotechnology, lactic acid bacteria fermentation technology has been applied to research related to traditional Chinese medicine (TCM), and fermented TCM using lactic acid bacteria as the fermentation strain has been widely reported. Traditional TCM fermentation often utilizes microorganisms from the natural environment, altering the medicinal properties of medicinal materials through their growth and metabolism, reducing toxicity, and improving efficacy and resource utilization. Modern TCM fermentation primarily uses strains commonly found in modern food fermentation industries, such as lactic acid bacteria, yeast, and Bacillus, as well as medicinal fungi like Ganoderma lucidum, Monascus purpureus, and Beauveria bassiana. Among these, lactic acid bacteria are widely used in TCM fermentation due to their high safety and market acceptance. Fermented lactic acid bacteria products (such as yogurt, cheese, kimchi, and natto) have functions such as improving the intestinal microenvironment, inhibiting the excessive growth of intestinal pathogens, protecting the intestinal barrier, regulating malabsorption, reducing lipid peroxidation, and inhibiting the release of inflammatory cytokines. Fermentation can combine the functions of lactic acid bacteria with traditional Chinese medicine (TCM), promoting the dissolution and transformation of the effective components of TCM and altering its functions. Furthermore, the intestinal microbiota-regulating function of lactic acid bacteria can assist in the efficacy of TCM, and the fermentation process can improve the bioavailability of TCM, allowing it to exert better medicinal effects.

[0004] With the expansion of the functional food market, lactic acid bacteria fermented foods have become one of the most popular functional foods. While *Anoectochilus roxburghii* (a type of orchid) is rich in bioactive substances, there are few deep-processed products, especially reports on the application of lactic acid bacteria fermentation. Therefore, using lactic acid bacteria to ferment *Anoectochilus roxburghii* to improve its sensory quality and enhance its functional activity has broad application prospects and market potential. Summary of the Invention

[0005] To address the shortcomings of lactic acid bacteria fermentation technology for Anoectochilus roxburghii, this invention provides a compound lactic acid bacteria starter and its application, a method for producing Anoectochilus roxburghii fermented liquid, and the product thereof. Using compound lactic acid bacteria to ferment Anoectochilus roxburghii liquid can not only maintain the food quality of Anoectochilus roxburghii, but also increase the in-situ expression of sensory attributes and enhance its functional biological activity, thereby promoting human health.

[0006] This invention provides a compound lactic acid bacteria starter culture, comprising Lactobacillus plantarum BXM2, Lactobacillus fermentum JIAN, and Lactobacillus paracasei YYS-69.

[0007] Furthermore, the mass ratio of *Lactobacillus plantarum* BXM2, *Lactobacillus fermentum* JIAN, and *Lactobacillus paracasei* YYS-69 is 1:1:1.

[0008] Furthermore, the *Lactobacillus plantarum* BXM2, *Lactobacillus fermentum* JIAN, and *Lactobacillus paracasei* YYS-69 are provided as bacterial powder, and the viable count of the *Lactobacillus plantarum* BXM2 bacterial powder is 1–5 × 10⁻⁶. 11 CFU / g, the viable count of the fermented Lactobacillus mucilaginosus JIAN powder is 1-5 × 10⁻⁶ CFU / g. 11 CFU / g, the viable count of the Lactobacillus paracasei YYS-69 powder is 1-5 × 10⁻⁶ CFU / g. 11 CFU / g. It should be noted that the above three types of bacteria are not limited to being provided in powder form; they can also be provided in liquid form.

[0009] This invention also provides the application of the above-mentioned compound lactic acid bacteria starter in the fermentation of traditional Chinese medicine, especially the application of fermenting Anoectochilus roxburghii.

[0010] Furthermore, the compound lactic acid bacteria starter culture includes at least one of the following functions:

[0011] (1) Increase the fermentation speed and increase the number of viable bacteria after fermentation;

[0012] (2) Increase antioxidant levels and total polyphenol and flavonoid content;

[0013] (3) Enhances hypoglycemic activity and inhibits α-amylase and glucosidase activity;

[0014] (4) Enhance sensory experience.

[0015] The present invention also provides a method for producing Anoectochilus roxburghii fermented liquid, which uses Anoectochilus roxburghii liquid as raw material and is produced by fermentation using the above-mentioned compound lactic acid bacteria fermentation agent.

[0016] Furthermore, the following steps are included:

[0017] (1) Using the extract of *Anoectochilus roxburghii* as raw material, the sugar content of the extract is adjusted to 50-60 g / L, and the sterilization conditions are 100-110℃ for 15-25 min; the preferred temperature is 105℃.

[0018] (2) Add the sterilized Anoectochilus roxburghii liquid from step (1) to the compound lactic acid bacteria fermentation agent;

[0019] (3) Fermentation conditions: fermentation temperature 35-40℃, fermentation time 40-50h.

[0020] Furthermore, the preparation method of the *Anoectochilus roxburghii* liquid is as follows: after cleaning the *Anoectochilus roxburghii*, add water and boil for 30 minutes, crush and pulp it, boil for another 30 minutes, and filter to obtain the processed *Anoectochilus roxburghii* liquid.

[0021] Preferably, the inoculum amount of the compound lactic acid bacteria starter is 0.02% of the mass of the Anoectochilus roxburghii liquid.

[0022] Preferably, the fermentation conditions are fermentation at 37°C for 45–48 hours.

[0023] The present invention also provides a *Anoectochilus roxburghii* fermentation liquid product, comprising *Anoectochilus roxburghii* fermentation liquid obtained by the above-described production method.

[0024] This invention compares the changes in physicochemical indicators, antioxidant indicators, polyphenol and flavonoid content, hypoglycemic function indicators, and sensory indicators of *Anoectochilus roxburghii* extract and its fermentation broth. The results show that the fermentation method in *Anoectochilus roxburghii* extract has the highest number of viable bacteria, the highest increase in free radical scavenging ability and polyphenol and flavonoid content, the best effect on enhancing hypoglycemic function, and the highest sensory evaluation score.

[0025] Compared with the prior art, the compound lactic acid bacteria starter provided by the present invention has the following beneficial effects:

[0026] This invention uses a compound lactic acid bacteria inoculation containing *Lactobacillus plantarum* BXM2, *Lactobacillus fermentatus* JIAN, and *Lactobacillus paracasei* YYS-69 for the fermentation of *Anoectochilus roxburghii*. This results in stronger acid production and a higher number of viable bacteria. Compared with single-strain fermentation, it significantly improves the free radical scavenging ability, polyphenol and flavonoid content, hypoglycemic activity, and sensory score of *Anoectochilus roxburghii*, giving the fermented *Anoectochilus roxburghii* liquid stronger nutritional value and providing better probiotic value for *Anoectochilus roxburghii* products. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 A comparison chart of DPPH free radical scavenging rates between *Anoectochilus roxburghii* extract and its fermentation broth;

[0029] Figure 2 A comparison chart of ABTS free radical scavenging rates between *Anoectochilus roxburghii* extract and its fermentation broth;

[0030] Figure 3 Comparison of the inhibitory effects of *Anoectochilus roxburghii* extract and its fermentation broth on α-amylase;

[0031] Figure 4 Comparison of the glucosidase inhibition effects of Anoectochilus roxburghii extract and its fermentation broth;

[0032] Figure 5 The image shows the sensory evaluation scores of *Anoectochilus roxburghii* extract and its fermentation broth. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] This invention provides a compound lactic acid bacteria starter culture, comprising Lactobacillus plantarum BXM2, Lactobacillus fermentum JIAN, and Lactobacillus paracasei YYS-69.

[0035] in,

[0036] Lactiplantibacillus plantarum BXM2 was deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 16436. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0037] Lactobacillus fermentum was deposited on April 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27027. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0038] Lactobacillus paracasei YYS-69 was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25837, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0039] It should be noted that in October 2022, the National Health Commission officially renamed "Lactobacillus plantarum" to "Lactobacillus plantarum" and "Lactobacillus fermentum" to "Lactobacillus fermentum". At the time of preservation, the names had not yet been changed. Therefore, "Lactobacillus plantarum BXM2" in the instruction manual and "Lactobacillus plantarum BXM2" in the preservation certificate are the same strain, and "Lactobacillus fermentum JIAN" and "Lactobacillus fermentum JIAN" in the preservation certificate are the same strain.

[0040] The compound lactic acid bacteria starter culture has at least one of the following functions:

[0041] (1) Increase the fermentation speed and increase the number of viable bacteria after fermentation;

[0042] (2) Increase antioxidant levels and total polyphenol and flavonoid content;

[0043] (3) Enhances hypoglycemic activity and inhibits α-amylase and glucosidase activity;

[0044] (4) Enhance sensory experience.

[0045] This invention also provides an operational example of a method for producing *Anoectochilus roxburghii* fermentation liquid, comprising the following steps:

[0046] (1) Using the extract of *Anoectochilus roxburghii* as raw material, the sugar content of the extract is adjusted to 50-60 g / L, and the sterilization conditions are 100-110℃ for 15-25 min;

[0047] (2) Add the compound lactic acid bacteria fermentation agent to the sterilized Anoectochilus roxburghii solution in step (1), wherein the inoculum amount of the compound lactic acid bacteria fermentation agent is 0.02% of the mass of the Anoectochilus roxburghii solution;

[0048] (3) Fermentation conditions: fermentation temperature 35-40℃, fermentation time 40-50h.

[0049] It should be noted that all reagents used in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] The culture media used in the strain screening and performance determination in the embodiments of the present invention are as follows:

[0051] MRS medium (g / L): yeast extract 5, peptone 10, beef extract 10, glucose 10, sodium acetate 5, diammonium hydrogen citrate 2, magnesium sulfate 0.6, manganese sulfate 0.3, Tween-80 1.

[0052] MRS medium plate (g / L): yeast extract 5, peptone 10, beef extract 10, glucose 10, sodium acetate 5, diammonium hydrogen citrate 2, magnesium sulfate 0.6, manganese sulfate 0.3, Tween-80 1, agar powder 10.

[0053] Golden Thread Orchid Fermentation Medium: Adjust the sugar content of the Golden Thread Orchid liquid to 50g / L, and sterilize it in a high-pressure steam cooker at 105℃ for 10min.

[0054] In one embodiment, the fermentation process of the strain fermenting Anoectochilus roxburghii is as follows: inoculate with compound lactic acid bacteria fermentation agent at a mass ratio of 0.02% and ferment at 37°C for 48 hours.

[0055] Example 1: Preparation of Lactobacillus plantarum BXM2 bacterial powder

[0056] 400 μL of live Lactobacillus plantarum BXM2 was inoculated into 20 mL of MRS liquid medium and activated at 37°C for 2 to 3 generations until the viable count reached 10⁻⁶. 9 When the viable bacterial count is above CFU / mL, centrifuge the culture medium at 5000–10000 rpm for 10–20 min, remove the supernatant, and add a lyophilization protectant containing 15% glycerol, 10% milk powder, and 2% monosodium glutamate under aseptic conditions. Emulsify with the lyophilization protectant for 15 min to obtain a bacterial suspension with a concentration of 10. 10 An emulsion with CFU / mL was pre-frozen at -40°C for 4 h and then freeze-dried at -35°C for 35 h to obtain a viable bacterial count of 1–5 × 10⁻⁶. 11 Freeze-dried Lactobacillus plantarum BXM2 active bacterial powder (CFU / g).

[0057] Example 2: Preparation of fermented Lactobacillus mucilaginosus JIAN powder

[0058] 400 μL of fermenting *Lactobacillus mucinus* JIAN was inoculated into 20 mL of MRS liquid medium and activated at 37°C for 2 to 3 generations until the viable count reached 10⁻⁶. 9When the viable bacterial count is above CFU / mL, centrifuge the culture medium at 5000–10000 rpm for 10–20 min, remove the supernatant, and add a lyophilization protectant containing 15% glycerol, 10% milk powder, and 2% monosodium glutamate under aseptic conditions. Emulsify with the protectant for 15 min to obtain a bacterial suspension with a concentration greater than 10. 10 An emulsion with CFU / mL was pre-frozen at -40°C for 4 hours and then freeze-dried at -35°C for 35 hours to obtain a viable bacterial count of 1–5 × 10⁻⁶. 11 Freeze-dried Lactobacillus mucinus JIAN active fermentation microbial powder (CFU / g).

[0059] Example 3: Preparation of Lactobacillus paracasei YYS-69 bacterial powder

[0060] 400 μL of Lactobacillus paracasei YYS-69 was inoculated into 20 mL of MRS liquid medium and activated at 37°C for 2 to 3 generations until the viable count reached 10⁻⁶. 9 When the viable bacterial count is above CFU / mL, centrifuge the culture medium at 5000–10000 rpm for 10–20 min, remove the supernatant, and add a lyophilization protectant containing 15% glycerol, 10% milk powder, and 2% monosodium glutamate under aseptic conditions. Emulsify with the protectant for 15 min to obtain a bacterial suspension with a concentration greater than 10. 10 An emulsion with CFU / mL was pre-frozen at -40°C for 4 hours and then freeze-dried at -35°C for 35 hours to obtain a viable bacterial count of 1–5 × 10⁻⁶. 11 Freeze-dried Lactobacillus paracasei YYS-69 bacterial powder, CFU / g.

[0061] Example 4: Preparation of Compound Lactic Acid Bacteria Fermentation Agent

[0062] The freeze-dried bacterial powders obtained in Examples 1-3 were mixed evenly at a mass ratio of 1:1:1 to obtain a compound lactic acid bacteria starter.

[0063] Example 5: Preparation of Anoectochilus roxburghii extract

[0064] Select undamaged, intact *Anoectochilus roxburghii* raw materials, wash away the dust and sand on the surface of the *Anoectochilus roxburghii* with clean water, rinse with purified water, add to water at a mass ratio of 1%, boil for 30 minutes, crush and pulp, boil again for 30 minutes, and filter to obtain *Anoectochilus roxburghii* liquid.

[0065] Example 6: Effects of Fermentation by Different Microbial Strains on Physicochemical Indicators

[0066] The *Lactobacillus plantarum* BXM2 powder, *Lactobacillus fermentatus* JIAN powder, *Lactobacillus paracasei* YYS-69 powder, any two of the three powders mixed in a 1:1 mass ratio to form a starter culture, and a compound lactic acid bacteria starter culture were inoculated at 0.02% by mass into the *Anoectochilus roxburghii* extract prepared in Example 5 and fermented at 37°C for 48 hours. The basic physicochemical properties of the fermentation broth were measured, and the results are shown in Table 1.

[0067] Among them, the starter culture formed by mixing BXM2 plantarum powder and JIAN fermentation mucinous bacteria powder in a 1:1 mass ratio is denoted as B+J; the starter culture formed by mixing BXM2 plantarum powder and YYS-69 paracasei bacteria powder in a 1:1 mass ratio is denoted as B+Y; the starter culture formed by mixing JIAN fermentation mucinous bacteria powder and YYS-69 paracasei bacteria powder in a 1:1 mass ratio is denoted as J+Y; and the compound lactic acid bacteria starter culture is denoted as compound bacteria, and the same applies below.

[0068] It should be noted that the initial inoculation amount of the above fermentation strains is equal, that is, the initial total number of viable cells is the same.

[0069] Table 1. Results of determination of basic physicochemical properties of Anoectochilus roxburghii and its fermentation broth.

[0070]

[0071] Note: Different letters indicate significant differences in the data within the same column. P <0.05).

[0072] Table 1 shows that the soluble solids content in the *Anoectochilus roxburghii* extract was the highest. After inoculation with lactic acid bacteria, the soluble solids content decreased to varying degrees. The samples fermented with the compound fermentation agent showed a 21.62% reduction in soluble solids, higher than the reduction achieved by mixing individual bacteria. BXM2 and YYS-69 showed the lowest reduction, at only 12.16%. Comparison of total acid and total sugar results among different samples revealed that the fermentation broth from the *Anoectochilus roxburghii* compound fermentation agent had the lowest total sugar content (32.01 g / L) and the highest total acid content (8.25 g / L). Furthermore, after 48 hours of fermentation, the viable bacteria count in the *Anoectochilus roxburghii* extract was highest in the compound fermentation group (9.24 lg CFU / mL), indicating that the lactic acid bacteria in the compound fermentation group exhibited the strongest growth capacity in the *Anoectochilus roxburghii* extract. In summary, *Lactobacillus plantarum* BXM2, *Lactobacillus fermentatus* JIAN, and *Lactobacillus paracasei* YYS-69 can all grow individually in *Anoectochilus roxburghii* solution. However, under the same inoculum amount, the three-strain compound fermentation group exhibits higher growth activity, stronger fermentation capacity, and stronger acid production capacity.

[0073] Example 7: Effects of Fermentation of Different Microbial Strains on Antioxidant Indicators

[0074] The antioxidant activity of each fermentation sample in Example 6 was evaluated based on two free radicals, DPPH and ABTS, and the scavenging rate of each sample against DPPH and ABTS free radicals was detected. The test method was as follows: 0.2 mL of the test sample or anhydrous ethanol control was added to 0.2 mL of DPPH solution, shaken to mix, and reacted at room temperature for 40 min in the dark. The absorbance (wavelength 517 nm) was measured, and the ratio of the absorbance to that of the blank control was the DPPH free radical scavenging rate. The results are shown in […]. Figure 1 Take 0.2 mL of the test sample and anhydrous ethanol control, add 4.8 mL of ABTS free radicals, react at room temperature in the dark for 30 min, and measure the absorbance at 734 nm. The ratio of the absorbance to the blank control absorbance is the ABTS free radical scavenging rate. The results are shown in [Figure number missing]. Figure 2 .from Figure 1 It can be seen that the DPPH free radical scavenging rate of each product was significantly improved after the fermentation of Anoectochilus roxburghii liquid, and the free radical scavenging rate of the compound bacteria fermentation group was significantly higher than that of the other groups. Figure 2 The ABTS assay for free radical scavenging showed that the ABTS free radical scavenging rate of the compound bacteria fermentation group was significantly the highest, followed by the *Anoectochilus roxburghii* extract fermented by BXM2. The addition of *Lactobacillus mucilaginosus* JIAN or *Lactobacillus paracasei* YYS-69 did not significantly improve the free radical scavenging rate. Lactic acid bacteria fermentation enhanced the free radical scavenging capacity of both DPPH and ABTS. This is likely due to the inherent antioxidant properties of the strains themselves, as well as the hydrolytic enzymes within the strains breaking down complex phenolic polymers into simpler forms, thus enhancing the antioxidant properties of the product. Compound bacteria fermentation further enhanced the growth and metabolic capacity of the strains, altering their growth and metabolic environment and further promoting the enhancement of antioxidant capacity.

[0075] Example 8: Effect of Fermentation by Different Microbial Strains on Polyphenol Flavonoid Content

[0076] Polyphenols and flavonoids in plants exist mainly in two forms: free and bound. Lactic acid bacteria can decompose bound polyphenols and flavonoids in plants into free forms and enhance their biological activity. This is because they can produce corresponding enzymes during fermentation, thereby improving their antioxidant activity. The total polyphenol (gallic acid equivalent) and total flavonoid (rutin equivalent) contents of each fermentation sample in Example 6 are shown in Table 2. The results show that the total polyphenol and total flavonoid contents of *Anoectochilus roxburghii* extract after lactic acid bacteria fermentation are increased to varying degrees, with the *Anoectochilus roxburghii* extract fermented with compound bacteria showing the highest total polyphenol and total flavonoid contents. Lactic acid bacteria can desugar glycosylated phenolic and glycoside substances and release free polyphenols and flavonoids from the plant cell wall. The compound bacterial fermentation of these three strains provides a more suitable environment for the growth and metabolism of the bacteria, resulting in vigorous physiological activity and promoting this process. Polyphenols and flavonoids are both functional components of Anoectochilus roxburghii. The compound lactic acid bacteria fermentation agent of this invention can significantly increase the total polyphenols and total flavonoids of Anoectochilus roxburghii after fermentation, and also improve the functionality and edible value of Anoectochilus roxburghii fermented products.

[0077] Table 2. Results of determination of total polyphenols and total flavonoids in Anoectochilus roxburghii and its fermentation broth

[0078]

[0079] Note: Different letters indicate significant differences in the data within the same column. P <0.05).

[0080] Example 9: Effects of Fermentation by Different Microbial Strains on Hypoglycemic Activity

[0081] Most antidiabetic drugs currently regulate blood sugar by inhibiting the activity of α-amylase and α-glucosidase, controlling postprandial blood glucose levels by inhibiting their activity and reducing glucose production and absorption. *Anoectochilus roxburghii* has been shown to have hypoglycemic activity. This study investigated the inhibitory effects of various fermentation samples on α-amylase and α-glucosidase activity in Example 6. The specific test method was as follows: 100 µL of α-amylase solution (40 mg / mL) and 50 µL of each fermentation sample were pre-incubated at 37°C for 10 min. Then, 100 µL of soluble starch solution was added to initiate the reaction. After 5 min, 750 µL of DNS solution was added, and the reaction was stopped by boiling in a water bath for 10 min. The mixture was then cooled to room temperature on ice. The reaction solution was diluted, and the absorbance was measured at 540 nm. The ratio of the absorbance to the blank control showed the inhibitory effect on amylase activity. The influence of the results was as follows: Figure 3Prepare a 0.5 mmol / L substrate PNPG solution, and add 40 μL of 0.4 U / mL glucosidase solution and 40 μL of each fermentation sample to a test tube. Heat in a water bath at 37°C for 5 min. Add 20 μL of the 0.5 mmol / L substrate PNPG solution, and treat the reaction system in a 37°C water bath for 30 min. The reaction is terminated with 50 μL of 0.2 mol / L Na₂CO₃ solution. Treat at room temperature (25°C) for 5 min, and measure the absorbance at 405 nm. The ratio of the absorbance to the blank control indicates the inhibitory effect on glucosidase activity. The influence of the results is as follows: Figure 4 .

[0082] Depend on Figure 3 It can be seen that the *Anoectochilus roxburghii* compound fermentation group exhibited the highest inhibitory activity against α-amylase, and at the same level, the inhibition rate was 221% higher than that of the *Anoectochilus roxburghii* solution before fermentation. Meanwhile, the *Anoectochilus roxburghii* solution fermented with a single strain BXM2 showed the highest inhibitory activity. Figure 4 It can be seen that the inhibitory effect of the compound bacteria fermentation group on glucosidase is significantly higher than that of other groups. Therefore, the compound lactic acid bacteria starter can significantly enhance the hypoglycemic activity of Anoectochilus roxburghii, laying the foundation for the development of functional hypoglycemic Anoectochilus roxburghii fermented products.

[0083] Example 10: Effects of Fermentation by Different Microbial Strains on Sensory Indicators

[0084] Sensory evaluation was primarily conducted by the laboratory's sensory evaluation team. The team consisted of 20 researchers selected through sensory evaluation training. During the evaluation process, each sample was scored based on its appearance, aroma, taste, and typicality according to the sensory evaluation table for *Anoectochilus roxburghii* extract and its fermentation liquid (Table 3). The results are shown below. Figure 5 The sensory evaluation results show that the sensory scores of all samples after lactic acid bacteria fermentation of *Anoectochilus roxburghii* liquid were significantly improved compared with those before fermentation. In particular, the *Anoectochilus roxburghii* liquid after fermentation with compound bacteria became more uniform in color, had a rich aroma, a mellow taste, a balanced sweet and sour flavor, and obvious fermentation characteristics. Its overall sensory score was significantly higher than the others.

[0085] Table 3 Sensory Evaluation Table of Anoectochilus roxburghii and its Fermentation Broth

[0086]

[0087] Based on the results of the above embodiments, the compound lactic acid bacteria starter provided by the present invention has the following functions and effects in the fermentation of *Anoectochilus roxburghii* liquid:

[0088] 1. It has strong growth ability in Anoectochilus roxburghii solution. Under the same inoculum amount, the compound fermentation agent has higher growth activity, stronger fermentation ability and stronger acid production ability compared with single strain fermentation and dual strain mixed fermentation.

[0089] 2. The *Anoectochilus roxburghii* extract fermented with the compound fermentation agent showed the strongest antioxidant activity compared to the extract before fermentation and the extract fermented with a single strain. It also exhibited the highest DPPH and ABTS free radical scavenging rates at the same concentration.

[0090] 3. After fermentation of Anoectochilus roxburghii with compound lactic acid bacteria starter, the total polyphenols and total flavonoids of Anoectochilus roxburghii can be significantly increased, which also improves the functionality and edible value of Anoectochilus roxburghii fermented products.

[0091] 4. Compound lactic acid bacteria starter can significantly enhance the hypoglycemic activity of Anoectochilus roxburghii, laying the foundation for the development of functional hypoglycemic Anoectochilus roxburghii fermented products;

[0092] 5. After fermentation with compound bacteria, the appearance of the Anoectochilus roxburghii liquid becomes uniform in color, with a rich aroma, mellow taste, and a balanced sweet and sour flavor. It has obvious fermentation characteristics and its overall sensory score is significantly higher than that of the Anoectochilus roxburghii liquid before fermentation and the Anoectochilus roxburghii liquid fermented with single bacteria and two bacteria.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound lactic acid bacteria starter culture, characterized in that: Including Lactobacillus plantarum ( Lactiplantibacillus plantarum BXM2, Lactobacillus fermentum ( Lactobacillus fermentum JIAN and Lactobacillus paracasei ( Lactobacillus paracasei YYS-69, the preservation number of the plant lactobacillus BXM2 is CGMCC No. 16436, the preservation number of the fermenting myxobacillus JIAN is CGMCC No. 27027, and the preservation number of the paracasei lactobacillus YYS-69 is CGMCC No. 25837.

2. The compound lactic acid bacteria starter culture according to claim 1, characterized in that: The mass ratio of *Lactobacillus plantarum* BXM2, *Lactobacillus fermentum* JIAN, and *Lactobacillus paracasei* YYS-69 is 1:1:

1.

3. The compound lactic acid bacteria starter culture according to claim 1, characterized in that: The *Lactobacillus plantarum* BXM2, *Lactobacillus fermentum* JIAN, and *Lactobacillus paracasei* YYS-69 are provided as bacterial powder, and the viable count of the *Lactobacillus plantarum* BXM2 bacterial powder is 1–5 × 10⁻⁶. 11 CFU / g, the viable count of the fermented Lactobacillus mucilaginosus JIAN powder is 1-5 × 10⁻⁶ CFU / g. 11 CFU / g, the viable count of the Lactobacillus paracasei YYS-69 powder is 1-5 × 10⁻⁶ CFU / g. 11 CFU / g.

4. The application of the compound lactic acid bacteria starter as described in any one of claims 1 to 3 in the fermentation of Anoectochilus roxburghii.

5. The application according to claim 4, characterized in that: The compound lactic acid bacteria starter culture has at least one of the following functions: (1) Increase the fermentation speed and increase the number of viable bacteria after fermentation; (2) Improve the antioxidant level and the content of total polyphenols and total flavonoids in the fermentation liquid of Anoectochilus roxburghii; (3) Enhance the hypoglycemic activity of the fermentation broth of Anoectochilus roxburghii and inhibit the activity of α-amylase and glucosidase in the fermentation broth of Anoectochilus roxburghii; (4) Improve the sensory evaluation score of the Anoectochilus roxburghii fermentation liquid.

6. A method for producing *Anoectochilus roxburghii* fermentation liquid, characterized in that: The product is produced by fermentation using *Anoectochilus roxburghii* extract as raw material and the compound lactic acid bacteria starter culture as described in any one of claims 1 to 3.

7. The production method according to claim 6, characterized in that: Includes the following steps: (1) Using the extract of *Anoectochilus roxburghii* as raw material, the sugar content of the extract is adjusted to 50-60 g / L, and the sterilization conditions are 100-110℃ for 15-25 min; (2) Add the compound lactic acid bacteria fermentation agent to the *Anoectochilus roxburghii* solution after sterilization in step (1); (3) Fermentation conditions: fermentation temperature 35-40℃, fermentation time 40-50h.

8. The production method according to claim 7, characterized in that: The inoculum amount of the compound lactic acid bacteria starter is 0.02% of the mass of the Anoectochilus roxburghii liquid.

9. A fermented liquid product of *Anoectochilus roxburghii*, characterized in that: It comprises the fermented liquid of *Anoectochilus roxburghii* prepared by the production method according to any one of claims 6 to 8.

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