Probiotic chewing tablet with hypoglycemic effect and preparation method thereof

Abstract

Diabetes is a global metabolic disease characterized by chronic hyperglycemia, which can easily lead to serious complications such as retinopathy, kidney failure, cardiovascular disease, etc. Traditional hypoglycemic drugs are prone to drug resistance, gastrointestinal reactions, hypoglycemia and other side effects, and are difficult to repair islet function and reverse the course of the disease. The patent provides a plant lactobacillus with hypoglycemic effect, which was preserved in China General Microbiological Culture Collection Center on March 9, 2026, with the preservation number of CGMCC No. 37876. The patent also provides a probiotic chewing tablet prepared by using the strain and a preparation process thereof. The probiotic chewing tablet provided by the patent has good preservation stability, can safely improve sugar metabolism and protect islet function, and makes up for the defects of existing drugs and products, and has important application value and market prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a probiotic chewable tablet with hypoglycemic function and its preparation method. Background Technology

[0002] Diabetes mellitus is a chronic disease characterized by persistently elevated blood glucose levels. It is becoming increasingly prevalent globally, posing a serious threat to human health and increasing the burden on healthcare. Due to impaired insulin secretion, peripheral tissues experience a significant reduction in glucose uptake and utilization, while hepatic glucose production is abnormally increased, leading to an imbalance between glycogen synthesis and breakdown, ultimately resulting in chronic hyperglycemia. Disorders of glucose metabolism can also lead to serious complications such as retinopathy, kidney failure, heart attack, and diabetic foot, severely damaging human health. In recent years, the number of people with diabetes has risen to 830 million. Besides autoimmune factors, an unhealthy diet and irregular lifestyle are the main reasons for the high incidence of diabetes. Furthermore, some people have low awareness of the causes, early symptoms, and importance of prevention and control of diabetes, lacking scientific awareness of diet, exercise, and blood glucose monitoring, resulting in a high rate of missed diagnoses in the early stages. By the time of diagnosis, pancreatic function damage or complications have already occurred, further complicating disease control.

[0003] Traditional oral hypoglycemic agents (such as sulfonylureas and biguanides) are prone to drug resistance with long-term use, and some drugs have significant side effects such as gastrointestinal irritation and hypoglycemia risk, making it difficult to achieve long-term safe blood sugar control. Existing drugs mostly focus on "controlling blood sugar" as their core objective, failing to fundamentally reverse insulin resistance or repair damaged pancreatic β-cell function, and thus unable to prevent disease progression and complications. Therefore, there is an urgent need for a novel, gentle, and safe intervention product for regulating glucose metabolism. Probiotics, with their benign regulatory effect on the body's metabolism, have become an important direction for solving the above-mentioned technical problems—the probiotic chewable tablets provided by this invention, which can regulate glucose metabolism disorders to achieve a blood sugar-lowering effect, can specifically repair the balance of glucose metabolism, and are expected to compensate for the shortcomings of existing intervention methods, delay disease progression, and have broad application prospects.

[0004] Currently, a few patents confirm the role of probiotics in lowering blood sugar. Patent CN114621896B discloses a strain of *Lactobacillus plantarum* that can lower fasting blood glucose in diabetic rats, but its efficacy was not compared with the hypoglycemic drug metformin. Patent CN120536282A discloses a strain of *Lactococcus lactis* and a strain of *Lactobacillus plantarum*, and cheese made from a compound probiotic preparation containing these bacteria has a hypoglycemic effect. This patent mainly utilizes the compound probiotics to exert its hypoglycemic effect. Although some literature reports on *Lactobacillus plantarum* with hypoglycemic effects, they have not used it in the preparation of chewable tablets.

[0005] Currently available probiotic products with glucose metabolism regulating effects suffer from drawbacks such as inconvenient consumption, low survival rate of live bacteria during storage, and unsatisfactory taste and acceptability, making it difficult to meet the needs of long-term consumption. To overcome these shortcomings, this patent provides a probiotic chewable tablet with a blood sugar lowering effect. By optimizing the formula and preparation process, the storage stability of probiotics in the chewable tablet is significantly improved, filling a gap in existing technology and possessing significant practical value and market potential. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a strain of *Lactobacillus plantarum* with hypoglycemic effects. This strain was deposited on March 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 37876, and the classification name is *Lactobacillus plantarum*.

[0007] Another aspect of the present invention provides a formula and preparation method for a hypoglycemic probiotic chewable tablet.

[0008] The basic formula for blood sugar lowering probiotic chewable tablets is as follows: 5%~15% probiotic freeze-dried powder, 8%~16% disintegrant, 0.2%~1.0% lubricant, 2.5%~12.5% ​​filler, 3%~15% sweetener, and 0%~8% prebiotic.

[0009] Preferably, the probiotic freeze-dried powder used has a live bacteria count of (2~4)×10⁻⁶. 12 CFU / g of Lactobacillus plantarum freeze-dried powder; Preferably, the sweetener is one or more of xylitol, aspartame, and erythritol, and the addition amount is 5% to 10%; the filler is soluble starch, and the addition amount is 5% to 10%; the lubricant is magnesium stearate, and the addition amount is 0.5% to 1.0%; and the disintegrant is microcrystalline cellulose, and the addition amount is 8% to 16%.

[0010] Preferably, the prebiotic is one or more of the following: resistant dextrin, fructooligosaccharide, trehalose, inulin, and galactooligosaccharide. Beneficial effects

[0011] The *Lactobacillus plantarum* strain obtained by screening in this invention possesses both excellent hypoglycemic regulatory activity and high biocompatibility. This strain exhibits high levels of α-glucosidase inhibitory activity, α-amylase inhibitory activity, and insulin sensitivity-enhancing ability in vitro, and can significantly improve cellular glucose metabolism disorders.

[0012] This solution scientifically combines functional probiotic powder with prebiotics to achieve synergistic effects and fully leverage the core function of lowering blood sugar. Supplemented with functional natural ingredients, the product not only precisely lowers blood sugar and maintains blood sugar stability but also boasts excellent flavor and sensory qualities. Furthermore, the product exhibits excellent storage stability, high food safety, and broad consumer acceptance, meeting the health management needs of individuals with high blood sugar and possessing significant potential for large-scale implementation and broad market application prospects. Attached Figure Description

[0013] Figure 1. Phylogenetic tree of Lactobacillus plantarum B2Z2 Figure 2. Effects of complex fatty acids on HepG2 cell survival and glucose consumption. Figure 3. Effect of lyophilized bacterial powder from fermentation supernatant on the survival rate of HepG2 cells. Figure 4. Effect of freeze-dried bacterial powder from fermentation supernatant on glucose consumption in HepG2 cells. Figure 5. Effects of different amounts of added fructooligosaccharide on the growth of the strain. Figure 6. Effect of xylitol addition on sensory evaluation Figure 7. Effect of soluble starch addition on sensory evaluation Figure 8. Effect of magnesium stearate addition on sensory evaluation Figure 9. Effect of microcrystalline cellulose addition on sensory evaluation. Figure 10. Effect of pressure on probiotic survival rate Detailed Implementation

[0014] To clearly illustrate the technical solution of this invention, a detailed description is provided below with reference to specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to the following embodiments. Any modifications, equivalent substitutions, or improvements made to the methods, steps, or conditions of this invention under the spirit and core concept of this invention should be included within the scope of protection of this invention. Unless otherwise explicitly stated, the raw materials, reagents, instruments, and equipment used in this embodiment are all commercially available conventional products; percentages not specifically indicated refer to volume percentages.

[0015] The culture medium formulations used in the following examples are as follows: The MRS medium formula is as follows: peptone 10.0 g / L, beef extract 10.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, ammonium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL / L, and agar 20 g / L to obtain the solid medium, pH 6.2±0.2. After preparation, it is autoclaved at 121℃ for 20 min.

[0016] Artificial gastric fluid: pepsin 3.5 g / L, sodium chloride 3 g / L, pH=3.0, sterilized by filtration through a 0.22 μm microporous membrane, stored at 4℃ for later use.

[0017] Artificial intestinal fluid: trypsin 1.0 g / L, sodium chloride 3 g / L, sodium bicarbonate 11 g / L, pH=8.0, sterilized by filtration through a 0.22 μm microporous membrane, stored at 4℃ for later use.

[0018] 1. Strains Isolation and Purification Take 1 g or 1 mL of samples from sources such as kimchi, yogurt, and cheese, add 10 mL of sterile water, and incubate overnight at 37°C with shaking. The enriched bacterial solution is then serially diluted with sterile water, 10 mL each time. -1 ~10 -6 Each gradient dilution was spread onto a solid culture medium. Based on differences in colony morphology, different single colonies were picked and numbered, and purified three times using the streak plate method to obtain pure cultures with consistent colony morphology, which were then stored.

[0019] After activation, the initially screened strains were plated on solid selection medium containing 0.5% calcium carbonate and incubated at 37°C for 48 hours. Colonies with obvious calcium dissolution zones were selected and subjected to three more streak plate purification and isolation cultures to finally obtain candidate pure strains.

[0020] 2. Initial screening of probiotics with hypoglycemic activity The inhibition rates of the strains against α-amylase and α-glucosidase were determined to screen probiotics that can inhibit the activity of key enzymes in the hydrolysis of carbohydrates in the intestine. By delaying the breakdown of starch and oligosaccharides into glucose and reducing the rate of glucose absorption in the small intestine, the in vitro hypoglycemic targets and hypoglycemic potential of the strains were identified, enabling rapid screening of superior probiotic strains with hypoglycemic effects.

[0021] 2.1 Determination of α-glucosidase activity inhibition rate The inhibitory activity of fermentation supernatant against α-glucosidase was determined using the PNPG method. The p-nitrophenol (PNP) generated by α-glucosidase hydrolyzing PNPG exhibits characteristic absorption at 405 nm; the inhibitor reduces PNP formation, thus decreasing absorbance.

[0022] Sample group: 50 μL of α-glucosidase and 25 μL of fermentation supernatant were added sequentially to a 96-well plate and incubated at 37 ℃ for 10 min. Then, 25 μL of 20 mmol / mL PNPG solution was added, and the reaction was stopped by adding 100 μL of 0.1 mol / L enzyme Na2CO3 after 20 min. The absorbance (A) was measured at 405 nm. Sample blank group (PBS instead of enzyme): B; Control group (PBS instead of sample): C; Blank group (PBS instead of sample and enzyme): D. The inhibition rate was calculated according to Formula 1.

[0023]

[0024] The results are shown in Table 1.

[0025]

[0026] The results of the inhibition rate determination of each strain on α-glucosidase show that B2Z2, ZW-1 and LYS-S have relatively high inhibition rates, with B2Z2 having the highest inhibition rate, and can be used for subsequent experimental strains.

[0027] 2.2 Determination of α-amylase activity inhibition rate Take 50 μL of 2.0% α-amylase solution, add 50 μL of fermentation supernatant, and react at 37℃ for 10 min; add 100 μL of 1% soluble starch solution preheated to 37℃, and continue reacting at 37℃ for 10 min; add 200 μL of DNS reagent, develop color in a boiling water bath for 5 min, cool and allow to stand, then dilute BS 5 times, and measure the absorbance at 540 nm, denoted as A. 样 Distilled water was used instead of α-amylase, and the assay was performed using the same method. This result was denoted as A. 对 The sample was replaced with distilled water, and the same method was used for determination; this was recorded as Amax. Both α-amylase and the sample were replaced with distilled water, and the same method was used for determination; this was recorded as Amin. The inhibition rate was calculated according to Formula 2.

[0028]

[0029] The inhibition rate of each strain against α-amylase is shown in Table 2.

[0030]

[0031] Based on the inhibition rate of α-amylase by each strain, B2Z2, LYS-S, ZW-1 and YS showed higher inhibition rates, and were selected as the strains for subsequent experiments.

[0032] 1. Acid and bile salt resistance tests The tolerance of the strains to acidic and bile salt environments was investigated. Acid tolerance test: The pH of MRS liquid medium was adjusted to 2.5 and 3.5 with 0.1 mol / L hydrochloric acid, sterilized at 121℃ for 20 min, and then cooled for later use. Bile salt tolerance test: Bovine bile salts were added to MRS medium to a final concentration of 0.15 g / 100 mL and 0.3 g / 100 mL, respectively, and sterilized using the same method.

[0033] The six activated probiotic strains were inoculated at a 10% inoculum into MRS media with different pH values ​​or bile salt concentrations and cultured statically at 37°C. Samples were taken from the acid-tolerant group at 0 h and 2 h, and from the bile salt-tolerant group at 0 h and 3 h. The viable bacterial count at each time point was determined using the plate count method, and the survival rate was calculated according to Formula 3. The results are shown in Tables 3 and 4.

[0034]

[0035] As shown in Tables 3 and 4, strain B2Z2 exhibits significantly better low pH tolerance and bile salt tolerance than the other two strains.

[0036] 2. Simulated gastric juice and simulated intestinal juice tolerance test Preparation of bacterial suspension: Activate the candidate bacterial strain according to the method described in 2.1 of Example 1. Take the activated probiotic bacterial suspension, centrifuge at 6000 r / min for 10 min, discard the supernatant, wash the bacterial precipitate 2-3 times with sterile physiological saline, and resuspend it in sterile physiological saline. Adjust the bacterial suspension concentration to approximately 10. 8 CFU / mL.

[0037] Tolerance test: 100 μL of the above bacterial suspension was added to 900 μL of preheated artificial gastric fluid and artificial intestinal fluid, respectively. After mixing, the mixture was incubated in a 37℃ incubator. The artificial gastric fluid treatment group was incubated for 3 h, and the artificial intestinal fluid treatment group was incubated for 4 h. After incubation, the bacterial suspension with appropriate dilution was plated and incubated at 37℃ for 24 h. The number of viable bacteria was counted, and the survival rate was calculated according to Formula 3. The results are shown in Table 5.

[0038]

[0039] Table 4 shows that the survival rates of the three candidate probiotic strains differed in simulated gastric and intestinal fluids. In simulated gastric fluid, LYS-S had the highest survival rate, followed by B2Z2, while ZW-1 had a lower survival rate. In simulated intestinal fluid, B2Z2 had the highest survival rate, while LYS-S had a lower survival rate. Tolerance under a single condition is insufficient to fully reflect the overall adaptability of the strains; further screening requires a comprehensive evaluation using multiple indicators.

[0040] 3. Surface property experiments 3.1 Determination of self-aggregation The activated bacterial strain was cultured at 37℃ for 18 h. The bacterial suspension was centrifuged at 6000 r / min for 10 min, and the bacterial pellet was collected. The pellet was washed with sterile physiological saline and resuspended. The concentration of the bacterial suspension was adjusted so that the initial absorbance value A0 at 600 nm was 0.8 ± 0.2. The bacterial suspension was incubated at 37℃. The supernatant was carefully aspirated at 3, 6, 9, 12, and 24 h, and its absorbance value (A0) at 600 nm was measured. t The self-aggregation rate of each strain was calculated according to Formula 4. The results are shown in Table 6.

[0041]

[0042] In the formula: A0 is the initial absorbance value of the bacterial suspension; A t The absorbance value is the value of the bacterial suspension after incubation for th time.

[0043]

[0044] Table 6 shows that the self-aggregation rate of the three candidate strains increased significantly over time, but the growth trends of different strains differed. During the 3-6 h culture period, the self-aggregation rates of B2Z2 and ZW-1 were relatively high, with B2Z2 leading the other strains in the early stages; by 24 h, the self-aggregation rates of all strains reached a high level.

[0045] 3.2 Hydrophobicity determination Mix 3 mL of bacterial suspension with 1 mL of xylene, vortex for 2 min to fully emulsify, and let stand at room temperature for 1 h to separate the two phases. Carefully aspirate the lower aqueous phase and measure its absorbance at 600 nm (A). t The hydrophobicity of each strain was calculated according to Formula 5, and the results are shown in Table 7.

[0046]

[0047] In the formula: A0 is the initial absorbance value of the bacterial suspension; A t The absorbance value of the aqueous phase after reaction with xylene.

[0048]

[0049] Hydrophobicity is an important indicator for evaluating the potential interaction between probiotics and intestinal mucosal epithelial cells. Generally, higher hydrophobicity helps bacteria adhere to the intestinal wall, thereby enhancing colonization ability. As shown in Table 7, there are significant differences in hydrophobicity among the three candidate strains. ZW-1 and B2Z2 showed superior hydrophobicity, which may be more conducive to intestinal colonization; while LYS-S exhibited weaker hydrophobicity.

[0050] 4. Overall Evaluation Six candidate probiotic strains were comprehensively evaluated using an entropy weight method combined with the TOPSIS method. Six indicators were selected: simulated gastric juice survival rate, simulated intestinal juice survival rate, acid tolerance, bile salt tolerance, hydrophobicity, and self-aggregation. The objective weight of each evaluation indicator was calculated using the entropy weight method, with the weight reflecting the indicator's contribution to the overall evaluation. The cumulative weight of gastrointestinal tolerance-related indicators (simulated gastric juice 20%, acid tolerance 20%, simulated intestinal juice 15%, bile salt tolerance 20%) reached 75%, while the cumulative weight of adhesion-related indicators (hydrophobicity 10%, self-aggregation 15%) was 25%. Using the TOPSIS (Topology Approximation to Ideal Solution Ranking) model, the relative closeness of each strain to the ideal solution was calculated. The strains were then comprehensively ranked based on their closeness, and the strain with the best overall performance was selected.

[0051] Based on the overall scores, the overall performance of the six probiotic strains was as follows: B2Z2 (0.82) > LYS-S (0.55) >> ZW-1 (0.45). The strain with the best overall performance was identified as B2Z2, which scored significantly higher than the other strains, demonstrating balanced and excellent performance.

[0052] 5. Strain identification DNA was extracted from strain B2Z2 and PCR amplification was performed according to the system shown in the table below. The PCR products were detected by 1% agarose gel electrophoresis, and the bands meeting the requirements were sent to a sequencing company for sequencing analysis. The obtained sequences were then compared for homology using NCBI BLAST to determine the strain classification.

[0053]

[0054] Amplification conditions: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ extension for 7 min.

[0055] The sequencing results were compared using BLAST on the NCBI website, and a phylogenetic tree of the strain was constructed based on the comparison results. Figure 1 As shown, strain B2Z2 was identified as Lactobacillus plantarum.

[0056] Preparation of lyophilized fermentation supernatant powder: Adjust the probiotic cell concentration to 1×10⁻⁶. 8 CFU / mL was inoculated into MRS culture medium at 4% (v / v) and incubated statically at 37°C for 24 h. The fermentation broth was centrifuged at 8000 rpm for 15 min at 4°C, and the supernatant was collected. The supernatant was then freeze-dried to obtain a lyophilized powder. Before use, the stock solution was prepared with serum-free DMEM medium.

[0057] 1. Preparation of free fatty acid (FFA) mixed modeling agent and determination of optimal modeling concentration The FFA-BSA inducer with an OA:PA ratio of 2:1 was prepared according to the literature. OA and PA were dissolved separately in sterile NaOH and water bath at 70°C to prepare 100 mmol / L monoacid stock solutions. While still hot, 10% defatted BSA solution preheated and sterilized at 55°C was added, and the mixture was incubated at 55°C for 1-2 h to ensure complete binding. The two systems were mixed at a volume ratio of 2:1, sterilized by filtering through a 0.22 μm filter membrane, and aliquoted. The solutions were stored at -80°C and diluted with DMEM before use.

[0058] HepG2 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h until adherence. The supernatant was discarded, and the cells were treated with DMEM medium containing different concentrations of FFA mixed modeling agent (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mmol / L). A control group (containing an equal volume of BSA) was also included. After 24 h of culture, cell viability was assessed using the CCK-8 assay. With the control group as 100% viability, cell survival rate was calculated at each concentration. Concentrations with a survival rate greater than 90% were selected as safe concentrations for subsequent experiments.

[0059] Cell culture supernatant was collected, and the residual glucose level in the culture medium was determined using a glucose oxidase kit. The glucose consumption of each cell group was calculated. Considering both cytotoxicity and glucose consumption capacity, the optimal FFA concentration was selected as the concentration that significantly induced insulin resistance and lipid metabolism disorders in HepG2 cells without significantly inhibiting cell viability.

[0060] Figure 2 The results showed that within the concentration range of 0.1–0.4 mmol / L, cell viability remained above 93%, with no significant difference compared to the control group, indicating that this concentration range had no significant effect on cell viability. Glucose consumption measurements showed that the glucose consumption of cells in the 0.4 mmol / L complex fatty acid treatment group was 5.52 ± 0.52 mmol / L, significantly lower than the control group. Therefore, 0.4 mmol / L was selected as the induction concentration for complex fatty acids in subsequent experiments.

[0061] 2. Effect of lyophilized fermentation supernatant on HepG2 cell viability Similar to the experiment on the effect of complex fatty acids on cell viability, the sample groups consisted of lyophilized probiotic fermentation supernatant, prepared into solutions of different concentrations (0, 0.5, 1, 2, 3, 4, 5, 6, 8 mg / mL) using serum-free DMEM medium. These solutions were added to each well, with six replicates per group. Concentrations with a viability >90% were selected as safe concentrations. Low, medium, and high concentrations were then used for subsequent glucose and lipid metabolism intervention experiments. The results are as follows: Figure 3 As shown.

[0062] The results are as follows Figure 3 As shown, the cell viability of B2Z2 samples was higher than 90% in the concentration range of 0.5~4 mg / mL, so 3 mg / mL was selected as the concentration for subsequent experiments.

[0063] 3. Effect of lyophilized fermentation supernatant on glucose consumption in HepG2 cells HepG2 cells were seeded in 96-well plates, with blank control, FFA model group, low / medium / high dose sample groups, and metformin positive control group included. After cell adhesion, except for the blank group, the others were treated with FFA for 24 h to establish a glucose metabolism disorder model. After successful modeling, the supernatant was discarded. The sample group and positive control group were added with culture medium containing the corresponding concentration of the sample, while the model group and control group were added with culture medium without the drug. Incubation continued for 24 h. After incubation, the cell supernatant was aspirated, and the glucose content of the supernatant was detected using the glucose oxidase method. Cell-free control wells were also included. The glucose consumption of each group was calculated by subtracting the glucose content of each drug-treated well from the glucose content of the cell-free wells.

[0064] The results are as follows Figure 4 As shown, the amount of glucose consumed by B2Z2 in cells increases with increasing concentration, and the consumption at high concentrations is higher than that in the control group (metformin group). Therefore, it can be concluded that the lyophilized sample of B2Z2 fermentation supernatant can improve insulin resistance by increasing glucose consumption.

[0065] 4. Effects of lyophilized fermentation supernatant on glycogen synthesis in HepG2 cells HepG2 cells were seeded in 6-well plates, and after grouping and drug administration as before, the cells were washed twice with pre-chilled PBS. 0.25% trypsin-EDTA digestion solution was added until the cells became rounded and detached. Serum-containing medium was immediately added to terminate digestion. Cells were collected and centrifuged at 1000 rpm for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in pre-chilled PBS and washed twice. After centrifugation and supernatant discarding, the cells were lysed on ice using a Triton X-100 for 1 h. Glycogen content was measured using a glycogen assay kit, and normalized using the BCA protein concentration standard. Results are expressed as mg / mg prot.

[0066] The results of protein concentration determination by BCA method showed that the glycogen content of the model group (Con group) was 0.566±0.028 mg / mgprot, the glycogen content of the positive control metformin group (Met group) was 0.857±0.038 mg / mgprot, and the glycogen content of the B2Z2 group (0.899±0.026 mg / mgprot) was higher than that of the metformin group. These results further confirmed the effect of probiotic fermentation supernatant freeze-dried sample on improving glucose metabolism disorder in HepG2 cells.

[0067] 1. Preparation of Lactobacillus plantarum powder 100 mL of *Lactobacillus plantarum* fermentation broth was placed in a centrifuge bottle and centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the precipitate was washed with sterile water and centrifuged twice. The supernatant was discarded to obtain bacterial precipitate. Then, a precipitate of 20% skim milk powder was added at a ratio of bacterial slurry to preservative (w / v). The precipitate was placed in a shaker at 37℃ and shaken at 100 rpm for 30 min to mix thoroughly. The lyophilized powder was prepared by freeze-drying. The freeze-drying conditions were as follows: pre-freezing at -50℃ for 4 h, followed by vacuuming for 1 h, then gradually increasing the temperature from -50℃ to 20℃ by 5℃ every 2 h, and running at 20℃ for 9 h, for a total freeze-drying time of 42 h. The viable cell concentration of the obtained *Lactobacillus plantarum* powder was 3.55 × 10⁻⁶. 12 CFU / g.

[0068] 2. Optimization of prebiotic types and dosage 2.1 Selection of Prebiotic Types The basic formula for compound probiotic chewable tablets was set as follows: Lactobacillus plantarum 10.0%, microcrystalline cellulose 12.0%, magnesium stearate 0.8%, soluble starch 8.0%, xylitol 10.0%, skim milk powder 53.2%, and prebiotics 6.0%. The selected prebiotics included resistant dextrin, fructooligosaccharides, trehalose, inulin, and galactooligosaccharides. In the blank test, resistant starch of the same mass was used instead of prebiotics. After the tablets were prepared, they were crushed, and 0.25 g of powder was added to every 100 mL of MRS medium. After static incubation at 40℃ for 24 h, the viable bacteria concentration was measured. The experimental results are shown in Table 9.

[0069]

[0070] The results showed that fructooligosaccharides were the most effective, achieving a yield of 12.7 × 10⁻⁶ when used as a prebiotic. 10 The CFU / mL level was significantly higher than that of other groups (p<0.05).

[0071] 2.2 Optimization of Fructooligosaccharide Addition The viable bacterial concentrations were measured after incubating at 40℃ for 24 hours with 0, 2%, 4%, 6%, and 8% fructooligosaccharides. Figure 5 .

[0072] Figure 5 The results showed that when the amount of fructooligosaccharide added was 6%, the viable bacteria count reached 15.5 × 10⁻⁶. 10 The CFU / mL concentration was significantly higher than that of other groups (p<0.05), indicating that fructooligosaccharides had the best effect on promoting probiotic growth when the addition amount was 6%.

[0073] 3. Optimization of the probiotic chewable tablet formula Based on the preliminary experimental results, the basic formula for the probiotic chewable tablets was set as described in section 2.1 of Example 4. Using taste as the evaluation index (Table 10), a single-factor experimental method was employed to investigate the effects of the added amounts of sweeteners, lubricants, fillers, and disintegrants on the sensory quality of the chewable tablets. The concentrations of each excipient in the chewable tablet formula were optimized using single-factor variables, and the optimization results were used in subsequent experiments.

[0074] The preparation process of probiotic chewable tablets is as follows: (1) Freeze-drying: freeze-dry Lactobacillus plantarum; (2) Pulverizing: mix the excipients and probiotic powder evenly; (3) Tableting: add lubricant, mix evenly, and then put into a tableting mold to press into tablets; (4) Evaluation: determine the optimal formula of chewable tablets based on the appearance, color, smell and taste of the chewable tablets.

[0075]

[0076] 3.1 Selection of sweetener types and optimization of addition amount This selection involved three sweeteners: aspartame, a synthetic sweetener, is high in sweetness and low in calories, but contraindicated for those intolerant to phenylalanine; xylitol, with a sweetness close to sucrose, not only enhances the sweetness and texture of the product but also improves oral health; and erythritol, with a weak sweetness, requires a large amount. Taking all factors into consideration, xylitol was ultimately chosen because its natural properties align with health needs, its sweetness is close to sucrose, it requires no large amounts, and its natural source makes it more readily accepted by consumers. Based on the basic formula, only the amount of xylitol added was changed to 5.0%, 7.5%, 10.0%, 12.5%, and 15.0%, and the final amount was determined after sensory evaluation. The sensory evaluation scores are as follows: Figure 6 As shown.

[0077] according to Figure 6 The sensory evaluation score was highest when the xylitol content was 7.5%.

[0078] 3.2 Optimization of filler addition amount Soluble starch has excellent water solubility and can be uniformly dispersed in the product system without affecting the efficacy of sweeteners or the product's taste. Furthermore, it is naturally sourced, inexpensive, and can also aid in thickening and improve product stability. Based on the basic formula, only the amount of soluble starch added was changed to 4.0%, 6.0%, 8.0%, 10.0%, and 12.0%, and the sensory evaluation results are as follows: Figure 7 As shown.

[0079] According to the sensory evaluation scores, the highest sensory evaluation score was achieved when the soluble starch content was 8.0%.

[0080] 3.3 Optimization of Lubricant Addition Amount Magnesium stearate, a commonly used pharmaceutical excipient, has multiple functions including lubrication, anti-sticking, and improving powder flowability. In direct compression tableting, it primarily acts as a flow aid, significantly improving tablet surface smoothness. However, in practical applications, the dosage must be strictly controlled; excessive use can easily lead to a decline in tablet quality. Based on the basic formulation, by changing only the dosage of magnesium stearate to 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%, the sensory evaluation results are as follows: Figure 8 As shown.

[0081] According to the sensory evaluation scores, the highest sensory evaluation score was achieved when the amount of magnesium stearate added was 0.8%.

[0082] 3.4 Optimization of disintegrant loading amount Microcrystalline cellulose is a hydrolysis product of natural cellulose and is a relatively good binder. Based on the basic formulation, only the amount of microcrystalline cellulose added was changed to 8.0%, 10.0%, 12.0%, 14.0%, and 16.0%, and the sensory evaluation results are as follows: Figure 9 As shown.

[0083] According to sensory evaluation scores, the highest sensory evaluation score was achieved when the amount of microcrystalline cellulose added was 12.0%.

[0084] In summary, increasing the amount of each excipient resulted in a trend of first increasing and then decreasing sensory scores. Based on the sensory evaluation scores, the optimal formula for probiotic chewable tablets is: 10% Lactobacillus plantarum powder, 12% microcrystalline cellulose, 0.8% magnesium stearate, 8% soluble starch, 7.5% xylitol, and 61.7% skim milk powder.

[0085] 4. The effect of stress on the survival rate of probiotic chewable tablets Pressure can cause probiotic cells to die, thereby reducing the live bacteria yield of the final product. This is due to two main reasons. First, physical damage: high pressure can directly destroy the bacterial cell wall and cell membrane structure, causing leakage of intracellular substances or cell lysis. Gram-negative bacteria, which lack cell wall protection, are more sensitive to this. Second, the synergistic effect of the process: the tableting process is usually accompanied by friction that generates local temperature rises. The synergistic effect of high temperature and high pressure can further exacerbate the inactivation of live probiotics.

[0086] Studies have found that the tablets cannot be formed under pressure below 8 kN. As the pressure increases, the survival rate of live bacteria in the chewable tablets decreases linearly, and the texture gradually changes from being similar to milk tablets to being similar to hard candy.

[0087] Tableting was performed using different pressures. Three probiotic chewable tablets from each batch were selected, crushed, and mixed with sterile water. The mixture was then serially diluted and spread onto MRS solid medium. After incubation at 37°C for 48 hours, the tablets were counted. The yield was calculated according to Formula 6. The results are as follows: Figure 10 As shown.

[0088]

[0089] Figure 10 The results showed that the survival rate of probiotics decreased continuously with increasing pressure, and the two were strongly negatively correlated. The survival rate remained between 78.50% and 92.90% at 8–12 kN, indicating good strain tolerance; however, the survival rate plummeted after exceeding 12 kN, reaching only 18.30% at 18 kN and further decreasing to 7.86% at 20 kN. At a pressure of 8 kN, the live bacteria concentration in the chewable tablets was 3.3 × 10⁻⁶. 11 CFU / g.

[0090] 5. Stability test of probiotic chewable tablets Stability test results showed that the probiotic chewable tablets of the present invention, after being stored at 37°C for 7 days, had a viable bacteria count of 2.48 × 10⁻⁶. 11 The CFU / g concentration and survival rate are 75%. The storage stability of the probiotic chewable tablets of this invention is superior to similar products on the market, which can effectively ensure the hypoglycemic physiological activity of the product.

Claims

1. A Lactiplantibacillus plantarum strain, characterized in that, This strain was deposited on March 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 37876.

2. The application of *Lactobacillus plantarum* as described in claim 1, characterized in that, The hypoglycemic effect is manifested in the following aspects: (1) In vitro assay, including the ability to inhibit the activity of α-amylase and α-glucosidase; (2) Cell experimental model verification, the model is the FFA-BSA induced HepG2 cell model, and the verification indicators include changes in HepG2 cell survival rate, glycogen synthesis and cell glucose consumption; In the in vitro experiments, *Lactobacillus plantarum* showed an inhibitory activity of 45%–55% against α-amylase and 20%–30% against α-glucosidase, both of which were superior to the positive control group (acarbose). In the HepG2 cell model, the amount of glucose consumed by cells from fermentation supernatant of different concentrations of *Lactobacillus plantarum* increased with increasing concentration, and the consumption at high concentrations was higher than that in the metformin group.

3. A hypoglycemic probiotic chewable tablet, characterized in that, The hypoglycemic probiotic chewable tablets are prepared by mixing and compressing the freeze-dried bacterial powder of Lactobacillus plantarum as described in claim 1 with other excipients.

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