Method for preparing synergistic hypoglycemic active composition based on specific symbiotic fermentation and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for preparing a synergistic hypoglycemic active composition based on specific symbiotic fermentation and application of the synergistic hypoglycemic active composition. The invention discloses a method for preparing a synergistic hypoglycemic active composition based on specific symbiotic fermentation and application thereof, and aims to solve the problems of complex process, high cost and incapability of synergistic interaction of active components caused by physical mixing or respective treatment of mulberry leaves and white kidney beans. The method comprises the following steps: mixing mulberry leaf powder and white kidney bean powder according to a ratio of 1: (0.5-2), inoculating a compound lactic acid bacteria agent compounded by lactobacillus plantarum and lactobacillus mucus, carrying out solid symbiotic fermentation at 30-37 DEG C for 60-84 hours, and carrying out extraction, centrifugation, concentration and freeze drying to obtain the composition. The obtained product is rich in 1-deoxynojirimycin, flavonoid aglycones, small molecule peptides and novel double-enzyme inhibition metabolites, has remarkable alpha-amylase and alpha-glucosidase synergistic inhibition activity, and can be used for preparing food, health food or medicine with a blood glucose reducing function.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a method for preparing a synergistically hypoglycemic active composition based on specific symbiotic fermentation and application thereof. BACKGROUND

[0002] The global prevalence of diabetes and its pre-stage has put an increasingly urgent technical demand on non-pharmaceutical management strategies centered on dietary intervention. In the field of research and application of functional ingredients from natural plant sources, 1-deoxynojirimycin contained in mulberry leaves and alpha-amylase inhibitors rich in white kidney beans have been widely recognized as natural hypoglycemic active substances with clear mechanisms of action, because they can effectively delay the digestion and absorption of dietary carbohydrates by inhibiting small intestinal brush border alpha-glucosidase and pancreatic alpha-amylase secretion, thereby regulating postprandial blood glucose peaks. Based on this, the combination of the two in functional foods or dietary supplements can theoretically achieve double blocking of the carbohydrate digestion pathway and has good application prospects. However, the existing technical path has been fundamentally disconnected between preparation process and mechanism of action for a long time in realizing this theoretical advantage.

[0003] The strategy commonly adopted by the industry and research institutions at present is to extract, purify or dry process mulberry leaves and white kidney beans separately, and then physically mix them in proportion to form the final product. Although this method has a certain feasibility in terms of operation, its internal logic is still at the linear superposition level of active ingredients in nature. Specifically, this process involves two independent pre-treatment, extraction and drying processes, resulting in high energy consumption, long cycle time and difficult cost compression. More importantly, physical mixing cannot trigger the interaction between the two plant substrates at the molecular level and cannot create the complex microenvironment required for microbial metabolism, thus completely losing the possibility of producing new active substances or enhancing the bioavailability of existing ingredients through biological transformation. On this basis, although some studies have attempted to ferment single raw materials (such as only mulberry leaves or only white kidney beans) to improve flavor, increase stability or release bound active ingredients, such single-substrate fermentation systems often face the problem of mismatch between the metabolic capacity of the strain and the chemical composition of the substrate - for example, certain lactic acid bacteria can effectively degrade polyphenol glycosides in mulberry leaves, but have difficulty in efficiently utilizing the high-protein substrate in white kidney beans; conversely, strains good at protein hydrolysis may lack the corresponding enzyme support for the complex flavonoid structure in mulberry leaves. This lack of substrate-bacterial population compatibility directly leads to low fermentation efficiency, poor product uniformity, and difficulty in simultaneously improving the synergistic effect of the two key active ingredients.

[0004] However, as the precision nutrition and functional food industry continues to deepen its understanding of the "multi-target, high synergistic, low-dose" product concept, the limitations of the aforementioned technical approaches have risen from the level of process economics to a fundamental bottleneck in the mechanism of action. The reason for this is that 1-deoxynojirimycin primarily acts on disaccharidases in the terminal small intestine, while α-amylase inhibitors act on the initial hydrolysis of starch in the upper small intestine; the two have a spatiotemporal connection at their physiological sites of action. If, within the same bioreaction system, the release, transformation, and stabilization of these two components can be simultaneously optimized through the co-metabolic activities of a specific microbial community, and the generation of secondary metabolites with auxiliary regulatory functions (such as small molecule peptides with ACE inhibitory or insulin-sensitizing activities) can be induced, it is hoped that a true synergistic hypoglycemic effect can be constructed at the molecular network level. However, the realization of this idea highly depends on the precise control of strain selectivity, substrate ratio window, and fermentation kinetic parameters in the dual-substrate symbiotic fermentation system. Current technologies have neither revealed the directional shaping effect of mulberry leaves and white kidney beans on the microbial community structure, nor established specific strain combinations capable of simultaneously and efficiently utilizing two types of heterogeneous substrates, nor verified whether non-additive biological activities beyond physical mixing necessarily occur in such symbiotic systems. Therefore, how to break free from the constraints of traditional stepwise processing and single fermentation paradigms, and construct a symbiotic fermentation system that simplifies the process and achieves synergistic enhancement of active ingredients and even the generation of new functional substances through microbial-mediated specific interactions, has become a key challenge and an urgent technical problem for those skilled in the art. Summary of the Invention

[0005] This invention provides a method for preparing a synergistic hypoglycemic active composition based on specific symbiotic fermentation and its application, aiming to solve the technical defects of existing technologies that involve separate processing or physical mixing of mulberry leaves and white kidney beans, resulting in complex processes, high costs, inability to synergistically enhance the active ingredients, and a lack of new functional substances. To achieve the above-mentioned objectives, this invention adopts the following technical solution.

[0006] In a first aspect, the present invention provides a method for preparing a mulberry leaf-white kidney bean symbiotic fermentation composition, the method comprising the following steps: firstly, substrate preparation, wherein dried and pulverized mulberry leaf powder and white kidney bean powder are uniformly mixed at a mass ratio of 1:(0.5-2), wherein a preferred mass ratio is 1:1; subsequently, deionized water is added to the mixed powder to adjust the overall moisture content to 45% to 55%, thereby obtaining a homogeneous solid fermentation substrate with suitable conditions for microbial growth; secondly, inoculation fermentation is performed, wherein a compound lactic acid bacteria starter is inoculated into the fermentation substrate, the inoculation amount being controlled at 5% to 8% (volume / weight), wherein the compound lactic acid bacteria starter consists of *Lactobacillus plantarum* and *Lactobacillus fermentum*. The mixture is prepared by combining two strains of live bacteria at a ratio of 1 to 3:1. Both strains are activated and cultured before being added as a cell suspension in the logarithmic growth phase. The symbiotic fermentation process is then carried out by placing the inoculated substrate in a constant temperature incubator and performing static solid-state fermentation at 30°C to 37°C for 60 to 84 hours, with 72 hours being the preferred fermentation time. Finally, a post-processing step is performed by adding three times the mass of deionized water to the fermentation product after fermentation and extracting it at 40°C for 2 hours. The mixture is then centrifuged at 8000 rpm for 15 minutes, and the supernatant is collected and concentrated to one-tenth of its original volume under reduced pressure. Finally, the mixture is freeze-dried to obtain a powdered symbiotic fermentation composition.

[0007] Secondly, the present invention provides a mulberry leaf-white kidney bean symbiotic fermentation composition prepared by the aforementioned method. This composition comprises 1-deoxynojirimycin, free flavonoid aglycones, small molecule peptides derived from white kidney bean protein hydrolysis, and metabolites with dual inhibitory activities against α-amylase and α-glucosidase. The composition contains at least 4.5 mg / g of 1-deoxynojirimycin, at least 55 mg / g of total flavonoid aglycones, and contains characteristic novel active peptides detectable by high-performance liquid chromatography-mass spectrometry, with a relative abundance significantly higher than that obtained by separate fermentation or natural fermentation.

[0008] Thirdly, this invention provides the application of the mulberry leaf-white kidney bean symbiotic fermentation composition in the preparation of a dual inhibitor of α-glucosidase and α-amylase. This dual inhibitor, by simultaneously acting on α-amylase in the upper small intestine and α-glucosidase at the brush border of the lower small intestine, effectively slows down the digestion rate of dietary carbohydrates and the absorption rate of monosaccharides, thereby reducing the amplitude of postprandial blood glucose peak fluctuations.

[0009] Fourthly, this invention provides the application of the mulberry leaf-white kidney bean symbiotic fermentation composition in the preparation of functional foods, health foods, or pharmaceuticals for managing postprandial blood glucose or weight. The functional food or health food uses this composition as the main active ingredient, supplemented with food-grade excipients, stabilizers, and flavoring agents, and is formulated into tablets, capsules, granules, or solid beverages. The pharmaceutical formulation is further combined with a pharmaceutically acceptable carrier and formulated into an oral solid dosage form using conventional pharmaceutical processes for the adjunctive treatment of type 2 prediabetes or mild hyperglycemia.

[0010] Furthermore, the particle size of the mulberry leaf powder and white kidney bean powder used in the substrate preparation step is controlled between 80 and 120 mesh to ensure uniform mixing of materials and sufficient contact and utilization of the substrate during subsequent fermentation. The viable cell concentrations of *Lactobacillus plantarum* and *Lactobacillus fermentum* in this compound lactic acid bacteria starter culture are each no less than 1 × 10⁻⁶. 9 The concentration was CFU / mL, and both were activated and cultured in MRS liquid medium at 37°C under anaerobic conditions for 12 hours before being mixed.

[0011] In a preferred embodiment of the present invention, the symbiotic fermentation process is carried out in a closed container, with a headspace of no less than 20% of the total volume reserved in the container to maintain a microaerobic environment. No turning or aeration is performed during fermentation; gas exchange is maintained solely by the porous structure of the substrate itself. The fermentation endpoint is determined by pH monitoring; fermentation is considered complete when the pH of the fermentation system stabilizes between 4.0 and 4.5 and remains stable for more than 6 hours.

[0012] The centrifugation operation in the above post-processing steps was completed using a continuous flow centrifuge. The supernatant was filtered through a 0.45-micron ceramic membrane and then concentrated to remove residual solid particles and large molecular impurities. The freeze-drying process was carried out under conditions of vacuum degree below 10 Pa and cold trap temperature not higher than -50°C, with a drying cycle of 24 to 36 hours. The moisture content of the obtained dry powder was controlled below 7%.

[0013] The new bioactive peptide has a molecular weight ranging from 800 to 1000 Daltons, and its amino acid sequence is rich in branched-chain amino acids and aromatic amino acids. In vitro ACE inhibition experiments have confirmed that it possesses angiotensin-converting enzyme inhibitory activity, with an IC50 value of [missing information]. 50 The value was below 100 μg / mL. This peptide was generated only in a symbiotic fermentation system where mulberry leaves and white kidney beans coexisted and mediated by a specific complex of lactic acid bacteria; it was not detected in single-substrate fermentation or physically mixed systems.

[0014] The α-glucosidase inhibitory activity of this composition is IC50. 50 The value is not higher than 40 μg / mL, and the IC50 value for α-amylase inhibitory activity is not higher than 40 μg / mL. 50The values ​​were no higher than 80 μg / mL, and both indicators were significantly better than those of physical mixtures or mixtures after separate fermentation under the same raw material ratio. This synergistic inhibitory effect stems from the simultaneous modification of two types of substrates by microbial metabolic activities during fermentation: on the one hand, β-glucosidase secreted by *Lactobacillus plantarum* catalyzes the hydrolysis of flavonoid glycosides in mulberry leaves into aglycones with high bioavailability, enhancing their affinity for α-glucosidase; on the other hand, alkaline protease produced by fermenting *Lactobacillus mucilaginosus* specifically cleaves white kidney bean globulin, releasing polypeptide fragments with higher α-amylase inhibitory activity. At the same time, the organic acid environment produced by the metabolism of the two strains stabilizes the chemical structure of 1-deoxynojirimycin, preventing its degradation during processing.

[0015] The recommended daily intake of the functional food prepared in this application is 1 to 3 grams, containing no less than 4.5 mg of 1-deoxynojirimycin and no less than 55 mg of total flavonoid aglycones. Preclinical animal experiments showed that after four weeks of continuous administration of this composition to rats with insulin resistance induced by a high-fat diet, the area under the postprandial blood glucose curve was reduced by more than 35% compared with the model control group, the fasting insulin level decreased by 28%, and there were no obvious liver and kidney toxicity symptoms.

[0016] The method described in this invention constructs a dual-substrate symbiotic microenvironment of mulberry leaves and white kidney beans, enabling specific lactic acid bacteria communities to synergistically metabolize heterogeneous plant substrates. This not only simplifies traditional stepwise extraction and mixing processes but also induces the generation of novel substances with multiple physiological activities at the molecular level, thus overcoming the limitations of physical mixing systems that rely solely on the superposition of components. This technical solution comprehensively covers the entire chain from raw material compatibility, strain selection, fermentation control to product application, possessing high systematicity, reproducibility, and industrial feasibility.

[0017] The beneficial effects of this invention

[0018] Compared with the prior art, the outstanding advantages and beneficial effects of the present invention are as follows:

[0019] 1. Significant synergistic effect: Through a specific dual substrate ratio and exclusive strain combination, the composition prepared by the present invention exhibits a significant synergistic effect on the inhibitory activities of α-glucosidase and α-amylase, and its effect is significantly better than that of physical mixtures of raw materials with the same ratio or products mixed after separate fermentation (see Table 1 of Example 1).

[0020] 2. Strong technological innovation: It pioneered a mulberry leaf-white kidney bean dual-substrate symbiotic fermentation system, which utilizes the interaction between strains and between strains and substrates to achieve simultaneous and efficient conversion of two raw materials in one reactor, simplifying the process flow.

[0021] 3. Excellent product activity: The fermentation products not only have high yields of DNJ and protein (α-AI source), but also produce new active peptides and significantly increase the content of flavonoid aglycones, giving the product multiple physiological activities. Detailed Implementation

[0022] This invention provides a method for preparing a synergistic hypoglycemic active composition based on specific symbiotic fermentation and its application. The technical solution of this invention will be described in detail below with reference to several specific embodiments to ensure that those skilled in the art can fully understand and reproduce all the technical content of this invention.

[0023] In one specific embodiment, the preparation method of the mulberry leaf-white kidney bean symbiotic fermentation composition includes four main process stages: substrate preparation, inoculation fermentation, symbiotic fermentation, and post-treatment. First, substrate preparation is carried out by selecting dried mulberry leaves and white kidney beans, pulverizing them separately and passing them through a standard sieve of 80 to 120 mesh to obtain mulberry leaf powder and white kidney bean powder with uniform particle size distribution. Then, the two powders are weighed at a mass ratio of 1:1 and placed in a three-dimensional mixer at a speed of 30 rpm for 30 minutes to ensure uniform dispersion of the materials at the microscopic scale. After mixing, deionized water is slowly added to the resulting powder, and the overall moisture content is adjusted to 50% under stirring conditions to form a solid fermentation substrate with suitable pore structure and water activity. This substrate is loosely granular, without obvious agglomeration, and has good air permeability and water retention capacity, providing a basic environment for subsequent microbial colonization and metabolic activities.

[0024] Further, inoculation and fermentation were carried out. The compound lactic acid bacteria starter culture used was prepared by mixing *Lactobacillus plantarum* and *Limosilactobacillus fermentum* at a viable count ratio of 2:1. Both strains were pre-activated in MRS liquid medium under anaerobic conditions at 37°C for 12 hours to bring them into the logarithmic growth phase. After activation, the viable cell concentrations of the two bacterial suspensions were measured to confirm that both were not less than 1 × 10⁻⁶. 9 CFU / mL. The two bacterial suspensions were then mixed in a specific ratio to obtain a compound lactic acid bacteria starter culture. This starter culture was then evenly sprayed into the aforementioned solid fermentation substrate at an inoculation rate of 6% (volume / weight), and the bacterial solution was thoroughly penetrated into the substrate by low-speed stirring. The inoculation process was completed in a sterile operating room, with the ambient temperature controlled at 25°C and the relative humidity maintained at 60% to maximize bacterial activity.

[0025] The symbiotic fermentation process then commenced. The inoculated substrate was transferred to covered polypropylene fermentation boxes, each containing 500 grams of wet substrate. A headspace of at least 20% of the total volume was reserved at the top of the container to create a microaerophilic fermentation environment. After sealing, the fermentation boxes were placed in a constant-temperature incubator at 35°C for static solid-state fermentation, without any turning, aeration, or water addition. During fermentation, the metabolic activities of the microorganisms gradually altered the physicochemical properties of the substrate. The pH value was monitored in real-time using a built-in pH electrode. The initial pH was approximately 6.2, and as organic acids such as lactic acid accumulated, the pH continuously decreased, reaching 4.3 at 48 hours of fermentation, and remained stable for the next 24 hours. Fermentation was considered complete when the pH remained between 4.0 and 4.5 for more than 6 hours, at which point the total fermentation time was 72 hours.

[0026] Finally, post-processing steps are performed. After fermentation, 1500g of deionized water is added to every 500g of wet fermentation product, and the mixture is extracted in a 40°C water bath for 2 hours, with gentle stirring at 100 rpm to promote the dissolution of water-soluble active ingredients. After extraction, the mixture is transferred to a continuous flow centrifuge and centrifuged at 8000 rpm for 15 minutes to separate the solid and liquid phases. The supernatant is collected and filtered through a 0.45-micron ceramic membrane filtration system to remove residual fine solid particles and large molecular colloidal impurities. The filtrate is then concentrated under reduced pressure at 60°C and a vacuum of -0.095 MPa until the volume is reduced to one-tenth of the original extract volume. The concentrate is immediately transferred to a freeze dryer and dried for 30 hours under a vacuum of less than 10 Pa and a cold trap temperature of -55°C, finally obtaining a light yellow to light brown loose powdery symbiotic fermentation composition. The moisture content of the dry powder was measured to be 4.2% using a moisture analyzer, which meets the stability requirements for subsequent formulation processing.

[0027] In a preferred embodiment of the present invention, the obtained symbiotic fermentation composition was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and confirmed to contain a variety of characteristic active ingredients. Among them, the content of 1-deoxynojirimycin (DNJ) was quantified as 4.82 mg / g using the external standard method; the content of total flavonoid aglycones (calculated as aglycones of quercetin, kaempferol, and isorhamnetin) was 58.6 mg / g. Furthermore, a characteristic peak was detected at a retention time of 23.7 min in the mass spectrum, with a mass-to-charge ratio (m / z) of 892.4, belonging to a small molecule peptide in the range of 500 to 2000 Daltons. This peptide was not detected in control samples fermented with single mulberry leaves, single white kidney beans, or a physical mixture of both, indicating that its formation strictly depends on the coexistence of mulberry leaves and white kidney beans in a symbiotic fermentation microenvironment mediated by specific complex lactic acid bacteria.

[0028] Furthermore, the enzyme inhibitory activity of this composition was evaluated in vitro. Using p-nitrobenzene-α-D-glucopyranoside (pNPG) as a substrate, its inhibitory activity against α-glucosidase was determined, and the IC50 was calculated. 50 The value was 36.7 μg / mL; using soluble starch as a substrate, its inhibitory activity against α-amylase was determined by the iodine-starch colorimetric method, and the IC50 value was [value missing]. 50 The value was 74.3 μg / mL. Both values ​​were significantly better than the corresponding values ​​of the physical mixture or the mixture of products after separate fermentation in the comparative example.

[0029] To verify the synergistic effect of the technical solution of the present invention, the following comparative examples are set up.

[0030] Comparative Example 1 (Physical Mixing Control): Mulberry leaf powder and white kidney bean powder were physically mixed at a mass ratio of 1:1 without fermentation.

[0031] Comparative Example 2 (mixed after separate fermentation): Mulberry leaf powder and white kidney bean powder were fermented separately under the fermentation conditions of Example 1 to prepare their respective freeze-dried powders, and then physically mixed in a 1:1 ratio.

[0032] Comparative Example 3 (Natural Fermentation Control): Except for not inoculating with exogenous bacteria, the other conditions were the same as in Example 1.

[0033] The efficacy experiment and data analysis were conducted on the key indicators of the products obtained in Example 1 and Comparative Examples 1-3 of the present invention, and the results are shown in the table below.

[0034]

[0035] As shown in the table above, only in the symbiotic fermentation system shown in Example 1, where mulberry leaves and white kidney beans coexist and *Lactobacillus plantarum* and *Lactobacillus fermentum* are combined in a specific ratio, can the simultaneous generation of high levels of 1-deoxynojirimycin, high-conversion flavonoid aglycones, and significant production of characteristic new peptides be achieved. In Comparative Example 1, because it was not fermented, the flavonoids remained in glycoside form, resulting in lower biological activity. Comparative Example 2, although fermented separately, lacked cross-metabolism between the two substrates, thus failing to induce the generation of new peptides. Comparative Example 3, due to its single bacterial species, could not simultaneously achieve the dual functions of glycoside hydrolysis and efficient protein hydrolysis, leading to an imbalance in enzyme inhibitory activity. Furthermore, the composition obtained in Example 1 was tested for in vitro ACE inhibitory activity. Using hippuryl-histyl-leucine (HHL) as a substrate, the amount of hippuric acid generated was determined by reversed-phase high-performance liquid chromatography, and the ACE inhibition IC50 of the composition was calculated. 50 The value was 86.4 μg / mL. After separation and purification, it was confirmed that the activity mainly originated from the aforementioned novel peptide with a molecular weight of approximately 1000 Daltons. Its ACE inhibition IC50, tested separately, showed a value of 86.4 μg / mL. 50 The value was 72.3 μg / mL, confirming its potential to inhibit angiotensin-converting enzyme.

[0036] At the application level, the symbiotic fermentation composition obtained in Example 1 was used to prepare functional foods. 100g of the composition powder was mixed with 40g of microcrystalline cellulose, 5g of silicon dioxide, 3g of magnesium stearate, and 2g of steviol glycosides. The mixture was then compressed into 1000 tablets, each containing 100mg of the composition. The recommended daily intake is 1 tablet three times a day, for a daily intake of 300mg of the composition, equivalent to approximately 1.45mg of 1-deoxynojirimycin and approximately 17.6mg of total flavonoid aglycones. For higher doses, the formulation can be adjusted to produce tablets containing 300mg of the composition per tablet, taken 1 to 3 tablets daily to meet a daily intake requirement of 1g to 3g.

[0037] Alternatively, capsule formulations can be prepared by filling the powdered composition into No. 0 gelatin capsules, with each capsule containing 300 mg. Granules are prepared by mixing the composition with maltodextrin in a 1:1 ratio, adding appropriate amounts of edible flavoring and citric acid, followed by fluidized bed granulation, drying, and packaging. Solid beverages are prepared by mixing the composition with erythritol, resistant dextrin, and natural fruit flavorings, with each 5-gram sachet containing 1 gram of the composition.

[0038] In terms of pharmaceutical formulation, the composition is mixed with pharmaceutically acceptable excipients such as lactose, pregelatinized starch, and croscarmellose sodium in conventional proportions, granulated with an appropriate amount of purified water, dried, and then granulated again. Magnesium stearate is added and the mixture is compressed into tablets containing 250 mg of the composition per tablet. This formulation is suitable for adjunctive treatment in patients with prediabetes or mild hyperglycemia. It is recommended to take it 30 minutes before meals to maximize its effect on regulating postprandial blood glucose fluctuations.

[0039] Animal experiments further verified the physiological efficacy of the composition of this invention. Male Sprague-Dawley rats, weighing 180±10 g, were selected and fed a high-fat diet (containing 20% ​​lard, 10% sucrose, and 2.5% cholesterol) for 8 weeks to establish an insulin resistance model. After successful modeling, the rats were randomly divided into a model control group, a positive drug control group (acarbose, 10 mg / kg body weight), and low, medium, and high dose groups of the composition of this invention (equivalent to 1, 2, and 3 times the human daily dose, respectively, converted according to body surface area). After continuous gavage administration for 4 weeks, an oral starch tolerance test (OSTT) was performed. The results showed that the area under the curve (AUC) of postprandial blood glucose in the high-dose group was reduced by 37.2% compared with the model control group, the fasting insulin level decreased by 29.1%, and the insulin sensitivity index was significantly improved. Liver and kidney function indicators (ALT, AST, BUN, Cr) were all within the normal range, and no obvious toxic reactions were observed.

[0040] In terms of mechanism of action, the synergistic hypoglycemic effect of the composition of this invention originates from the integrated regulation of multiple components and multiple targets. β-glucosidase secreted by *Lactobacillus plantarum* specifically hydrolyzes flavonoid-O-glycosides such as rutin and isoquercitrin in mulberry leaves, releasing aglycones such as free quercetin. These aglycones, due to their reduced polarity, are more readily bound to the active pocket of α-glucosidase, enhancing inhibitory efficacy. Simultaneously, alkaline protease expressed by *Lactobacillus fermentum* recognizes specific cleavage sites (such as Lys-X or Arg-X) in white kidney bean globulin, releasing polypeptide fragments rich in hydrophobic amino acids. These fragments have higher affinity and inhibitory efficiency for α-amylase. The short-chain fatty acids such as lactic acid and acetic acid produced by the co-metabolism of both strains not only lower the system pH and inhibit the growth of other microorganisms, but also stabilize the piperidine ring structure of 1-deoxynojirimycin through hydrogen bonding, preventing ring-opening degradation. In addition, during fermentation, mulberry leaf polyphenols and white kidney bean protein hydrolysates may undergo covalent binding mediated by non-enzymatic Maillard reaction intermediates to form a heteropeptide-polyphenol complex with a new spatial conformation. Such substances may further enhance the allosteric inhibition of digestive enzymes.

[0041] In summary, this invention constructs a highly specific symbiotic fermentation system by precisely controlling the ratio of mulberry leaves and white kidney beans, the types and proportions of compound lactic acid bacteria, and the moisture, temperature, time, and microaerobic environment during solid-state fermentation. This system not only achieves efficient enrichment and activation of traditional active ingredients but also induces the generation of new substances with multiple physiological functions within the microbial-substrate interaction network. The entire process requires no organic solvent extraction or high-temperature, high-pressure treatment, resulting in low energy consumption, high safety, and all steps can be completed in existing food or pharmaceutical GMP workshops, demonstrating excellent prospects for industrial scale-up. The obtained composition exhibits clear synergistic effects in the dual inhibition of α-glucosidase and α-amylase, ACE inhibition, and improvement of insulin sensitivity, providing solid technical support for its application in functional foods, health foods, and adjunctive hypoglycemic drugs.

Claims

1. A method for preparing a mulberry leaf-white kidney bean symbiotic fermentation composition, characterized in that, Includes the following steps: Mulberry leaf powder with a particle size of 80 to 120 mesh and white kidney bean powder are mixed evenly at a mass ratio of 1:(0.5 to 2), and water is added to adjust the moisture content to 45% to 55% to obtain a solid fermentation substrate. A compound lactic acid bacteria starter culture composed of *Lactobacillus plantarum* and *Lactobacillus fermentatus* at a live cell ratio of (1 to 3):1 is inoculated into the substrate at an inoculation amount of 5% to 8% (volume / weight), wherein both strains are added in the form of logarithmic growth phase cell suspension. The inoculated substrate is subjected to static solid fermentation at 30°C to 37°C for 60 to 84 hours. The fermentation is carried out in a closed container with a headspace of not less than 20% of the total volume. After fermentation, three times the mass of deionized water is added and extracted at 40°C for 2 hours. The supernatant is collected by centrifugation, filtered through a 0.45-micron ceramic membrane, concentrated under reduced pressure to one-tenth of the original volume, and then freeze-dried to obtain a powdered symbiotic fermentation composition.

2. The preparation method according to claim 1, characterized in that, The mass ratio of mulberry leaf powder to white kidney bean powder is 1:

1.

3. The preparation method according to claim 1, characterized in that, The viable cell concentrations of *Lactobacillus plantarum* and *Lactobacillus fermentum* in the compound lactic acid bacteria starter are each not less than 1×10⁻⁶. 9 The concentration of CFU / mL was determined, and both ingredients were activated by MRS liquid culture at 37°C under anaerobic conditions for 12 hours before being mixed.

4. The preparation method according to claim 1, characterized in that, The symbiotic fermentation was carried out at a temperature of 35°C for 72 hours. The fermentation endpoint was determined by pH value: fermentation ended when the pH value of the system stabilized between 4.0 and 4.5 for more than 6 hours.

5. The preparation method according to claim 1, characterized in that, The centrifugation operation uses a continuous flow centrifuge with a speed of 8000 rpm and a centrifugation time of 15 minutes; the freeze drying is carried out under conditions of vacuum degree below 10 Pa and cold trap temperature not higher than -50℃, with a drying cycle of 24 to 36 hours, and the resulting dry powder has a moisture content of less than 7%.

6. A mulberry leaf-white kidney bean symbiotic fermentation composition prepared by the method according to any one of claims 1 to 5, characterized in that, The compound comprises 1-deoxynojirimycin, free flavonoid aglycones, and small molecule peptides with a molecular weight of 500 to 2000 Daltons, wherein the content of 1-deoxynojirimycin is not less than 4.5 mg / g, the content of total flavonoid aglycones is not less than 55 mg / g, the amino acid sequence of the small molecule peptides is rich in branched-chain amino acids and aromatic amino acids, and it has angiotensin-converting enzyme inhibitory activity, IC50. 50 The value is below 100 micrograms per milliliter.

7. The symbiotic fermentation composition according to claim 6, characterized in that, The small molecule peptides are small molecule peptides with a molecular weight of 800 to 1000 Daltons.

8. The symbiotic fermentation composition according to claim 6, characterized in that, The composition exhibits IC50 inhibitory activity against α-glucosidase. 50 The IC50 value for the inhibitory activity against α-amylase is not higher than 40 μg / mL. 50 The value is not higher than 80 micrograms / ml.

9. The use of the mulberry leaf-white kidney bean symbiotic fermentation composition according to any one of claims 6 to 8 in the preparation of a dual inhibitor of α-glucosidase and α-amylase.

10. The use of the mulberry leaf-white kidney bean symbiotic fermentation composition according to any one of claims 6 to 8 in the preparation of functional foods, health foods or pharmaceuticals for managing postprandial blood glucose or weight.

Images