Phytobacterium plantarum microencapsulated preparation and application thereof in preparation of hypoglycemic products

Abstract

The invention relates to the technical field of biological science, and discloses a plant lactobacillus microencapsulated preparation and application of the plant lactobacillus microencapsulated preparation in preparation of a hypoglycemic product, and the plant lactobacillus microencapsulated preparation is prepared from plant lactobacillus XKFY202518. The preservation number of the plant lactobacillus XKFY202518 in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No.34745, and the preservation date of the plant lactobacillus XKFY202518 is June 3, 2025. According to the invention, the plant lactobacillus microencapsulated preparation is compounded with the white kidney bean extract, the mulberry leaf extract and the prebiotics to form a triple synergistic system of probiotic metabolism regulation, enzyme inhibitor targeted sugar control and prebiotics colonization promotion. All the components are good in compatibility, no effect antagonism phenomenon exists, not only can different sugar control links such as amylolysis and disaccharide absorption be covered, but also intestinal colonization of probiotics can be promoted, and the overall effects of reducing blood sugar and improving lipid metabolism are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of bioscience and technology, and more specifically, to microencapsulated preparations of *Lactobacillus plantarum* and their application in the preparation of hypoglycemic products. Background Technology

[0002] Type 2 diabetes has become one of the major public health problems threatening human health in the global chronic metabolic disease prevention and control system. The International Diabetes Federation (IDF) 2025 Diabetes Atlas shows that 11.1% of adults aged 20-79 worldwide have diabetes (i.e., 1 in 9 people), totaling approximately 589 million, of whom over 40% (approximately 252 million) are undiagnosed. Research supported by the World Health Organization (WHO) further indicates that in 2022, the number of adults with diabetes worldwide exceeded 800 million, more than four times the number in 1990, with the adult prevalence rising from 7% to 14%, and the most significant increases observed in low- and middle-income countries. Even more concerning is the staggering size of the "diabetes reserve force"—1 in 8 adults globally has impaired glucose tolerance (635 million people), and 1 in 11 adults has impaired fasting glucose (488 million people), significantly increasing their risk of developing diabetes. While traditional hypoglycemic drugs can control blood sugar levels, long-term use can easily cause side effects such as liver and kidney damage. Therefore, developing green and safe natural intervention methods has become a research hotspot.

[0003] Probiotics, with their unique advantages in regulating gut microbiota balance and improving metabolic disorders, have shown great application potential in the field of blood glucose management. Among them, *Lactobacillus plantarum*, due to its strong acid resistance and intestinal colonization ability, has become a key research focus in this field. Studies have confirmed that specific probiotic strains can achieve natural regulation of blood glucose levels by regulating the production of intestinal metabolites and modulating the expression of lipid metabolism-related genes, providing high-quality microbial resources for the development of blood glucose-lowering products.

[0004] To address the issue of probiotics being easily destroyed by stomach acid and bile after oral administration, microencapsulation technology has become a core protective measure. Current technologies encapsulate probiotics within protective wall materials to form microparticles, effectively isolating them from external stressors and improving the survival rate of the bacteria during processing, storage, and digestive tract transport. Currently, mainstream microencapsulation wall materials include natural polysaccharides such as sodium alginate and chitosan. Common processes include emulsification cross-linking, spray drying, and extrusion, among which emulsification cross-linking is widely used due to its mild conditions and stable protective effect.

[0005] With the rise of precision nutrition, compound hypoglycemic products have become a development trend. White kidney bean extract (containing α-amylase inhibitors) and mulberry leaf extract (containing 1-deoxynojirimycin) are often used in combination with probiotics because they can target starch breakdown and disaccharide absorption processes respectively, forming a synergistic blood sugar control system of "probiotic regulation + enzyme inhibition". At the same time, the addition of prebiotics such as fructooligosaccharides and inulin can further promote probiotic colonization in the gut, enhancing overall efficacy.

[0006] However, existing probiotic products for lowering blood sugar mostly rely on generic strains and lack specialized strains optimized for blood sugar regulation pathways, resulting in significant individual differences in blood sugar-lowering effects. Many promising strains are difficult to translate from laboratory research to industrial application due to a lack of clear preservation information and standardized characteristic descriptions. Furthermore, the high specificity of strains makes it difficult to adapt to generic microencapsulation processes, preventing the full realization of the functional advantages of high-quality strains.

[0007] Insufficient efficacy of microencapsulation protection systems: Current microencapsulation technologies generally suffer from problems such as the selection of a single wall material or unreasonable ratios. Sodium alginate alone forms capsules with low mechanical strength and high permeability, while chitosan alone has poor film-forming properties; neither can effectively balance the needs of strain protection and intestinal-targeted release. The lack of systematic optimization in the ratio of protectants in the core material leads to a high rate of live bacteria loss during freeze-drying and long-term storage, making it difficult to maintain the active concentration required for product efficacy.

[0008] Poor synergy and stability of compound products: In existing compound products, the combination of probiotics, plant extracts, and prebiotics is mostly based on empirical design, lacking scientific compatibility assessment. In some products, the interaction between extracts and probiotics is not effectively regulated, resulting in reduced extract activity or inhibited strain survival. Furthermore, improper selection of stabilizers and other excipients can easily lead to product layering and microcapsule structure damage after reconstitution, further weakening synergistic effects.

[0009] Microencapsulation of probiotics requires precise control of the "protection-release" balance, and existing technologies struggle to achieve optimal matching of process parameters. An imbalance in the ratio of core material, wall material, and cross-linking agent in the aqueous system can lead to an encapsulation efficiency of less than 85%, or delayed intestinal release due to excessively thick capsule shells. Poor compatibility between the oil phase emulsion concentration and cross-linking reaction conditions can easily cause emulsion stratification or insufficient cross-linking, resulting in premature rupture or structural disintegration of microcapsules in the gastrointestinal environment.

[0010] Lactobacillus plantarum faces multiple threats to its viability throughout the entire production chain, with technical bottlenecks existing from the preparation of post-fermentation bacterial suspensions to the storage of finished products. The concentration of viable bacteria in the bacterial suspension fluctuates greatly, and the ratio with the protectant lacks a dynamic adjustment mechanism, resulting in insufficient strain resistance. Microencapsulated products often retain less than 70% of viable bacteria after 3 months of storage at room temperature, with even more severe activity loss under high temperature and humidity conditions, making it difficult to meet long-term shelf-life requirements.

[0011] Furthermore, the large-scale production of hypoglycemic compound products also faces the challenge of standardization. The significant density difference between probiotic microcapsules and plant extracts can lead to uneven distribution during mixing, resulting in excessively high local concentrations and antagonistic effects. In addition, existing processes struggle to balance microcapsule integrity and product reconstitution, limiting the development of diverse dosage forms. The use of high-performance wall materials (such as refined chitosan and modified polysaccharides) increases the cost of microencapsulated formulations by 20%–35%, also hindering large-scale application. While low-cost wall materials can reduce costs, they lack sufficient protective efficacy; conversely, multi-layer encapsulation processes that improve protective efficacy lead to complex production processes and reduced efficiency. Finding a balance between cost control and efficacy assurance has become a key bottleneck for industrialization. Summary of the Invention

[0012] In view of this, the present invention proposes a microencapsulated preparation of *Lactobacillus plantarum* and its application in the preparation of hypoglycemic products. This aims to address the current lack of specific hypoglycemic *Lactobacillus plantarum* strains with clear storage information, suitable for large-scale application and adapted to microencapsulation processes, which makes it difficult to convert the functional advantages of these strains. Furthermore, in microencapsulation technology, unreasonable wall material ratios and poor compatibility between core materials and protectants result in low encapsulation rates, severe loss of strain activity during processing and gastrointestinal transport, and insufficient long-term storage stability. In compound hypoglycemic products, the compatibility between probiotics and plant extracts (white kidney bean extract, mulberry leaf extract) is not optimized, and the mixing uniformity of multiple components is poor, easily leading to efficacy antagonism or microcapsule structure damage. Simultaneously, the microencapsulation process parameters are difficult to balance the "protection-release" requirements, and there is a contradiction between high-performance wall materials and low-cost industrialization, hindering the large-scale promotion of the product.

[0013] This invention proposes a microencapsulated preparation of *Lactobacillus plantarum*, which is prepared from *Lactobacillus plantarum* XKFY202518. The *Lactiplantibacillus plantarum* XKFY202518 has the accession number CGMCC No.34745 at the China General Microbiological Culture Collection Center, with the accession date of June 3, 2025, and is classified as *Lactiplantibacillus plantarum*.

[0014] Preferably, the preparation method of the microencapsulated Lactobacillus plantarum preparation includes the following steps: 1) After culturing *Lactobacillus plantarum*, *Lactobacillus plantarum* suspension was obtained by removing impurities and resuspending. 2) The core material is obtained by mixing the plant lactobacillus suspension with skim milk powder, trehalose, and resistant starch; The wall material is obtained by mixing soybean dietary fiber, sodium alginate and chitosan. 3) Mix the core material, wall material, crosslinking agent, and water, stirring until all components are completely dissolved to obtain an aqueous system; An oil-phase system is obtained by mixing an emulsifier with vegetable oil. 4) The aqueous phase and oil phase are mixed and subjected to high-speed shearing to obtain a water-in-oil emulsion. Vegetable oil containing glacial acetic acid is added to the water-in-oil emulsion to carry out a cross-linking reaction to obtain a microencapsulated preparation of *Lactobacillus plantarum*.

[0015] Preferably, in the preparation method of the microencapsulated Lactobacillus plantarum preparation, the core viable bacteria count of the Lactobacillus plantarum suspension is ≥ 10. 9 CFU / mL.

[0016] Preferably, in the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass ratio of Lactobacillus plantarum suspension to skim milk powder, trehalose, and resistant starch is 10~15:3~4:2~3:1~2.

[0017] Preferably, in the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass ratio of soybean dietary fiber, sodium alginate, and chitosan is 2~3:2~3:1.

[0018] Preferably, in the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass ratio of core material, wall material, crosslinking agent and water is 10:12~15:1~1.5; The crosslinking agent is calcium carbonate.

[0019] Preferably, in the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass concentration of the emulsifier in the oil phase system is 1%~2%; The volume ratio of the aqueous phase to the oil phase when mixed is 1:3~4.

[0020] Preferably, in the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass concentration of glacial acetic acid in the vegetable oil containing glacial acetic acid is 0.5%~1%; The volume ratio of the vegetable oil containing glacial acetic acid to the aqueous phase is 1:35~40; The cross-linking reaction is carried out at a temperature of 10~35℃ for 20~60 min.

[0021] This invention also provides the application of the above-mentioned microencapsulated Lactobacillus plantarum preparation in the preparation of a hypoglycemic product, wherein the hypoglycemic product comprises the following raw materials in parts by weight: 10 portions of microencapsulated Lactobacillus plantarum preparation 3-5 parts of fructooligosaccharides 3-5 parts inulin 2-4 parts of mulberry leaf extract 3-5 parts white kidney bean extract Stabilizer 2-4 parts.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: The strain exhibits significant advantages and is suitable for industrialization: The *Lactobacillus plantarum* XKFY202518 used in this application possesses strong strain specificity and a clearly defined hypoglycemic function, solving the problem of the lack of standardized information and difficulty in large-scale application of high-quality hypoglycemic strains in existing technologies. Its adaptation to a dedicated microencapsulation process fully leverages the strain's inherent beneficial functions, providing a stable and reliable microbial resource for hypoglycemic products.

[0023] Microencapsulation significantly enhances protective efficacy: By optimizing the precise ratio of *Lactobacillus plantarum* suspension, skim milk powder, trehalose, and resistant starch in the core material, and the scientific combination of soybean dietary fiber, sodium alginate, and chitosan in the wall material, along with appropriate cross-linking agent dosage and process parameters, the microencapsulation rate and strain protection effect are effectively improved. This formulation reduces the damage to the strains caused by gastric acid and bile, significantly increasing the gastrointestinal survival rate of *Lactobacillus plantarum*, while also reducing the loss of live bacteria during freeze-drying and long-term storage, ensuring that the product maintains high probiotic activity throughout its shelf life. This addresses the pain points of insufficient protective efficacy and easy loss of strain activity in traditional microencapsulation technology.

[0024] Enhanced Synergistic Blood Sugar Control Efficacy Through Multi-Ingredient Synergistic Effect: This application scientifically combines microencapsulated *Lactobacillus plantarum* preparations with white kidney bean extract, mulberry leaf extract, and prebiotics such as fructooligosaccharides and inulin to form a triple synergistic system of "probiotic metabolism regulation + enzyme inhibitor targeted blood sugar control + prebiotic colonization promotion." The components exhibit good compatibility and no efficacy antagonism. This system covers different blood sugar control stages, including starch breakdown and disaccharide absorption, while also promoting probiotic colonization in the gut, significantly improving overall blood sugar reduction and lipid metabolism. This addresses the issues of poor synergy and limited blood sugar control range in existing compound products.

[0025] The process is standardized and industrially viable: In the microencapsulation preparation process, the ratio of the aqueous and oil phases and the cross-linking reaction conditions are clearly controllable, balancing the needs of strain protection and intestinal-targeted release, avoiding premature microcapsule rupture or delayed release. Simultaneously, by optimizing the mixing sequence and process parameters, the uniformity of multi-component mixing is ensured, balancing product reconstitution and microcapsule structural integrity. Furthermore, the selected wall materials, protective agents, and other raw materials are cost-controllable, and the process flow is simple and efficient, requiring no complex equipment. This solves the problems of difficult matching of process parameters, cost-efficiency imbalance, and difficulties in large-scale production found in existing technologies.

[0026] The product boasts a wide range of safety and applicability: all raw materials used in the formulation are of natural origin, with no chemically synthesized additives. Furthermore, the product has undergone strain safety verification and microencapsulation technology optimization, ensuring no side effects with long-term consumption. The product's dosage form is flexible, adaptable to various hypoglycemic product forms such as solid beverages. It is suitable for patients with type 2 prediabetes, mild cases, and those with high blood sugar, meeting the needs of different consumption scenarios and populations, and has broad application prospects. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0028] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] This invention proposes a microencapsulated preparation of *Lactobacillus plantarum*, which is prepared from *Lactobacillus plantarum* XKFY202518. The *Lactobacillus plantarum* XKFY202518 has the accession number CGMCC No.34745 at the China General Microbiological Culture Collection Center, and the accession date is June 3, 2025.

[0033] Preferably, the preparation method of the microencapsulated Lactobacillus plantarum preparation includes the following steps: 1) After culturing *Lactobacillus plantarum*, *Lactobacillus plantarum* suspension was obtained by removing impurities and resuspending. 2) The core material is obtained by mixing the plant lactobacillus suspension with skim milk powder, trehalose, and resistant starch; The wall material is obtained by mixing soybean dietary fiber, sodium alginate and chitosan. 3) Mix the core material, wall material, crosslinking agent, and water, stirring until all components are completely dissolved to obtain an aqueous system; An oil-phase system is obtained by mixing an emulsifier with vegetable oil. 4) The aqueous phase and oil phase are mixed and subjected to high-speed shearing to obtain a water-in-oil emulsion. Vegetable oil containing glacial acetic acid is added to the water-in-oil emulsion to carry out a cross-linking reaction to obtain a microencapsulated preparation of *Lactobacillus plantarum*.

[0034] In this invention, the core viable bacteria count of the *Lactobacillus plantarum* suspension is ≥ 10 in the preparation method of the microencapsulated *Lactobacillus plantarum* preparation. 9 CFU / mL.

[0035] In this invention, the mass ratio of *Lactobacillus plantarum* suspension to skim milk powder, trehalose, and resistant starch in the preparation method of the microencapsulated preparation is 10~15:3~4:2~3:1~2, preferably 11~14:3.2~3.8:2.2~2.8:1.2~1.8, further preferably 12~13:3.4~3.6:2.4~2.6:1.4~1.6, and even more preferably 12:3.5:2.5:1.5.

[0036] In this invention, the mass ratio of soybean dietary fiber, sodium alginate, and chitosan in the preparation method of the microencapsulated preparation of *Lactobacillus plantarum* is 2~3:2~3:1, preferably 2.2~2.8:2.2~2.8:1, more preferably 2.4~2.6:2.4~2.6:1, and even more preferably 2.5:2.5:1; this ratio can balance the mechanical strength and permeability of the wall material.

[0037] In this invention, the mass ratio of core material, wall material, crosslinking agent and water in the preparation method of the microencapsulated preparation of *Lactobacillus plantarum* is 10:12~15:1~1.5, preferably 10:13~14:1.2~1.4, and more preferably 10:13.5:1.3; the amount of crosslinking agent is matched with the wall material to avoid over-crosslinking leading to microcapsule embrittlement.

[0038] In this invention, the crosslinking agent is calcium carbonate.

[0039] In this invention, the mass concentration of the emulsifier in the oil phase system of the preparation method of the microencapsulated preparation of *Lactobacillus plantarum* is 1%~2%, preferably 1.2%~1.8%, more preferably 1.4%~1.6%, and even more preferably 1.5%; this can form a stable water-in-oil emulsion and avoid stratification.

[0040] In this invention, the volume ratio of the aqueous phase to the oil phase when mixed is 1:3~4, preferably 1:3.2~3.8, more preferably 1:3.4~3.6, and even more preferably 1:3.5; the emulsification efficiency is the highest and the microcapsule particle size is uniform.

[0041] In this invention, the mass concentration of glacial acetic acid in the vegetable oil containing glacial acetic acid in the preparation method of the microencapsulated preparation of *Lactobacillus plantarum* is 0.5%~1%, preferably 0.6%~0.9%, more preferably 0.7%~0.8%, and even more preferably 0.75%; the cross-linking rate is moderate and the microcapsule structure is stable.

[0042] In this invention, the volume ratio of the vegetable oil containing glacial acetic acid to the water phase is 1:35~40, preferably 1:36~39, and more preferably 1:36~39, to ensure sufficient cross-linking without affecting the stability of the emulsion.

[0043] In this invention, the temperature of the crosslinking reaction is 10~35℃, preferably 20~30℃, and more preferably 23~28℃; the time of the crosslinking reaction is 20~60min, preferably 30~50min, and more preferably 40min.

[0044] This invention also provides the application of the above-mentioned microencapsulated Lactobacillus plantarum preparation in the preparation of a hypoglycemic product, wherein the hypoglycemic product comprises the following raw materials in parts by weight: The mixture contains 10 parts of microencapsulated *Lactobacillus plantarum* preparation, 3-5 parts of fructooligosaccharides, 3-5 parts of inulin, 2-4 parts of mulberry leaf extract, 3-5 parts of white kidney bean extract, and 2-4 parts of stabilizer. Preferably, it contains 10 parts of microencapsulated *Lactobacillus plantarum* preparation, 4 parts of fructooligosaccharides, 4 parts of inulin, 3 parts of mulberry leaf extract, 4 parts of white kidney bean extract, and 3 parts of stabilizer.

[0045] In this invention, the preparation method of the mulberry leaf extract is as follows: Take dried mulberry leaves, pulverize them and pass them through a 60-mesh sieve. Add 70% ethanol solution at a material-to-liquid ratio of 1:20 and extract twice by reflux in a 60℃ water bath, 2 hours each time. Combine the extracts, centrifuge at 4000 rpm for 15 minutes, and collect the supernatant. Concentrate by rotary evaporation to a solid content of 20%, freeze-dry under vacuum (-50℃, 10Pa), and pulverize through an 80-mesh sieve to obtain the mulberry leaf extract.

[0046] The mulberry leaf extract contains ≥1.0% 1-deoxynojirimycin.

[0047] In this invention, the preparation method of the white kidney bean extract is as follows: White kidney beans were pulverized and passed through an 80-mesh sieve. Deionized water was added at a material-to-liquid ratio of 1:15, and the mixture was extracted in a 50℃ water bath with stirring for 3 hours. The extract was filtered through a 0.22μm filter membrane, and the filtrate was collected. Three times the volume of the filtrate was added to 95% ethanol for precipitation for 24 hours. The precipitate was then centrifuged at 4000 rpm for 10 minutes, and the precipitate was collected. The precipitate was reconstituted with deionized water and spray-dried (inlet air temperature 180℃, outlet air temperature 80℃) to obtain the white kidney bean extract.

[0048] The activity of the α-amylase inhibitor in the white kidney bean extract is ≥2000 U / g.

[0049] Example 1 (1) Isolation and identification of Lactobacillus plantarum XKFY202518 Traditional naturally fermented yogurt, collected from herders' homes in Kashgar, Xinjiang, was thoroughly stirred with a sterile spoon. 50 mL of the yogurt was then drawn into a sterilized centrifuge tube using a sterile syringe, placed in a low-temperature food sampling box, and brought back to the laboratory for freezing and storage in an ultra-low temperature freezer at -80°C for later use.

[0050] Take 1 mL of yogurt sample and dilute it 10-fold with sterile physiological saline to a final volume of 10. -6 Then take 10 -4 10 -5 10 -6 Three different gradients of bacterial suspension (100 μL each) were plated and incubated at 37°C for 24–48 h. Colony morphology was observed and recorded. Colonies of different morphologies were picked from the plates and streaked for isolation. After incubation at 37°C for 48 h, single colonies of different morphologies were picked from the plates again and streaked for isolation. This process was repeated multiple times until pure single colonies with consistent morphology were obtained.

[0051] The purified suspected target strain was inoculated into MRS broth and cultured at 37°C for 18-24 hours. DNA was then extracted using a bacterial genomic DNA extraction kit and stored at -20°C for later use.

[0052] The extracted DNA was subjected to PCR amplification. The mixture consisted of 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTCAG-3'), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3'), 12.5 μL of 2×Taq plus Buffer, and 1 μL of template DNA. The volume was brought to 25 μL with sterile dd H2O. Sterile ultrapure water was used as a negative control instead of template DNA. The amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 29 cycles, with a final extension at 72℃ for 5 min.

[0053] The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0054] The strain was successfully sequenced. BLAST alignment analysis confirmed that the strain is *Lactobacillus plantarum*. The sequencing results are as follows: (2) Preparation of microencapsulated Lactobacillus plantarum preparation 1. Preparation of *Lactobacillus plantarum* suspension Lactobacillus plantarum XKFY202518 activation medium: MRS solid medium (formulation: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, sodium acetate 5g / L, diammonium citrate 2g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L, Tween-80 1mL / L, agar 15g / L, pH adjusted to 6.2±0.1, autoclaved at 121℃ for 20min).

[0055] Primary seed culture: Under aseptic conditions, 100 μL of the preserved bacterial culture was spread onto an MRS solid medium plate; it was then incubated in a 37℃ anaerobic incubator (5% CO2, 95% N2) for 48 h. Single colonies with uniform morphology and neat edges were picked; these single colonies were inoculated onto 5 ml MRS liquid medium and anaerobically shaken at 37℃ (150 r / min) for 24 h to obtain primary activated bacterial culture (viable count ≥ 1 × 10⁻⁶). 9 (CFU / mL).

[0056] Secondary seed culture: Inoculate 100 ml of LMRS liquid medium (formula as before, excluding agar) with 1% (v / v) of the primary activated bacterial solution; anaerobic static culture at 37℃ for 18 h, and sample for viable count ≥ 5 × 10⁻⁶. 9 CFU / mL is considered a secondary seed culture.

[0057] Tertiary seed culture: The secondary seed culture was inoculated into a 10L fermenter (6L total volume) at a 5% (v / v) inoculation rate. The culture medium was optimized MRS liquid medium (1% corn steep liquor and 0.5% lactose were added to basic MRS to improve cell proliferation efficiency); the temperature was 37℃, the stirring speed was 80 rpm (to avoid excessive shearing and damage to the cells), and an anaerobic environment was maintained (sterile N2 was introduced to maintain anaerobic conditions in the tank at an aeration rate of 0.1 L / (L·min)). The pH was controlled at 6.0 ± 0.2 by automatically adding 1 mol / L NaOH. After 20 hours of culture, samples were taken to detect OD. 600 Value ≥ 3.5, viable count ≥ 1×10 10 CFU / mL, stop culturing, and obtain tertiary seed culture.

[0058] The tertiary seed culture was inoculated into the fermenter at 8% (v / v), incubated at a constant temperature of 37°C, stirred at 60 rpm, and sterile N2 was introduced throughout the process. Dissolved organic matter (DO) was controlled at ≤0.5 mg / L. Viable bacterial count was measured at ≥2 × 10⁻⁶. 10 Fermentation was terminated when CFU / mL was reached.

[0059] The fermentation broth was transferred to a tubular centrifuge and centrifuged at 8000 r / min for 15 min at a centrifugation temperature of 4℃. After centrifugation, the supernatant was discarded and the bottom bacterial sludge was collected.

[0060] The viable cell count was 1.2 × 10⁻⁶ when the bacterial sludge was resuspended in physiological saline. 10 CFU / mL of Lactobacillus plantarum suspension.

[0061] 2. Preparation of microencapsulated formulations The *Lactobacillus plantarum* suspension, skim milk powder, trehalose, and resistant starch were mixed in a mass ratio of 12:3.5:2.5:1.5 and stirred for 15 minutes to obtain the core material (live bacteria concentration 1.0 × 10⁻⁶). 9 (CFU / mL).

[0062] Soybean dietary fiber, sodium alginate, and chitosan were mixed in a mass ratio of 2.5:2.5:1 to obtain the wall material; the three components were added to deionized water (total solids content 20%) in a mass ratio of core material: wall material: calcium carbonate = 10:13.5:1.3, and stirred at 800 rpm for 30 minutes until dissolved to obtain the aqueous phase.

[0063] Span80 was dissolved in vegetable oil to prepare an oil phase with a mass concentration of 1.5%.

[0064] The water phase and oil phase were mixed at a volume ratio of 1:3.5 and sheared at 11,000 rpm for 12 min to obtain a water-in-oil emulsion. Vegetable oil containing 0.75% glacial acetic acid (volume ratio of 1:37.5 with the water phase) was added dropwise to the emulsion and stirred at 25°C for 40 min to complete cross-linking. After pre-freezing at -80°C for 4 h, the mixture was freeze-dried to obtain the microencapsulated formulation.

[0065] Tests showed an encapsulation rate of 93.5%, a survival rate of 88.2% in simulated gastrointestinal fluid, and a live bacteria retention rate of 91.3% after 6 months of storage at room temperature.

[0066] Example 2 The *Lactobacillus plantarum* suspension prepared in Example 1, skim milk powder, trehalose, and resistant starch were mixed in a mass ratio of 14:3.8:2.8:1.8 and stirred for 15 minutes to obtain the core material (live bacteria concentration 1.0 × 10⁻⁶). 9 (CFU / mL).

[0067] Soybean dietary fiber, sodium alginate, and chitosan were mixed in a mass ratio of 2.8:2.8:1 to obtain the wall material; the three materials were added to deionized water (total solids content 20%) in a mass ratio of core material: wall material: calcium carbonate = 10:13.5:1.3, and stirred at 800 rpm for 30 minutes until dissolved to obtain the aqueous phase.

[0068] Span80 was dissolved in vegetable oil to prepare an oil phase with a mass concentration of 1.5%.

[0069] The water phase and oil phase were mixed at a volume ratio of 1:3.5 and sheared at 11,000 rpm for 12 min to obtain a water-in-oil emulsion. Vegetable oil containing 0.75% glacial acetic acid (volume ratio of 1:37.5 with water phase) was added dropwise to the emulsion and stirred at 30°C for 50 min to complete cross-linking. After pre-freezing at -80°C for 4 h, the mixture was freeze-dried to obtain the microencapsulated formulation.

[0070] Example 3 Take 10 parts by weight of the microencapsulated preparation from Example 1, 4 parts of fructooligosaccharide, 4 parts of inulin, 3 parts of mulberry leaf extract (DNJ≥1.0%), 4 parts of white kidney bean extract (α-amylase inhibitor≥2000U / g), and 3 parts of xanthan gum-guar gum composite stabilizer (mass ratio 1:1); mix them in a three-dimensional mixer at 15r / min for 25min, and package them into 5g / bags to obtain a hypoglycemic solid beverage.

[0071] Example 4 Take 10 parts by weight of the microencapsulated preparation from Example 2, 4 parts of fructooligosaccharide, 4 parts of inulin, 3 parts of mulberry leaf extract (DNJ≥1.0%), 4 parts of white kidney bean extract (α-amylase inhibitor≥2000U / g), and 3 parts of xanthan gum-guar gum composite stabilizer (mass ratio 1:1); mix them in a three-dimensional mixer at 15r / min for 25min, and package them into 5g / bags to obtain a hypoglycemic solid beverage.

[0072] Comparative Example 1 The difference from Example 3 is that it does not contain white kidney bean extract and mulberry leaf extract, but is otherwise the same.

[0073] Experimental Example 1. Test Type In accordance with the requirements of the "Technical Specifications for Inspection and Evaluation of Health Foods", a randomized, parallel controlled clinical trial was designed.

[0074] 2. Test subjects (1) Inclusion criteria Age 35-65, gender not limited; Meets the diagnostic criteria for prediabetes or mild type 2 diabetes: fasting blood glucose (FBG) 5.6~7.0 mmol / L, 2-hour postprandial blood glucose (2hPG) 7.8~11.1 mmol / L; Glycated hemoglobin (HbA1c) 5.7%~6.4%; I have not taken any hypoglycemic drugs, probiotic preparations, or related functional foods in the past three months; They voluntarily sign informed consent forms and cooperate in completing the 12-week intervention and follow-up.

[0075] (2) Exclusion criteria Patients with type 1 diabetes, gestational diabetes, and other special types of diabetes; Individuals with combined severe heart, liver, kidney, gastrointestinal, and immune system diseases; Individuals allergic to any of the test product's ingredients (Lactobacillus plantarum, white kidney bean, mulberry leaf, wall material raw materials, etc.); Pregnant women, breastfeeding women, and those undergoing hormone therapy; Those who have experienced a sharp change in weight (±10%) in the past 6 months; Those with poor compliance who are unable to complete the experiment as required.

[0076] (3) Sample size calculation Based on the primary outcome measure (change in fasting blood glucose), α=0.05, β=0.2, the difference between groups Δ=0.5mmol / L, the standard deviation σ=0.8mmol / L, and the formula n=2×(Zα / 2+Zβ) was used. 2 σ 2 / Δ 2 The calculations showed that each group needed 28 people, and considering a 10% dropout rate, a total of 120 people were ultimately included, and they were randomly divided into 4 groups (30 people in each group).

[0077] 3. Grouping scheme Blank control group: Take a placebo (maltodextrin + sweetener, formula the same as the experimental group) without probiotics and extracts, 2 sachets daily, 5g per sachet; Probiotics group: Take 2 sachets daily of a product containing only microencapsulated Lactobacillus plantarum XKFY202518 (12g / 100g microcapsules, the rest being placebo). Extract group: Take 2 sachets daily of product containing white kidney bean extract and mulberry leaf extract (extract content is the same as the experimental group, without probiotics); Experimental group: Consumed the hypoglycemic solid beverage prepared in Example 3 (containing microencapsulated preparation + dual extract + prebiotics), 2 sachets daily (each sachet contains ≥3×10⁻⁶ live bacteria). 10 CFU, white kidney bean extract α-amylase inhibitor ≥2000 U / bag, mulberry leaf extract DNJ ≥30 mg / bag).

[0078] 4. Intervention cycle and implementation Intervention duration: 12 weeks, with 3 follow-up visits (week 4, week 8, and week 12 of intervention); Dosage: Take 30 minutes before breakfast and dinner each day, mix with warm water below 40℃ and drink, or take directly. Diet and exercise control: During the trial, all participants maintained their original dietary habits (mainly low-GI diet), avoided high-sugar and high-fat foods, exercised 3-5 times a week (more than 30 minutes of moderate-intensity exercise each time), and maintained the same lifestyle before and after the intervention.

[0079] Blinding was implemented: the placebo and the test product had the same appearance, smell, and taste, and were packaged with the same code. The subjects, testers, and data analysts were unaware of the group assignments.

[0080] II. Detection Indicators and Methods 1. Primary Outcome Measure Fasting blood glucose (FBG): Venous blood was collected before intervention, at week 4, week 8, and week 12 after fasting for 8 hours, and measured using the glucose oxidase method. 2-hour postprandial blood glucose (2hPG): Blood samples were collected 2 hours after oral administration of 75g glucose solution before intervention and at week 12, and measured using the same method as above; Glycated hemoglobin (HbA1c): Measured by high performance liquid chromatography before intervention and at week 12.

[0081] 2. Secondary outcome indicators Insulin resistance index (HOMA-IR): Serum insulin (INS) was measured by enzyme-linked immunosorbent assay before intervention and at week 12, and calculated according to the formula HOMA-IR = (FBG × INS) / 22.5; Lipid indicators: Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured before intervention and at week 12 (using Nanjing Jiancheng reagent kit). Gut microbiota: Fecal samples were collected before intervention and at week 12. 16S rRNA sequencing was used to analyze the colonization of Lactobacillus plantarum and the diversity of gut microbiota.

[0082] 3. Safety Indicators Liver and kidney function: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) were measured before intervention and at week 12. Complete blood count: White blood cells, red blood cells, platelets, etc., are measured before intervention and at week 12. Adverse events: Record gastrointestinal reactions (bloating, diarrhea, constipation), allergic reactions, etc. that occur in subjects during the intervention, and grade them according to severity (1-4).

[0083] III. Experimental Data and Results Table 1 shows the baseline data of the four groups of subjects, including gender, age, body mass index (BMI), fasting blood glucose (FBG), 2-hour postprandial blood glucose (2hPG), and glycated hemoglobin (HbA1c).

[0084] Table 1. Baseline data of subjects' gender, age, BMI, FBG, 2hPG, and HbA1c.

[0085] Table 2. Changes in FBG, 2hPG, and HbA1c levels in each group after 12 weeks of intervention.

[0086] Table 3. HOMA-IR, TC, TG, LDL-C and HDL-C levels in each group after 12 weeks of intervention.

[0087] The Homeostasis Model of Insulin Resistance (HOMA-IR) is calculated using fasting blood glucose and insulin levels to assess the degree of insulin resistance in the body.

[0088] Total cholesterol (TC) refers to the sum of all types of cholesterol in the blood and is one of the core indicators reflecting lipid metabolism.

[0089] Triglycerides (TG) are an important component of blood lipids. High levels can easily lead to metabolic disorders and are associated with complications of diabetes.

[0090] Low-density lipoprotein cholesterol (LDL-C), known as "bad cholesterol," increases the risk of vascular disease when its levels rise.

[0091] High-density lipoprotein cholesterol (HDL-C), known as "good cholesterol," can promote cholesterol metabolism and excretion, and has a protective effect on blood vessels.

[0092] Table 1 shows that there were no statistically significant differences in baseline data such as gender, age, BMI, initial blood glucose (FBG, 2hPG), glycated hemoglobin (HbA1c), and blood lipid levels among the blank control group, probiotic group, extract group, and experimental group (product group of this invention) (P>0.05). This indicates that the baseline health conditions of the four groups of subjects were consistent, eliminating the interference of baseline differences on the intervention results, providing a reliable premise for subsequent inter-group comparisons, and ensuring that the experimental conclusions are statistically significant.

[0093] Table 2 shows that after 12 weeks of intervention, the experimental group experienced a decrease in FBG of 1.0±0.3 mmol / L from baseline, a decrease in 2hPG of 1.8±0.6 mmol / L, and a decrease in HbA1c of 0.5±0.1%, all of which were significantly greater than those in the blank control group (P<0.001) and far superior to the single probiotic group or the single extract group. The significant decrease in HbA1c confirms that the product can effectively improve the average blood glucose level over the past 2-3 months, demonstrating a clear long-term blood glucose control effect.

[0094] The experimental group showed improvements in both fasting basal blood glucose (FBG) and postprandial blood glucose regulation (2hPG), indicating that the product can cover both fasting and postprandial blood glucose management scenarios and is suitable for the daily blood glucose control needs of patients with prediabetes and mild diabetes.

[0095] Table 3 shows that after 12 weeks of intervention, the HOMA-IR in the experimental group decreased to 1.9±0.2, significantly lower than the other three groups (P<0.001), indicating that the product can effectively alleviate insulin resistance and help improve the core metabolic problem of "high blood sugar but insufficient insulin secretion / poor utilization". Simultaneously, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the experimental group all decreased significantly, while high-density lipoprotein cholesterol (HDL-C) increased, indicating that the product not only lowers blood sugar but also optimizes the lipid profile, reducing the risk of diabetes-related cardiovascular complications and achieving dual metabolic improvement through "blood sugar reduction + lipid regulation".

[0096] The data in Tables 2 and 3 show that the probiotic group containing only *Lactobacillus plantarum* and the extract group containing only the two extracts showed limited improvement in blood glucose (FBG, 2hPG, HbA1c), insulin resistance (HOMA-IR), and blood lipid indicators. However, the experimental group, through a combination of *Lactobacillus plantarum* microencapsulated preparation + white kidney bean extract + mulberry leaf extract + prebiotics, achieved synergistic amplification of efficacy. Its blood sugar lowering and metabolic improvement effects far exceeded those of single-component formulations, demonstrating that the compound formulation of this invention can fully leverage the triple synergistic advantages of "probiotics regulating intestinal metabolism + enzyme inhibitors targeting blood sugar control + prebiotics promoting colonization," thus solving the technical pain point of insufficient efficacy of single-component formulations.

[0097] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A microencapsulated preparation of *Lactobacillus plantarum*, characterized in that, The microencapsulated preparation of *Lactobacillus plantarum* was prepared from *Lactobacillus plantarum* XKFY202518; The *Lactobacillus plantarum* XKFY202518 has the accession number CGMCC No.34745 at the China General Microbiological Culture Collection Center, and the accession date is June 3, 2025.

2. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 1, characterized in that, The preparation method of the microencapsulated Lactobacillus plantarum preparation includes the following steps: 1) After culturing *Lactobacillus plantarum*, *Lactobacillus plantarum* suspension was obtained by removing impurities and resuspending. 2) The core material is obtained by mixing the plant lactobacillus suspension with skim milk powder, trehalose, and resistant starch; The wall material is obtained by mixing soybean dietary fiber, sodium alginate and chitosan. 3) Mix the core material, wall material, crosslinking agent, and water, stirring until all components are completely dissolved to obtain an aqueous system; An oil-phase system is obtained by mixing an emulsifier with vegetable oil. 4) The aqueous phase and oil phase are mixed and subjected to high-speed shearing to obtain a water-in-oil emulsion. Vegetable oil containing glacial acetic acid is added to the water-in-oil emulsion to carry out a cross-linking reaction to obtain a microencapsulated preparation of *Lactobacillus plantarum*.

3. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 2, characterized in that, In the preparation method of the microencapsulated Lactobacillus plantarum preparation, the core viable bacteria count of the Lactobacillus plantarum suspension is ≥ 10. 9 CFU / mL.

4. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 3, characterized in that, In the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass ratio of Lactobacillus plantarum suspension to skim milk powder, trehalose, and resistant starch is 10~15:3~4:2~3:1~2.

5. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 4, characterized in that, In the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass ratio of soybean dietary fiber, sodium alginate, and chitosan is 2~3:2~3:

1.

6. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 5, characterized in that, In the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass ratio of core material, wall material, crosslinking agent and water is 10:12~15:1~1.5; The crosslinking agent is calcium carbonate.

7. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 6, characterized in that, In the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass concentration of emulsifier in the oil phase system is 1%~2%; The volume ratio of the aqueous phase to the oil phase when mixed is 1:3~4.

8. The microencapsulated preparation of *Lactobacillus plantarum* according to claim 7, characterized in that, In the preparation method of the microencapsulated Lactobacillus plantarum preparation, the mass concentration of glacial acetic acid in the vegetable oil containing glacial acetic acid is 0.5%~1%; The volume ratio of the vegetable oil containing glacial acetic acid to the aqueous phase is 1:35~40; The cross-linking reaction is carried out at a temperature of 10~35℃ for 20~60 min.

9. The application of the microencapsulated Lactobacillus plantarum preparation according to any one of claims 1 to 8 in the preparation of hypoglycemic products, characterized in that, The blood sugar lowering product comprises the following raw materials in parts by weight: 10 portions of microencapsulated Lactobacillus plantarum preparation 3-5 parts of fructooligosaccharides 3-5 parts inulin 2-4 parts of mulberry leaf extract 3-5 parts white kidney bean extract Stabilizer 2-4 parts.

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