Lactobacillus buchneri with blood glucose-lowering activity and application thereof in fermented milk

Abstract

The application discloses a lactobacillus buchneri with blood sugar reducing activity and application of the lactobacillus buchneri in fermented milk, the strain is lactobacillus buchneri PB6, which is preserved in the Guangdong Microbial Culture Collection Center on May 19, 2025, and the preservation number is GDMCC No: 66352, the strain has blood sugar reducing activity, safety and probiotic property, has good tolerance to gastrointestinal environment, has excellent self-aggregation capacity, antioxidant capacity, and can inhibit the growth of common pathogenic bacteria, and is helpful to maintaining the balance of intestinal flora. The application of the lactobacillus buchneri PB6 in fermented milk can improve the sensory quality and rheological properties of the fermented milk, and has blood sugar reducing activity, rich organic acids and volatile flavor substances, and provides a basis for the application of the strain in functional food.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus bruneri PB6 derived from Guizhou Suantang and its application in fermented milk. Background Technology

[0002] Diabetes can lead to a variety of serious complications, including cardiovascular and cerebrovascular diseases, neuropathy, retinopathy, nephropathy, and diabetic foot, seriously threatening patients' lives and health. Type 2 diabetes (T2DM) patients account for 90% of all cases. T2DM is characterized by insulin resistance and glucose and lipid metabolism disorders, and the number of patients is constantly rising. Although existing drugs can control the disease to some extent, they still have significant side effects such as hypoglycemia and gastrointestinal discomfort, and there is an urgent need for safe and effective alternatives.

[0003] In recent years, the potential of lactic acid bacteria as a novel treatment option for diabetes has attracted increasing attention. Studies have shown that specific strains (such as *Lactobacillus plantarum* and *Lactobacillus paracasei*) and their metabolites (including extracellular polysaccharides and organic acids) have been proven to have hypoglycemic functions. Their mechanisms of action may involve regulating gut microbiota balance, improving insulin signaling, modulating glucose metabolism pathways, and exerting antioxidant and anti-inflammatory effects.

[0004] *Lactobacillus bruneri* is a non-spore-forming, non-motile, Gram-positive lactic acid bacterium, morphologically short rod-shaped, belonging to heterofermentative lactic acid bacteria. Its end products include lactic acid, acetic acid, and other compounds. Currently, *Lactobacillus bruneri* is mainly used in silage fermentation. This strain can tolerate the high temperature and high acidity environment during silage processing, effectively inhibiting mold growth, lowering pH, and increasing the total number of lactic acid bacteria, demonstrating excellent environmental adaptability and fermentation regulation capabilities. However, research on other functions of *Lactobacillus bruneri* is still relatively limited. Chinese invention patent application number 202410991998.X discloses that *Lactobacillus bruneri* ZD62 has a uric acid-lowering function.

[0005] Furthermore, there is currently limited research in China on the hypoglycemic effects of lactic acid bacteria dairy products, and the material basis and mechanism of their hypoglycemic effect remain unclear. Studies on the hypoglycemic mechanisms of other strains have mostly focused on single mechanisms, without comprehensive investigation of multiple mechanisms. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a Lactobacillus brucellosis PB6 with hypoglycemic activity and its application in fermented milk.

[0007] In a first aspect, the present invention provides a strain of *Lactobacillus buchneri* with hypoglycemic activity, wherein the strain is *Lactobacillus buchneri* PB6, deposited on May 19, 2025, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 66352. The supernatant, cell lysate, and bacterial suspension of this strain showed inhibition rates of 32.02%, 63.10%, and 46.36% against α-glucosidase, respectively, and inhibition rates against α-amylase of 44.94%, 6.97%, and 15.22%, respectively. The GLP-1 secretion level was 24.11 pmol / L.

[0008] In a second aspect of the invention, a safety evaluation of the aforementioned *Lactobacillus brucellosis* PB6 is provided, including antibiotic susceptibility testing, hemolytic activity testing, indole testing, nitroreductase testing, and amino decarboxylase testing of the strain. This demonstrates that *Lactobacillus brucellosis* PB6 is sensitive to ampicillin, kanamycin, streptomycin, and erythromycin, does not hemolyze, is negative in the indole test, is negative in the nitroreductase test, and is negative in the amino decarboxylase test, and has no oral toxicity in mice, thus classifying it as safe and non-toxic. Therefore, this strain is safe.

[0009] In a third aspect of the invention, the tolerance of the *Lactobacillus brucellosis* PB6 strain to a simulated gastrointestinal fluid environment is provided. *Lactobacillus brucellosis* PB6 exhibits a survival rate greater than 80% after 3 hours of exposure to simulated gastric fluid at different pH levels (pH=2, 3), a survival rate greater than 100% after 6 hours of exposure to simulated intestinal fluid, and a survival rate greater than 89% after 3 hours of exposure at a 0.3% bile salt concentration.

[0010] In a fourth aspect of the invention, the *Lactobacillus brunelli* PB6 strain is provided with self-aggregation, hydrophobicity, and antioxidant capabilities. After 24 hours of cultivation, the self-aggregation capacity of PB6 reached 55.47%. After 1 hour of cultivation, the hydrophobicity of PB6 reached 84.72%. The PB6 supernatant exhibited resistance to DPPH, ·OH, and O2. － The clearance rates were 92.61%, 77.14%, and 55.56%, respectively.

[0011] In a fifth aspect of the invention, the inhibitory effect of the *Lactobacillus bruneri* PB6 strain on common pathogenic bacteria is provided. The inhibition zone size (mm) of the *Lactobacillus bruneri* PB6 supernatant against *Escherichia coli* is 15.70–17.26, against *Staphylococcus aureus* is 12.00–12.36, and against *Salmonella typhimurium* is 13.24–13.80.

[0012] In a sixth aspect of the invention, the effects of the *Lactobacillus brunelli* PB6 strain on liver glucose metabolism are provided. Treatment with PB6 increases glucose consumption and glycogen synthesis in IR-HepG2 cells, increases the activity of pyruvate kinase (PK), the rate-limiting enzyme in glycolysis, decreases the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in gluconeogenesis, and restores the expression of glucose metabolism-related genes. Some results from the supernatant treatment are superior to those from the positive control (metformin) treatment.

[0013] In a seventh aspect of the invention, the effects of the *Lactobacillus brunelli* PB6 strain on hormone secretion in the gut-pancreatic axis are provided. Treatment with PB6 promotes the secretion of glucagon-like peptide-1 (GLP-1) and the expression of genes related to GLP-1 secretion, with the bacterial suspension showing the most significant effect.

[0014] In an eighth aspect of the invention, a method for preparing the Lactobacillus brucellosis PB6 direct-inoculation starter culture is provided, which yields a viable count of 1.5 × 10⁻⁶. 11 CFU / g direct-inoculation starter culture.

[0015] In a ninth aspect of the invention, the application of the direct-inoculation starter culture in fermented milk is provided, resulting in a viable count of 2.5 × 10⁻⁶ cells / ml. 9 The fermented milk contained CFU / mL, with an α-amylase inhibition rate of 88.98% and an α-glucosidase inhibition rate of 55.98%. It exhibited 34 volatile flavor compounds and contained various organic acids such as citric acid, tartaric acid, and succinic acid. Lactic acid content reached as high as 30 mg / mL, and acetic acid content reached 10.96 mg / mL. The supernatant of the fermented milk showed positive effects on DPPH, ·OH, and O2. － The removal rates of · were 98.87%, 98.69% and 76.78%, respectively, and it had an inhibitory effect on Escherichia coli, Staphylococcus aureus and Salmonella typhimurium. The addition of PB6 can improve the sensory quality and rheological properties of fermented milk.

[0016] To achieve the above objectives, the present invention adopts the following technical solution:

[0017] This invention isolated and preserved a novel *Lactobacillus buchneri* strain with hypoglycemic potential from a Guizhou sour soup sample, naming it *Lactobacillus buchneri* PB6. This strain exhibits good gastrointestinal tolerance and can exert its hypoglycemic potential, specifically demonstrating high inhibition rates of α-amylase and α-glucosidase, and effectively promoting the secretion of GLP-1 by intestinal STC-1 cells. Using this strain does not induce drug resistance or cause acute toxicity.

[0018] The biological characteristics of strain *Lactobacillus brunelli* PB6 are as follows: When streaked onto MRS agar plates and incubated at 37°C for 48–72 h, the colonies are round, raised, with smooth, regular edges, and are milky white with a moist, smooth surface. Gram staining and microscopic examination reveal it to be a Gram-positive bacterium, with short rod-shaped cells and no spores (e.g., ...). Figure 1 (As shown). Physiological and biochemical tests showed that the strain could produce acid using maltose, galactose, and glucose, and was facultatively anaerobic. Based on its 16S rDNA and physiological and biochemical characteristics, PB6 was identified as *Lactobacillus bruschetta*. Its 16S rDNA sequence is shown in SEQ NO: 1.

[0019] The application of Lactobacillus brucellosis PB6 as a starter in fermented milk or in the preparation of drugs for the prevention and treatment of diabetes.

[0020] The application of the Lactobacillus bruneri direct-inoculation starter in the preparation of fermented milk.

[0021] Preferably, the fermenting agent is a mixture of Lactobacillus brucellosis PB6 and a traditional fermenting agent.

[0022] Preferably, the conventional fermentation agent is Streptococcus thermophilus DMST-H2 and Lactobacillus delbrueckii DMLD-H1.

[0023] Preferably, the mass ratio of Lactobacillus brucellosis PB6 to the conventional starter culture is 1:4-3:2; and the mass ratio of Streptococcus thermophilus DMST-H2 to Lactobacillus delbrueckii DMLD-H1 is 1:(0.5-2.0).

[0024] Preferably, the method for preparing the fermented milk is as follows: by weight, 2-3 parts of erythritol, 0.5-1.5 parts of xylitol, and 0.01-0.02 parts of steviol glycosides are added and the volume is adjusted to 100 parts with whole milk. The mixture is heated at 90-95°C for 5±3 minutes, cooled, and then a starter culture is added for fermentation to obtain sugar-free fermented milk.

[0025] Preferably, the fermentation conditions are: fermentation at 43±5℃ for 6~8 h, followed by fermentation at 4℃ for 12~24 h.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] (1) This invention discloses a strain of *Lactobacillus bruneri* PB6, screened from the traditional Chinese fermented food red sour soup. Its biological characteristics are more compatible with the dietary structure and intestinal microecological environment of Chinese people than imported strains, exhibiting higher colonization potential and physiological activity. This strain possesses hypoglycemic activity; specifically, the fermentation supernatant, bacterial cell lysate, and bacterial suspension all effectively inhibit α-amylase and α-glucosidase activity and promote GLP-1 secretion. Its hypoglycemic activity is higher than that of the commercial strain *Lactobacillus rhamnosus* LRa66. This strain can effectively delay the decomposition and absorption of carbohydrates, thereby stabilizing postprandial blood glucose and achieving long-term blood glucose stability.

[0028] (2) This invention has demonstrated the safety of this strain through a series of safety experiments, and acute toxicology has shown that it is practically non-toxic. This bacterium has good tolerance to the gastrointestinal environment, excellent self-aggregation ability and antioxidant capacity, and can inhibit the growth of common pathogenic bacteria, which helps to maintain the balance of intestinal flora.

[0029] (3) The Lactobacillus brucellosis PB6 of the present invention can improve liver glucose metabolism, specifically by promoting glucose consumption in IR-HepG2 cells, increasing glycogen synthesis, increasing pyruvate kinase (PK) activity, decreasing phosphoenolpyruvate carboxykinase (PEPCK) activity, upregulating the expression of AMPK, PI3K, IRS1, AKT, and GYS2 genes, and downregulating the expression of GSK3β, FOXO1, and PEPCK genes, thereby activating the liver AMPK / PI3K / AKT pathway. In some groups, the effect is even better than that of the positive control metformin.

[0030] (4) The Lactobacillus Brucella PB6 of the present invention can promote the secretion of hormones in the gut-pancreatic axis. Specifically, it significantly upregulates the expression of the pro-glucagon gene and its key cleavage enzyme (PCSK1) in STC-1 cells, thereby effectively promoting the secretion of biologically active GLP-1. The effect of PB6 bacterial suspension treatment is better than that of the positive control acarbose.

[0031] (5) This invention discloses the application of Lactobacillus brucellosis PB6 in fermented milk. The fermented milk produced exhibits outstanding processing characteristics and physiological activity, specifically high α-amylase inhibition rate, α-glucosidase inhibition rate and antioxidant capacity, rich in organic acids and volatile flavor substances, and has excellent rheological properties, antioxidant capacity, antibacterial capacity and storage stability. Its comprehensive sensory and functional indicators are close to or even exceed those of fermented milk made from commercial strains. Moreover, no sucrose is added, so it can be used as a high-quality raw material for functional foods, providing consumers with a new dietary choice that combines nutrition, flavor and physiological benefits.

[0032] The Lactobacillus buchneri PB6 strain was deposited on May 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 66352. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0033] The Lactobacillus delbrueckii DMLD-H1 strain was deposited on April 16, 2019, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 60645). The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0034] The Streptococcus thermophilus DMST-H2 strain was deposited on April 16, 2019, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 60642. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0035] Figure 1 These are Gram-stained microscopic images of Lactobacillus brunetti PB6 (left) and morphological images of pour culture (right).

[0036] Figure 2 This is a phylogenetic tree diagram of Lactobacillus brunelli strain PB6.

[0037] Figure 3 These are images showing the growth of E. coli 25922 (left) and Lactobacillus brunelli PB6 (right) on blood agar plates.

[0038] Figure 4 This is a diagram of the indole experiment results; from left to right, they are E. coli 25922, Lactobacillus brunelli PB6, and a blank control.

[0039] Figure 5 This is a diagram of the nitroreductase experiment results; from left to right, they are E. coli 25922, Lactobacillus brunelli PB6 (3 tubes), and a blank control.

[0040] Figure 6 The results of the amino decarboxylase experiment are shown in the figure. From top to bottom, the results are for the culture medium with (a) lysine, (b) tyrosine, (c) arginine, and (d) histidine. The left column is the blank control group, the middle column is the experimental group with the positive control of Escherichia coli ATCC 25922, and the right column is the experimental group with Lactobacillus brucellosis PB6.

[0041] Figure 7This is a graph showing the gastric juice tolerance results of Lactobacillus bruneri PB6.

[0042] Figure 8 This is a graph showing the bile salt tolerance results of Lactobacillus brucellosis PB6.

[0043] Figure 9 The images show the self-aggregation ability (left) and hydrophobicity test results (right) of Lactobacillus brunelli PB6 and Lactobacillus rhamnosus LRa66.

[0044] Figure 10 The graph shows the antioxidant capacity of Lactobacillus brucellosis PB6 and Lactobacillus rhamnosus LRa66. Among them, (a) is the DPPH free radical scavenging rate, (b) is the hydroxyl free radical scavenging rate, and (c) is the superoxide anion free radical scavenging rate.

[0045] Figure 11 This is a graph showing the effect of Lactobacillus brucellosis PB6 on glucose consumption in HepG2 cells.

[0046] Figure 12 This is a graph showing the effect of Lactobacillus bruneri PB6 on glycogen synthesis in HepG2 cells.

[0047] Figure 13 The graph shows the effects of Lactobacillus brucellosis PB6 on key enzymes of glucose metabolism in HepG2 cells. The left graph shows pyruvate kinase (PK) activity, and the right graph shows phosphoenolpyruvate carboxykinase (PEPCK) activity.

[0048] Figure 14 This is a graph showing the effect of Lactobacillus bruneri PB6 on the expression of glucose metabolism-related genes in HepG2 cells. The top row, from left to right, contains AMPK, PI3K, IRS-1, and AKT, while the bottom row, from left to right, contains GSK3β, GYS2, FOXO1, and PEPCK.

[0049] Figure 15 This is a graph showing the effect of Lactobacillus brucellosis PB6 on GLP-1 secretion levels.

[0050] Figure 16 This is a graph showing the effect of Lactobacillus brunelli PB6 on the expression of genes related to the gut-pancreatic axis in STC-1 cells. The left graph is pro-glucagon, and the right graph is PCSK1.

[0051] Figure 17 This is a graph showing the sensory evaluation results of fermented milk with different compound ratios.

[0052] Figure 18 This is a viscosity curve of fermented milk.

[0053] Figure 19The graph shows the dynamic time-varying curves of the viscoelasticity of fermented milk. The left graph shows the change of the storage modulus (G') of fermented milk over time, and the right graph shows the change of the loss modulus (G") of fermented milk over time.

[0054] Figure 20 This is a graph showing the organic acid content of fermented milk.

[0055] Figure 21 The graph shows the results of the antioxidant capacity test of fermented milk, where (a) is the DPPH free radical scavenging rate, (b) is the hydroxyl free radical scavenging rate, and (c) is the superoxide anion free radical scavenging rate.

[0056] Figure 22 The graph shows the results of stability testing of fermented milk during storage, where (a) is pH value, (b) is titratable acidity, (c) is water-holding capacity, and (d) is the result of changes in viable cell count over storage time. Detailed Implementation

[0057] The present invention will now be described and illustrated in detail with reference to specific embodiments.

[0058] Example 1: Isolation and screening of lactic acid bacteria with hypoglycemic activity, carried out according to the following steps:

[0059] (1) Select Guizhou sour soup samples, use physiological saline with a mass concentration of 0.9% for serial dilution, select appropriate gradients to spread on MRS solid medium, incubate at 37℃ for 2 days, pick strains with different colony morphologies to streak on solid medium for purification, pick single colonies to expand culture in MRS liquid medium, and then preserve with 50% glycerol in a -80℃ refrigerator.

[0060] (2) The supernatant of 41 single colonies from Guizhou were used for initial screening of the inhibition rates of α-amylase and α-glucosidase. 10-15 strains with better effects were selected and re-screened using the same indicators in bacterial suspension and bacterial cell lysate. The experiments on promoting GLP-1 secretion in STC-1 cells were further performed using these strains. The results are shown in Tables 1-3. A strain with significant effect, PB6, was screened out. The overall hypoglycemic activity of this strain was higher than that of the commercial strain LRa66, which has been proven to have hypoglycemic activity. The inhibition rates of α-glucosidase in the supernatant, bacterial cell lysate and bacterial suspension of this strain were 32.02%, 63.10% and 46.36%, respectively, and the inhibition rates of α-amylase were 44.94%, 6.97% and 15.22%, respectively. The GLP-1 secretion amount was 24.11 pmol / L. Compared with LRa66, PB6 showed a 102.15% higher α-glucosidase inhibition rate in its supernatant, a 27.11% higher combined inhibition rate of α-amylase and α-glucosidase in its bacterial suspension and cell lysate, and a 22.82% higher GLP-1 secretion rate. PB6 was then identified.

[0061] The α-glucosidase inhibition experiment was performed as follows: 50 μL of PBS solution was mixed with 50 μL of PNPG solution (1.5 mol / L), and then 25 μL of fermentation supernatant, bacterial suspension, or cell lysate was added. After mixing, the mixture was reacted at 37℃ for 10 min. Then, 30 μL of 0.2 U / mL α-glucosidase solution was added, and the reaction was continued at 37℃ for 30 min. Finally, 50 μL of Na2CO3 solution (0.2 mol / L) was added to terminate the reaction. The OD was measured using a microplate reader. 405nm .

[0062]

[0063] A: OD value containing the sample and α-glucosidase solution; B: OD value containing the sample and without α-glucosidase solution; C: OD value without the sample and containing α-glucosidase solution; D: OD value without the sample and without α-glucosidase solution.

[0064] The α-amylase inhibition experiment was performed as follows: 0.25 mL of fermentation supernatant, bacterial suspension, or cell lysate was mixed with 0.25 mL of α-amylase solution (1 mg / mL) and reacted at 37°C for 10 min. After the reaction, 0.5 mL of 1.5% soluble starch solution (preheated to 37°C) was added, and the mixture was reacted at 37°C for 5 min. Then, 1 mL of DNS chromogenic reagent was added, mixed, boiled in boiling water for 5 min, cooled with ice water, appropriately diluted with PBS solution, and allowed to stand at room temperature for 30 min. The OD was then measured using a microplate reader. 450nm .

[0065]

[0066] A: OD value containing the sample and α-amylase solution; B: OD value containing the sample and without α-amylase solution; C: OD value without the sample and containing α-amylase solution; D: OD value without the sample and without α-amylase solution.

[0067] The STC-1 cell GLP-1 secretion experiment: STC-1 cell lines were seeded in 6-well plates and cultured for 48-72 h until the density reached 2×10⁶ cells / wells. 5 Each / well, using Ca-free 2+ and Mg 2+ The cells were washed twice with PBS buffer and cultured in DMEM without glucose and L-glutamine for 30 min. The bacterial strain was washed twice with PBS, centrifuged (8000 rpm, 4℃, 10 min) to collect the pellet, and the bacterial cells were resuspended in the above DMEM medium. The bacterial cell concentration was adjusted to 10-. 8CFU / mL. Incubate at 37℃ for 4 h. Collect the supernatant and centrifuge (5900 rpm, 4℃, 10 min). Determine the GLP-1 content according to the ELISA kit method. Use the supernatant of STC-1 cells without bacterial strain as a blank control.

[0068] The method for preparing the supernatant is as follows: after liquid culture at 37℃ for 12~36 h, the bacterial culture is centrifuged (8000 rpm, 4℃, 10 min), the supernatant is taken and filtered through a 0.22 μm filter membrane.

[0069] The bacterial suspension was prepared as follows: After liquid culture at 37℃ for 12-36 h, the bacterial suspension was centrifuged (8000 rpm, 4℃, 10 min), and the bacterial sludge was collected. The sludge was washed 2-3 times with 0.9% physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0070] The method for preparing the bacterial cell lysate is as follows: After liquid culture at 37℃ for 12-36 h, the bacterial culture is centrifuged (8000 rpm, 4℃, 10 min), the bacterial sludge is collected, washed 2-3 times with 0.9% physiological saline, and the bacterial concentration is adjusted to 1×10⁻⁶. 9 Cells were lysed at CFU / mL under ice bath conditions at 200 W power for 15 min pulse, with a lysing time of 3-8 s (3 s working, 8 s stopping). The lysate was centrifuged (11000 rpm, 10 min), and the supernatant was collected and filtered through a 0.22 μm filter membrane.

[0071] Table 1. Results of the α-amylase and α-glucosidase inhibition experiments in the supernatant.

[0072]

[0073] Table 2. Results of the α-amylase and α-glucosidase inhibition experiments in bacterial suspension and bacterial cell lysate.

[0074]

[0075] Table 3 Results of GLP-1 secretion experiment in STC-1 cells

[0076]

[0077] Gram staining and microscopic examination revealed that PB6 is a Gram-positive bacterium with short rod-shaped cells and no spores (e.g., ...). Figure 1 (As shown). Physiological and biochemical tests showed that the strain could produce acid using maltose, galactose, and glucose, and was facultatively anaerobic. Based on 16S rDNA and physiological and biochemical characteristics, PB6 was identified as *Lactobacillus bruschetta*. Its 16S rDNA sequence is shown in SEQ NO: 1, and the phylogenetic tree is shown below. Figure 2 As shown.

[0078] The MRS liquid culture medium is as follows (by mass): 1 part casein digest, 1 part beef extract powder, 0.4 parts yeast extract powder, 0.2 parts triammonium citrate, 0.5 parts sodium acetate, 0.02 parts magnesium sulfate heptahydrate, 0.005 parts manganese sulfate tetrahydrate, 0.2 parts dipotassium hydrogen phosphate, 2 parts glucose, 0.1 parts Tween-80, and distilled water to a final volume of 100 parts. The pH before sterilization is 5.7 ± 0.2, and the medium is autoclaved at 121°C for 15 min.

[0079] The PBS solution is as follows: by mass ratio, 0.8 parts NaCl, 0.02 parts KH2PO4, 0.115 parts Na2HPO4, and distilled water is added to bring the total to 100 parts, and the mixture is stirred until homogeneous.

[0080] This embodiment uses in vitro α-amylase, α-glucosidase inhibition rate and GLP-1 secretion as screening criteria to screen lactic acid bacteria with hypoglycemic activity from Guizhou sour soup. The screened strains were morphologically observed and physiologically and biochemically identified, and 16S rDNA was identified. The identification results showed that PB6 belongs to Lactobacillus buchneri and was named L buchneri PB6.

[0081] Example 2: Safety evaluation of Lactobacillus bruneri PB6

[0082] The strains frozen at -80℃ were inoculated into seed culture medium and activated 2-3 times to obtain seed culture solution. The seed culture medium was MRS liquid culture medium, which was sterilized at 121℃ for 15 min.

[0083] 2.1 Antibiotic susceptibility testing of strains

[0084] The minimum inhibitory concentrations (MICs) of *Lactobacillus bruneri* PB6 against ampicillin, kanamycin, streptomycin, and erythromycin were determined using a micro-dilution method. The detection concentrations for ampicillin were 0.125–128 μg / mL, for kanamycin 0.5–512 μg / mL, for streptomycin 0.5–512 μg / mL, and for erythromycin 0.0625–64 μg / mL. The antibiotics were first diluted twofold to twice their detection concentration. 100 μL of the antibiotic solution was added to wells 2–12 of a sterile 96-well plate, followed by 100 μL of MH medium containing PB6 bacterial culture in each well, and mixed thoroughly with a sterile pipette tip. The positive control consisted of 100 μL of the same concentration of the test bacterial culture mixed with 100 μL of distilled water, and the negative control consisted of 100 μL of MH medium mixed with 100 μL of distilled water. After incubation at 37 ℃ for 24 h, the OD was measured using a microplate reader. 600nmThe results of the PB6 drug resistance test were obtained by comparing the results with the corresponding critical values ​​specified in the guidelines of the European Food Safety Authority. The results are shown in Table 4. Lactobacillus brucellosis PB6 is sensitive to ampicillin, kanamycin, streptomycin, and erythromycin, and is therefore safe.

[0085] Table 4. Minimum inhibitory concentrations of antibiotics against Lactobacillus brunelli PB6

[0086]

[0087] Note: EFSA is the abbreviation for European Food Safety Authority.

[0088] The MH culture medium consisted of the following components by mass: 30 parts beef (from which extract powder was extracted), 0.15 parts soluble starch, 1.75 parts casein hydrolysate, and distilled water to a final volume of 100 parts. The pH before sterilization was 7.3 ± 0.2, and the medium was autoclaved at 121°C for 15 min.

[0089] 2.2 Hemolysis test

[0090] Activated Lactobacillus bruneri PB6 and the quality control strain Escherichia coli ATCC 25922 were streaked onto Columbia blood agar using sterilized inoculation needles, with a blank control also performed. The cultures were incubated at 37°C for 48 h, and the presence or absence of obvious hemolysis zones around the colonies was observed and photographed. Results are as follows: Figure 3 As shown, the right side shows the growth of *Lactobacillus bruneri* PB6 on a blood agar plate, while the left side shows the growth of the quality control strain *Escherichia coli* on a blood agar plate. The quality control strain used in this example exhibits hemolytic activity, with a hemolytic zone appearing around it. Compared to the quality control strain, *Lactobacillus bruneri* PB6 did not form a significant hemolytic zone, meaning that PB6 will not cause hemolytic harm to humans and is safe.

[0091] 2.3 Indole Experiment

[0092] The activated *Lactobacillus bruneri* PB6 seed fermentation broth and the positive control strain *Escherichia coli* broth were inoculated into peptone water medium at a rate of 3% (v / v), with a blank control also included. The mixture was incubated at 37°C for 72 h. After adding 8–10 drops of indole reagent, the experimental results were observed. A red ring at the interface between the two liquid layers indicated a positive result; no color change indicated a negative result. The experimental results are as follows: Figure 4 As shown, after the addition of indole reagent, a distinct red ring appeared at the interface between the two liquid layers in the peptone water medium inoculated with the positive control *E. coli*, indicating a positive indole test result. No obvious color change was observed in the peptone water medium inoculated with PB6 or the blank control, indicating a negative indole test result.

[0093] The peptone aqueous culture medium is prepared by adding 1 part bacteriological peptone, 0.5 parts sodium chloride, and distilled water to a total of 100 parts by mass ratio, stirring evenly, adjusting the pH of the culture medium to 7.8, and autoclaving at 121°C for 15 min.

[0094] 2.4 Nitroreductase Experiment

[0095] Three copies of activated *Lactobacillus bruneri* PB6 and the positive control strain *Escherichia coli* were inoculated into nitroreductase detection medium at an inoculation rate of 3% (v / v). A blank control group was also set up. The medium was incubated at 37°C for 72 h. Two to three drops of α-naphthylamine solution and p-aminobenzoic acid solution were added sequentially to the medium, and after gentle shaking, the color change of the medium was observed and photographed. A red color in the medium indicated a positive nitroreductase detection result; otherwise, it was negative. Results are as follows: Figure 5 As shown, the nitroreductase experiment of Lactobacillus brunelli PB6 was negative, proving that PB6 does not contain nitroreductase and will not reduce nitrate to nitrite.

[0096] The nitroreductase detection medium is prepared by adding 1 part bacteriological peptone, 0.1 parts potassium nitrate, and distilled water to a total of 100 parts by mass ratio, stirring until homogeneous, adjusting the pH of the medium to 7.4, dispensing into Erlenmeyer flasks, and autoclaving at 121°C for 15 minutes.

[0097] 2.5 Amino decarboxylase experiment

[0098] Activated *Lactobacillus bruneri* PB6 and the positive control strain *Escherichia coli* were inoculated at a rate of 3% (v / v) into amino decarboxylase medium and subcultured 5-7 times at 37°C to induce amino decarboxylase production. 100 μL of each medium was spread onto amino decarboxylase medium containing histidine, arginine, tyrosine, and lysine at a final concentration of 0.5%, and incubated at 37°C for 3 days. The production of histamine, putrescine, spermidine, and tyramine was then detected. A change in medium color from yellow to purple indicated a positive result; otherwise, a negative result was observed. The experimental results are as follows: Figure 6 As shown, the culture medium containing the four amino acids added to Escherichia coli was purple, indicating a positive result; while the culture medium containing Lactobacillus brucellosis PB6 was yellow, indicating a negative result, consistent with the blank control result. This indicates that PB6 does not produce amino decarboxylase during its proliferation and metabolism, thus avoiding the formation of biogenic amines.

[0099] The amino decarboxylase medium is composed of, by mass ratio, 0.5 parts of tryptone, 0.8 parts of beef extract, 0.4 parts of yeast extract, 0.05 parts of Tween-80, 0.02 parts of magnesium sulfate, 0.005 parts of manganese sulfate, 0.004 parts of ferric sulfate, 0.01 parts of calcium carbonate, and 0.006 parts of bromocresol purple. Distilled water is added to make up 100 parts, and the mixture is stirred evenly. The pH value of the medium is adjusted to 5.3, and it is sterilized at 121 °C under high pressure for 15 min.

[0100] 2.6 Oral acute toxicity experiment of mice

[0101] Lactobacillus buchneri PB6 is made into a bacterial liquid of 1×10 8 CFU / mL, and the Guangzhou Customs Technology Center is entrusted to conduct the oral acute toxicity experiment of PB6 on mice in accordance with the "National Food Safety Standard Acute Oral Toxicity Experiment" (Experimental Animal Use License No. SYXK (Guangdong) 2018-0086). Twenty SPF-grade Kunming mice (half male and half female, weighing 20.0 - 22.0 g, Experimental Animal Production License No. SCXK (Guangdong) 2018-0002, Quality Certificate No. 44007200105037) are purchased from the Guangdong Provincial Medical Experimental Animal Center. The room temperature is 22 ± 1 °C, and the humidity is 62 ± 5%. 50.00 g of the bacterial liquid is weighed and mixed with ultrapure water to 100 mL to make the experimental liquid. The experimental liquid is administered orally to the mice once by gavage at a dose of 0.2 mL / 10 g body weight. The mice are fasted for 4 h before gavage and allowed to drink water freely. Normal diet is given 1 h after gavage; the experimental observation period is 14 days. Record the poisoning symptoms, number of deaths, and death time of the mice. The mice are weighed once a week during this period and the results are recorded. As shown in Table 5, 14 days after oral administration of the PB6 bacterial liquid, no clinical abnormalities, eye abnormalities, or signs of death were found in the mice. After the experiment, the mice were dissected, and no abnormalities were seen macroscopically. The body weight of the mice gradually increased during the study period. The acute oral toxicity LD50 of the oral liquid containing PB6 for male and female KM mice > 10.0 g / kg body weight, belonging to the actually non-toxic level.

[0102] Table 5 Results of the acute oral toxicity experiment of the PB6 bacterial liquid on mice

[0103]

[0104] Example 2 shows that Lactobacillus buchneri PB6 does not produce harmful metabolites, has no oral toxicity to mice, and is safe and non-toxic.

[0105] Example 3: Probiotic evaluation of Lactobacillus buchneri PB6

[0106] 3.1 Experiment on tolerance to artificial gastrointestinal environment

[0107] The activated Lactobacillus bruneri PB6 seed fermentation broth was inoculated into simulated gastric juice at pH 2.0 and 3.0 at an inoculation rate of 10% (v / v), with an equal volume of distilled water as a control group. The mixture was incubated at 37℃, and samples were taken at 0 h, 1 h, 2 h, and 3 h to count the viable cells. Figure 7 As shown, Lactobacillus brunetti PB6 survived well in artificial gastric fluids at different pH levels. Within 1-3 hours, the survival rate of PB6 increased in the pH 3.0 environment, and the survival rate was still above 80% after 3 hours in gastric fluid at pH 2.0. This indicates that the PB6 strain has strong acid resistance and can adapt to the acidic gastric fluid environment and proliferate normally.

[0108] The artificial gastric fluid was prepared by adding 0.3 parts pepsin to 100 parts PBS buffer solution and adjusting the pH to 2.0 or 3.0 with 1N HCl. After complete dissolution, the solution was filtered through a 0.22 μm microporous membrane for sterilization.

[0109] The activated Lactobacillus bruneri PB6 seed fermentation broth was inoculated into artificial intestinal fluid at an inoculation rate of 10% (v / v), with an equal volume of distilled water as a control group. The cultures were incubated at 37°C, and samples were taken at 0 h, 2 h, 4 h, and 6 h to count the viable bacteria. As shown in Table 6, Lactobacillus bruneri PB6 could proliferate normally after treatment in simulated intestinal fluid, and its survival rate in artificial intestinal fluid after 2–6 h of exposure exceeded 100%, similar to the control group.

[0110] Preparation of the artificial intestinal fluid: 0.1 parts trypsin by mass, add PBS buffer solution to 100 parts, adjust the pH to 8.0 with 1N NaOH, dissolve thoroughly, and then filter sterilize using a microporous membrane with a pore size of 0.22 μm.

[0111] Table 6. Changes in the survival rate of Lactobacillus bruneri PB6 in artificial intestinal fluid.

[0112]

[0113] The activated Lactobacillus bruneri PB6 seed fermentation broth was inoculated at a rate of 10% (v / v) into MRS broth medium containing 0.15% and 0.3% ox bile salts, with an equal volume of distilled water as a control. The culture was incubated at 37°C, and samples were taken at 0 h, 1 h, 2 h, and 3 h to count the viable cells. The experimental results are as follows: Figure 8 As shown, PB6 can grow normally in low concentrations of bile salts, and after 3 hours of culture in 0.3% bile salts, the survival rate is still over 89%, proving that PB6 can tolerate higher concentrations of bile salts. Combined with the above experiments, this demonstrates that PB6 has strong gastrointestinal tolerance and can smoothly pass through the human gastrointestinal tract to exert its beneficial effects.

[0114] 3.2 Determination of self-coagulation ability and hydrophobicity

[0115] Centrifuge the overnight culture (8000 rpm, 4℃, 10 min) to collect the bacterial pellet, wash twice with sterile 0.9% physiological saline, and resuspend the bacterial cells in sterile 0.9% physiological saline to OD. 600nm The concentration was approximately 0.6. After standing at 37°C for 24 hours, samples were taken at 4, 8, and 24 hours to measure the OD. 600nm

[0116]

[0117] Among them, A t A0 and OD at time t and 0, respectively. 600nm value.

[0118] Activated Lactobacillus rhamnosus LRa66 (purchased from Microcare Probiotics Co., Ltd.) was compared with Lactobacillus bruneri PB6 using the same method described above. Figure 9 As shown in (a), the self-aggregation capacity of both strains showed an increasing trend over time. At 24 h, the self-aggregation capacity of PB6 exceeded 55%, which was higher than that of Lactobacillus rhamnosus LRa66.

[0119] Centrifuge the overnight culture (8000 rpm, 4℃, 10 min) to collect the bacterial pellet, wash twice with sterile 0.9% physiological saline, and resuspend the bacterial cells in sterile 0.9% physiological saline to OD. 600nm To obtain an OD of approximately 0.5, take 3 mL of the adjusted bacterial suspension, add 1 mL of xylene, vortex for 2 min to mix thoroughly, and let stand at room temperature. Samples were taken from the aqueous phase at 0.5 h and 1 h to detect the OD. 600nm .

[0120]

[0121] Among them, A t A0 and OD at time t and 0, respectively. 600nm value.

[0122] The activated Lactobacillus rhamnosus LRa66 was compared with Lactobacillus brunelli PB6 using the same method described above. The results are as follows: Figure 9 As shown in (b), PB6 has stronger hydrophobicity than LRa66, reaching 81.80% hydrophobicity in 0.5 h and remaining stable after 1 h.

[0123] Lactobacillus bruneri PB6 is superior to Lactobacillus rhamnosus LRa66 in both self-aggregation and hydrophobicity, showing stronger intestinal colonization potential. The strong hydrophobicity proves that PB6 can more easily penetrate the mucus layer and attach to intestinal epithelial cells, while the strong self-aggregation helps the strain form a stable biofilm in the intestine.

[0124] 3.3 Antioxidant capacity determination

[0125] 3.3.1 DPPH free radical scavenging experiment

[0126] The concentration of the activated Lactobacillus bruneri PB6 bacterial culture was adjusted to OD. 600nm Within 1, the DPPH free radical scavenging capacity of the bacterial suspension and supernatant were measured separately. 2 mL of the sample to be tested was mixed with 2 mL of 0.2 mmol / L DPPH ethanol solution, and the mixture was reacted in the dark at room temperature for 30 min, followed by centrifugation (3500 rpm, 10 min). The OD of the supernatant was then measured. 517nm .

[0127]

[0128] Among them, A r The absorbance of the sample was measured after mixing it with DPPH ethanol solution. A s To measure absorbance using an equal volume of anhydrous ethanol instead of DPPH ethanol solution, A t The absorbance was measured using PBS buffer instead of the sample. The DPPH radical scavenging rate of *Lactobacillus rhamnosus* LRa66 was determined using the same method described above. Figure 10 As shown in (a), Lactobacillus blight PB6 has strong antioxidant capacity, and the antioxidant capacity of the supernatant is stronger than that of the bacterial suspension. The DPPH scavenging rate of both PB6 and LRa66 supernatants exceeded 90%, and the DPPH scavenging rate of PB6 bacterial suspension was higher than that of LRa66, exceeding 41%.

[0129] 3.3.2 Hydroxyl radical scavenging experiment

[0130] The concentration of the activated Lactobacillus bruneri PB6 bacterial culture was adjusted to OD. 600nm Within a range of 1, the hydroxyl radical scavenging capacity of the bacterial suspension and supernatant was measured separately. 0.05 mL of 9 mmol / L ferrous sulfate solution, 0.5 mL of 8.8 mmol / L hydrogen peroxide solution, 0.4 mL of the test sample, 0.05 mL of 9 mmol / L salicylic acid ethanol solution, and 1.9 mL of distilled water were added sequentially and mixed thoroughly. After standing for 30 min at room temperature in the dark, the OD was measured. 510nm .

[0131]

[0132] Among them, A s To measure the absorbance after adding ferrous sulfate solution and reacting with the sample, A b To measure absorbance using an equal volume of water instead of ferrous sulfate solution, A c The absorbance was measured by replacing the sample with an equal volume of water. The hydroxyl radical scavenging rate of *Lactobacillus rhamnosus* LRa66 was determined using the same method described above. Figure 10 As shown in (b), the antioxidant capacity of Lactobacillus brunelli PB6 supernatant is stronger than that of bacterial suspension, and the scavenging rate of hydroxyl radicals of PB6 bacterial suspension exceeds 30%, which is significantly higher than that of LRa66, exceeding 48.6%.

[0133] 3.3.3 Superoxide anion scavenging experiment

[0134] The concentration of the activated Lactobacillus bruneri PB6 bacterial culture was adjusted to OD. 600nm Within 1, the scavenging capacity of superoxide anions in bacterial suspension and supernatant was tested. 2 mL of 150 mmol / L Tris-HCl solution (pH 8.2) and 0.5 mL of the sample were added, preheated at 25°C for 10 min, then 0.5 mL of 1.2 mmol / L pyrogallol aqueous solution (preheated at 25°C) was added. After incubating in a water bath at the same temperature for 20 min, the mixture was centrifuged (3500 rpm, 10 min) and the OD was measured. 325nm .

[0135]

[0136] Among them, A j To measure the absorbance after adding pyrogallol aqueous solution and the sample to be tested to a warm water bath, A k A. To measure absorbance using PBS buffer instead of pyrogallol aqueous solution. m The absorbance was measured using PBS buffer instead of the sample to be tested. n The absorbance was measured using PBS buffer instead of pyrogallol aqueous solution and the test sample. The superoxide anion scavenging rate of *Lactobacillus rhamnosus* LRa66 was determined using the same method described above. Figure 10 As shown in (c), the PB6 bacterial suspension achieved a superoxide anion scavenging rate of 75.08%, which exceeded that of Lactobacillus rhamnosus LRa66 by 18.6%.

[0137] In summary, PB6 bacterial suspension outperforms LRa66 in DPPH radical scavenging, hydroxyl radical scavenging, and superoxide anion scavenging, exhibiting better antioxidant activity.

[0138] 3.4 Pathogen Inhibition Experiment

[0139] Staphylococcus aureus ATCC 12598, Escherichia coli ATCC 25922, and Salmonella typhimurium ATCC 14028 were used as indicator strains. The concentrations of the three strains were diluted to OD using sterilized LB medium. 600nm The value was 0.5 ± 0.01. LB medium with an agar content of 0.75% was prepared, sterilized, and cooled to approximately 40°C for later use. 160 μL of diluted bacterial suspension was added to 100 mL of the above LB medium, mixed well, poured onto a plate, and stored at 4°C. Using a 10 mm diameter punch, wells were made on the plates containing the three bacteria. 100 μL of *Lactobacillus brucellosis* PB6 fermentation supernatant was added to each well, and after thorough diffusion, the plates were incubated upside down at 37°C for 6–10 h. The size and diameter of the inhibition zone were observed and recorded. The results are shown in Table 7. The supernatant of PB6 showed a certain inhibitory effect on common pathogenic bacteria *Staphylococcus aureus*, *Escherichia coli*, and *Salmonella typhimurium*. The inhibitory effect of PB6 supernatant on *Escherichia coli* was slightly stronger than that on *Staphylococcus aureus* and *Salmonella typhimurium*.

[0140] Table 7. Size of inhibition zones of Lactobacillus bruneri PB6 against different indicator bacteria.

[0141]

[0142] The above LB agar medium, by weight, consists of 1 part sodium chloride, 0.5 parts yeast extract, 1 part bacteriological peptone, 0.75 parts agar, and distilled water to a final volume of 100 parts. The pH is 7.0 ± 0.2. The medium is then autoclaved at 121°C for 15 min.

[0143] Example 3 shows that PB6 can tolerate the gastrointestinal environment, has strong self-cohesion, hydrophobicity and antioxidant capacity, can inhibit the growth of common pathogenic bacteria, and has the potential to be a probiotic.

[0144] Example 4: Hypoglycemic Activity of Lactobacillus Brucella PB6

[0145] 4.1 Effects of PB6 on glucose metabolism-related indicators

[0146] HepG2 cells are a human hepatocellular carcinoma-derived cell model that retains many key biological characteristics of hepatocytes and are commonly used in in vitro studies related to glucose homeostasis. An insulin resistance (IR) model was established using 8 mM glucosamine induction. Measurements were taken from PB6 supernatant, bacterial cell lysate, and bacterial suspension (1×10⁻⁶ cells). 7 The effect of CFU / mL on glucose metabolism-related indicators.

[0147] Cells were divided into a normal treatment group (Normal) and an insulin resistance model group (IR-HepG2).

[0148] Normal cell treatment group: HepG2 cell suspension after desulfurization and counting, at 2.5 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates at a rate of 100 μL per well and cultured for 12 h until the cells adhered. After washing with PBS, the cells were starved for 12 h in DMEM medium without serum and antibiotics. After aspirating the medium, the cells were washed with PBS and cultured in DMEM medium for 24 h. Then, normal cells were treated with 1 mmol / L metformin (Met), different concentrations of fermentation supernatant and cell lysate, and PB6 cells for 24 h.

[0149] Insulin resistance model group: HepG2 cell suspension after detoxification and counting, at 2.5 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates at a rate of 100 μL per well and cultured for 12 h until the cells adhered. After washing with PBS, the cells were starved in DMEM medium without serum and antibiotics for 12 h. After aspirating the medium, the cells were washed with PBS and induced with 8 mM glucosamine for 24 h. Then, the cells were treated with 1 mmol / L metformin, different concentrations of fermentation supernatant and cell lysate, and PB6 cells for 24 h.

[0150] The preparation methods of fermentation supernatant and cell lysate of different concentrations are as follows: Fermentation supernatant and cell lysate are obtained according to the method described in Example 1, pre-frozen at -80℃ for 3-4 h, freeze-dried in a vacuum freeze dryer for 48 h, and then prepared into solutions of different concentrations with DMEM culture medium and filtered through a 0.22 μm filter membrane.

[0151] 4.1.1 Effects of PB6 on glucose consumption and glycogen synthesis

[0152] After sample processing, the glucose content in the culture medium was determined using a glucose content assay kit. The results are as follows: Figure 11As shown, the intervention of the samples had varying degrees of effect on glucose consumption in both normal cells and IR-HepG2 cells. Compared with the normal control group (Con) cells, the glucose consumption of IR group cells was reduced. After intervention with PB6 fermentation supernatant, the glucose consumption of IR-HepG2 cells was significantly restored, even higher than that of normal cells treated with the same concentration. The PB6 fermentation supernatant at 100 μg / mL had the best effect on IR-HepG2 cells, with a glucose consumption of 17.22 mmol / L, which was 25.97% higher than that of the IR group. Compared with metformin, it was more effective in promoting glucose consumption in IR-HepG2 cells. Bacterial cell lysate significantly increased glucose consumption in normal cells, but the effect of 20–60 μg / mL lysate on restoring glucose consumption in IR-HepG2 cells was less pronounced than that of the supernatant. 80 μg / mL PB6 lysate showed the best effect in treating IR-HepG2 cells, achieving a glucose consumption of 17.53 mmol / L, an increase of 28.14% compared to the IR group. In contrast, PB6 treatment of IR-HepG2 cells resulted in a glucose consumption of 16.57 mmol / L, an increase of 8.87%, indicating that the glucose consumption capacity of HepG2 cells decreased after glucosamine induction, while PB6 treatment improved the glucose consumption capacity of the IR group cells.

[0153] Glycogen synthesis and breakdown are pathways of hepatic glucose metabolism. Insulin resistance leads to decreased insulin sensitivity, impaired hepatic glycogen synthesis, and increased glucose output. After sample processing, the cellular glycogen content was measured using a glycogen assay kit. The results are as follows: Figure 12 As shown, compared with the normal group, the glycogen content of cells in the IR group decreased by 25.31%. Supernatant, bacterial cell lysate, and bacterial suspension all significantly increased the glycogen content of IR-HepG2 cells. Among them, the effects of supernatant and bacterial suspension were more significant than those of the Met group, with glycogen contents of 29.24 mg / mgprot and 28.66 mg / mgprot, respectively, representing increases of 178.48% and 173.17% compared to the IR group, and increases of 23.82% and 21.46% compared to the Met group. This indicates that PB6 helps increase the glucose consumption capacity of hepatocytes.

[0154] 4.1.2 Effects of PB6 on key enzymes of glucose metabolism

[0155] Glycolysis is the core pathway of glucose catabolism, and pyruvate kinase (PK) participates in coordinating the rate-limiting step. After sample processing, cellular PK enzyme activity was measured using a PK kit, and the results are as follows: Figure 13As shown in (a), the PK activity in the IR group was 83.26% lower than that in the normal group. Compared with the IR group, all three sample treatments significantly increased PK enzyme activity. Among them, the bacterial suspension had the most significant effect, with PK enzyme activity reaching 54.03 U / gprot, which was 45.71 U / gprot higher than that in the IR group. This indicates that PB6 can promote glycolysis and thus enhance glucose metabolism by increasing PK enzyme activity, but the effect is not as good as that in the Met group.

[0156] The liver is the primary organ for gluconeogenesis, and phosphoenolpyruvate carboxykinase (PEPCK) is one of the key enzymes regulating gluconeogenesis. It converts oxaloacetate to phosphoenolpyruvate, which is the first step in gluconeogenesis. Therefore, controlling PEPCK enzyme activity can effectively regulate gluconeogenesis. After sample processing, cellular PEPCK enzyme activity was measured using a PEPCK kit. The results are as follows: Figure 13 As shown in (b), the PEPCK activity in the IR group was 51.47% higher than that in the normal group. Compared with the IR group, all three sample treatments significantly reduced PEPCK enzyme activity, with the supernatant showing a more significant effect than the Met group, reducing PEPCK enzyme activity to 34.81 U / 104 cells, a decrease of 26.33% compared to the Met group. This indicates that PB6 can inhibit gluconeogenesis by inhibiting PEPCK enzyme activity.

[0157] 4.1.3 Effects of PB6 on the expression of genes related to glucose metabolism

[0158] The PI3K / AKT-dependent signaling pathway of insulin action is a major pathway in hepatic glucose metabolism, playing a crucial role in regulating the expression of genes related to hepatic gluconeogenesis, glycolysis, and lipid synthesis. Adenosine monophosphate-activated protein kinase (AMPK) plays a key role in glucose homeostasis regulation and can affect the expression of the PI3K / AKT signaling pathway. IRS-1, the main substrate protein produced after insulin receptor phosphorylation, binds to and activates PI3K. PI3K activation is a critical step in glucose uptake and insulin-induced glucose transport. PI3K activates the downstream protein AKT, and activated AKT phosphorylates GSK3β, thereby inhibiting GSK3β, enhancing GYS2, and increasing glycogen synthesis. Activated AKT also phosphorylates forkhead protein (FOXO1), thereby promoting FOXO1 export to the extranuclear region. Since FOXO1 retention in the nucleus promotes the expression of gluconeogenesis genes, the removal of FOXO1 inhibits the expression of gluconeogenic enzymes such as PEPCK, reducing gluconeogenesis.

[0159] RNA was extracted from HepG2 cells using an RNA extraction kit, and the RNA concentration was measured using a Nanodrop 2000c microspectrophotometer. cDNA amplification templates were prepared using a reverse transcription kit, mixed with primers, and analyzed using real-time quantitative PCR. Gene-specific primer designs are shown in Table 8. The relative expression levels of target genes were determined according to a 2:1 ratio. -(ΔΔCt) Perform calculations and representation. The results are as follows: Figure 14 Intervention with Met and PB6 supernatant, bacterial cell lysate, and bacterial suspension resulted in varying degrees of activation of cytokines related to glucose metabolism in IR-HepG2 cells. Compared with the Con group, the IR group showed varying degrees of decrease in AMPK, PI3K, IRS-1, AKT, and GYS2, while significantly increasing GSK3β, FOXO1, and PEPCK. After intervention with PB6 supernatant, bacterial cell lysate, and bacterial suspension, the expression levels of AMPK, PI3K, IRS1, AKT, and GYS2 increased, while GSK3β, FOXO1, and PEPCK decreased. Among these, the PB6 bacterial suspension had a more significant restorative effect on AMPK and IRS-1, and the supernatant had a more significant effect on GYS2 than Met. Compared with the IR group, the bacterial suspension upregulated AMPK expression by 397%, IRS1 expression by 177%, and the supernatant upregulated GYS2 expression by 236%. The results showed that PB6 alleviates insulin resistance by regulating the glucose metabolism pathway AMPK / PI3K / AKT, thereby promoting glycogen synthesis and inhibiting gluconeogenesis.

[0160] Table 8 Primer sequences for glucose metabolism-related genes

[0161]

[0162] 4.2 Effects of PB6 on gut-pancreatic axis related parameters

[0163] Studies have shown that lactic acid bacteria can stimulate intestinal L cells to secrete glucagon-like peptide-1 (GLP-1). GLP-1 can compete with glucagon for binding sites on its associated receptors, inhibiting the rise in blood glucose concentration. GLP-1 can also improve insulin sensitivity and inhibit pancreatic β-cell apoptosis. STC-1 cells are a mouse enterocrine tumor cell line with many characteristics of natural enterocrine cells and are often used to screen substances that regulate gastrointestinal hormone secretion in vitro. In this example, STC-1 cells were used to determine the effect of PB6 on intestinal-pancreatic axis related indicators.

[0164] 4.2.1 Effects of PB6 on GLP-1 secretion

[0165] For specific procedures, refer to Example 1. The PB6 supernatant (150 μg / mL) and cell lysate (150 μg / mL) were also treated using the same method. Experimental results are as follows: Figure 15 As shown, all three samples could increase GLP-1 secretion to varying degrees. The bacterial suspension treatment was more effective than the positive control acarbose (0.25 mg / mL), reaching 24.04 pmol / L, which was 23.28% higher than the acarbose group, proving that PB6 can promote intestinal GLP-1 secretion.

[0166] 4.2.2 Effects of PB6 on the expression of gut-pancreatic axis-related genes

[0167] The PCSK1 gene is a rate-limiting gene controlling GLP-1 secretion. It cleaves the expression product of the pro-glucagon gene to obtain the corresponding GLP-1 fragment. The method for determining the relative expression level of the gene is described in 4.1.3, and the primer sequences for the relevant genes are shown in Table 9. Experimental results are as follows... Figure 16 Compared with the blank control group, PB6 bacterial suspension significantly increased the expression levels of pro-glucagon and PCSK1 genes in STC-1 cells. The increase in pro-glucagon expression was more significant than that in the positive control, with the relative expression level of pro-glucagon increasing by 206% compared to the acarbose group. However, PCSK1 gene was not detected after treatment with supernatant and bacterial cell lysate. The results indicate that PB6 bacteria can promote the expression of proglucagon gene and PCSK1 gene, which cleaves this gene, thereby promoting GLP-1 secretion.

[0168] Table 9 Primer sequences for gut-pancreatic axis-related genes

[0169]

[0170] Example 4 explored the hypoglycemic activity of PB6 using a dual model. An insulin resistance model was established to determine the effect of PB6 on hepatic glucose metabolism. PB6 could increase glucose consumption, glycogen content, and PK enzyme activity in IR-HepG2 cells, inhibit PEPCK enzyme activity, and regulate the expression of AMPK / PI3K / AKT-related genes in the glucose metabolism pathway. The effect of PB6 on gut-pancreatic axis hormone secretion was determined using STC-1 cells. PB6 cells could promote GLP-1 secretion and the relative expression of gut-pancreatic axis genes.

[0171] Example 5: Application of Lactobacillus brucellosis PB6 in fermented milk

[0172] Preparation of cryoprotectant: 10 parts by weight of trehalose and 10 parts by weight of skim milk powder were diluted to 100 parts by distilled water and sterilized at 121°C for 15 min to obtain cryoprotectant solution.

[0173] Fermentation agent preparation: A suitable amount of PB6 bacterial suspension, which had undergone secondary activation in optimized culture medium (4℃, 8000 rpm, 15 min), was centrifuged to obtain bacterial sludge precipitate. The protectant and bacterial sludge were mixed at a ratio of 1:1 (by weight), and pre-frozen at -80℃ for 3-4 h. The pre-frozen bacterial sludge precipitate was then freeze-dried in a vacuum freeze dryer for 48 h. The freeze-dried bacterial powder was counted according to national standards. The results showed that the viable count of *Lactobacillus brucellosis* PB6 bacterial powder was 1.5 × 10⁻⁶. 11 CFU / g, stored at -20℃ for later use. The traditional starter culture (SL) consists of lyophilized Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2 powders, freeze-dried and then compounded at a 1:1 mass ratio, with a viable count of 10 for each. 11 CFU / g. The control group (CON) used Colson fermentation primordial, purchased from Guangzhou Yukaishen Biotechnology Co., Ltd., with a viable count of 1.5 × 10⁻⁶. 11 CFU / g, store in a -20℃ refrigerator for later use.

[0174] Preparation of fermented milk: By weight, 2.5 parts erythritol, 1 part xylitol, and 0.015 parts steviol glycosides are added and the volume is adjusted to 100 parts with whole milk. The mixture is heated at 90℃ for 5 min, cooled to room temperature, and then the above-mentioned starter culture is added. Fermentation is carried out at 43℃ for 6-8 h, and then placed in a refrigerator at 4℃ for 12-24 h for post-fermentation.

[0175] 5.1 Determination of the optimal compound ratio of fermentation inoculum

[0176] Lactobacillus brucellosis PB6 was mixed with traditional starter culture at mass ratios of 1:4, 1:2, 1:1, 3:2, and 2:1. The starter culture was inoculated at a rate of 0.0025–0.005 parts of bacterial powder per 100 parts of the above-mentioned milk. Fermentation was carried out at 43°C for 6–8 h, followed by ripening at 4°C for 12 h. The quality of the fermented milk was analyzed, and pH, titratable acidity, water-holding capacity, and sensory properties were measured to determine the optimal blend ratio (LB).

[0177] The pH measurement method is as follows: Take an appropriate amount of fermented milk sample into an Erlenmeyer flask and measure the pH using a calibrated pH meter.

[0178] The titration acidity determination method is as follows: Accurately weigh approximately 10.0 g of fermented milk sample, record the sample mass m, add 20 mL of boiled and cooled distilled water, add 1-2 drops of phenolphthalein indicator (10 g / L), mix well, and titrate with NaOH standard solution (0.1 mol / L) until the solution turns pink and does not fade within 30 s. Record the volume V of NaOH standard solution consumed at this time. The calculation formula is as follows:

[0179]

[0180] The method for determining water-holding capacity is as follows: Take an appropriate amount of fermented milk sample, denoted as m1, centrifuge (4000 rpm, 10 min), pour off the separated whey, weigh and record the mass of the remaining precipitate m2, and calculate using the following formula:

[0181]

[0182] The sensory evaluation method is as follows: The sensory evaluation team consists of 20 members, including undergraduate students, graduate students, and teachers of different ages and genders who have received training. The fermented milk is evaluated from three aspects: color, taste and aroma, and texture. The maximum score is 100 points. The specific sensory evaluation indicators are based on RHB 104-2020 "Sensory Evaluation Guidelines for Fermented Milk" with slight modifications, as shown in Table 10.

[0183] Table 10 Sensory Rating Table

[0184]

[0185] Table 11 Effects of different compound ratios on the basic physicochemical properties and sensory properties of fermented milk

[0186]

[0187] Note: The compound ratio in the table is the ratio of Lactobacillus brucellosis PB6:(Streptococcus thermophilus DMST-H2:Lactobacillus delbrueckii DMLD-H1=1:1).

[0188] Table 11 shows that a high proportion of *Lactobacillus brucellosis* results in a lower sensory score, insufficient flavor, poor coagulation state, and lower water-holding capacity, which is detrimental to the storage stability of the fermented milk. Conversely, a low proportion of *Lactobacillus brucellosis* results in a flavor that meets requirements, but with insufficient acidity. When the blending ratio is 1:1, the fermented milk exhibits good coagulation state, moderate acidity, and a maximum water-holding capacity of 80.04 ± 0.06%, achieving the highest overall sensory score. Sensory evaluations of fermented milk with different blending ratios are as follows: Figure 17 Sensory evaluation revealed that the fermented milk was in excellent condition in terms of color, aroma, taste, and texture, and was relatively balanced. A 1:1 ratio of bacterial strains was selected as the optimal ratio.

[0189] 5.2 Determination of the hypoglycemic activity of fermented milk

[0190] The fermented milk was divided into three groups: the optimal compound group (LB), the *Streptococcus thermophilus* DMST-H2: *Lactobacillus delbrueckii* DMLD-H1 = 1:1 group (SL), and the commercial strain group (CON). The inhibition rates of α-glucosidase and α-amylase were determined using the supernatant of the fermented milk, following the method described in Example 1. The results of the hypoglycemic activity of the fermented milk are shown in Table 12. It can be seen that the inhibition rates of α-glucosidase and α-amylase in the LB group reached 55.98% and 88.98%, respectively. Compared with the SL and CON groups, the inhibition rates of α-glucosidase in the LB group increased by 51.01% and 141.61%, respectively, and the inhibition rates of α-amylase in the LB group increased by 191.45% and 283.86%, respectively. This indicates that the fermented milk with added *Lactobacillus delbrueckii* PB6 can better inhibit the activities of α-glucosidase and α-amylase, exhibiting outstanding hypoglycemic activity.

[0191] Table 12. Hypoglycemic Activity of Fermented Milk

[0192]

[0193] The method for preparing the supernatant of the fermented milk is as follows: Stir the yogurt evenly, centrifuge (8000 rpm, 4℃, 10 min), take the supernatant, and filter it through a 0.22 μm filter membrane.

[0194] 5.3 Determination of Rheological Properties of Fermented Milk

[0195] The rheological properties of the samples were measured using an Anton Paar MCR 702e high-end microrheometer (Austria) with a CP25 rotor (1 mm gap) installed at 25 ± 1 °C. 2 mL of the stirred fermented milk sample was pipetted onto the rheometer plate, and the shear rate range was 0.1–100 s⁻¹. -1 The apparent viscosity of the samples was used as an indicator, and data were collected using the logarithmic sampling method. A PP25 rotor was used, with a gap of 1 mm. The strain amplitude in the linear viscoelastic region was selected as a fixed amplitude of 1%. The scanning frequency was 0.1~100 rad / s, and the viscoelastic properties of different fermented milks were determined using storage modulus and loss modulus as indicators.

[0196] The viscosity curves of the three groups of fermented milk are as follows: Figure 18 As shown, in the range of 0.1~100 s -1Within a certain shear rate range, the viscosity of all three groups of fermented milk samples showed a trend of first increasing and then decreasing with increasing shear rate, indicating that the fermented milk is a shear-thinned pseudoplastic liquid. Among them, the CON group had the highest viscosity, the SL group had the lowest viscosity, and the LB group was in between. This suggests that adding PB6 to the traditional starter culture will make the viscosity curve closer to that of commercial strains, which helps to improve the rheological properties of the fermented milk.

[0197] The viscoelastic dynamic time change curves of the three groups of fermented milk are as follows: Figure 19 As shown, within the frequency scanning range of 0.1–10 Hz, the storage modulus (a) and loss modulus (b) of the three groups of fermented milk all showed an increasing trend with increasing frequency. Within the range of 0.1–10 Hz, the storage modulus (G') of the three groups of fermented milk was higher than the loss modulus (G"). G' represents the elastic component of the sample, and G" represents the viscous component, indicating that elastic properties dominate and a relatively stable three-dimensional gel network structure is formed inside the fermented milk. In comparison, the CON group had the highest G' and G" values, the SL group had the lowest G' and G" values, and the LB group was in between, indicating that the addition of PB6 helps to improve the rheological properties of the fermented milk.

[0198] 5.4 Determination of Organic Acid Content in Fermented Milk

[0199] Organic acids in fermented milk supernatant were determined by high performance liquid chromatography (HPLC). The chromatographic column was an FMH-1138-KONU 59952FM, 300 × 7.8 mm (Guangzhou Philomens Co., Ltd.). The column temperature was 60℃, the detection wavelength was 210 nm, and the injection volume was 10 μL. A 0.005 mol / L H₂SO₄ solution was prepared as the mobile phase, filtered through a 0.22 μm filter membrane, and degassed by ultrasonication.

[0200] Dilute the standards (oxalic acid, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, formic acid, acetic acid, propionic acid, and isobutyric acid) with ultrapure water to a final volume of 100 mL to prepare a mixed standard stock solution, and store at 4°C for later use. Serially dilute the stock solution with ultrapure water to prepare mixed standard working solutions of 0.15, 0.3, 0.5, 1, 1.5, and 2 mg / mL, and prepare single standard solutions of specific concentrations. Filter these solutions through a 0.22 μm aqueous filter to remove impurities before analysis. Dilute the fermented milk supernatant with ultrapure water by a certain factor, filter it through a 0.22 μm aqueous filter to remove impurities, and then analyze it.

[0201] The organic acid content of the three groups of fermented milk is as follows: Figure 20As shown, the lactic acid content of fermented milk in the LB group was 30.26 mg / mL, which was significantly higher than that in the SL and CON groups. This indicates that Lactobacillus brucellosis PB6 can efficiently convert lactose into lactic acid through glycolysis, which can quickly lower the pH, shorten the fermentation time, and produce good curdling effect, thus laying the foundation for the sour taste of yogurt. Meanwhile, PB6 fermentation produces a variety of organic acids such as citric acid, tartaric acid, lactic acid, and acetic acid, giving the fermented milk a richer fermented flavor. The contents of citric acid, tartaric acid, lactic acid, and acetic acid in the fermented milk of the LB group were 6.54, 8.68, 30.26, and 10.96 mg / mL, respectively, all significantly higher than those in the SL and CON groups. Citric acid increased by 6.04 and 5.36 mg / mL, respectively; tartaric acid increased by 4.35 and 3.82 mg / mL, respectively; lactic acid increased by 29.84 and 27.14 mg / mL, respectively; and acetic acid increased by 9.67 and 8.91 mg / mL, respectively. Acetic acid accounted for a large proportion of the total content. Acetic acid can effectively inhibit yeast, mold, and some putrefactive bacteria, significantly extending the shelf life of yogurt. In addition, acetic acid is also an important short-chain fatty acid in the intestine, which is beneficial to human health.

[0202] 5.5 Determination of total free amino acids in fermented milk

[0203] The total free amino acid content in fermented milk supernatant was determined using the ninhydrin method. 0.8 g of ninhydrin was added to 80 mL of anhydrous ethanol and 10 mL of glacial acetic acid. 1.0 g of chromium oxide was weighed and added to 1 mL of distilled water. After dissolving by shaking, the two reagents were mixed together to obtain the cadmium-ninhydrin reagent. 1 mL of leucine solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L were added to 2 mL of the cadmium-ninhydrin reagent. After mixing, the mixture was incubated in a water bath at 84℃ for 5 min, cooled to room temperature, and the OD was measured. 570nm Plot a standard curve. After appropriately diluting the supernatant of the fermented milk, take 1 mL and mix it with 2 mL of cadmium-indene tricopper reagent. Incubate at 84 ℃ for 5 min, and measure the OD after cooling to room temperature. 570nm The amino acid content was calculated using a standard curve. The results are shown in Table 13. The free amino acid concentration in the LB group was 4.16 mmol / L, while the free amino acid concentrations in the SL and CON groups were relatively close, around 2.3 mmol / L. The free amino acid concentration in the LB group was significantly higher than that in the SL and CON groups, indicating that the addition of PB6 promotes the enzymatic hydrolysis of proteins in milk, breaking down large protein molecules into smaller amino acids and peptides, increasing the free amino acid content, making it easier for the body to absorb, and improving the biological value of fermented milk.

[0204] Table 13 Total Free Amino Acids in Fermented Milk

[0205]

[0206] 5.6 Determination of volatile flavor compounds in fermented milk

[0207] Volatile flavor compounds in fermented milk were determined using solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS). 5–10 g of the fermented milk sample was placed in a headspace extraction vial. The extraction head was inserted into the headspace vial, and the SPM fiber was advanced. The extraction temperature was 55 °C, equilibration was maintained for 10 min, and the extraction time was 50 min with magnetic stirring. After extraction, the extraction head was inserted into the GC inlet, and the extraction head was advanced. Desorption was performed at 250 °C for 3 min, followed by detection using a GC-MS system. GC conditions were as follows: inlet liner diameter 0.75 mm, helium as carrier gas at a flow rate of 1.2 mL / min; inlet temperature 240 °C, split ratio 5:1; initial column oven temperature 40 °C for 3 min, increased to 100 °C at a rate of 8 °C / min, then to 150 °C at a rate of 5 °C / min, and finally to 240 °C at a rate of 15 °C / min and held for 2.5 min. MS conditions were as follows: electron ionization mode (230 °C, 70 eV), ion scan range of 28–500 amu.

[0208] The results of the detection of flavor substances in the three groups of fermented milk are shown in Tables 14 and 15. In terms of types and composition, a total of 34 volatile flavor substances were detected in the LB group, which is the most abundant. This indicates that Lactobacillus brucellosis PB6 has strong metabolic diversity and can generate a more complex flavor spectrum.

[0209] The relative contents of aldehydes, esters, and alcohols in the three groups of fermented milk were all low. The LB group of fermented milk contained 0.30% aldehydes, 0.33% esters, and 0.83% alcohols. The SL group of fermented milk contained 0.32% aldehydes, and no esters or alcohols were detected. The Control group of fermented milk did not contain any aldehydes, esters, or alcohols, possibly because their chemical properties are unstable and they are converted into alcohols or acids during the post-fermentation process.

[0210] Ketones are key aromatic compounds in fermented milk, primarily contributing fruity and creamy aromas. Four ketones were detected across the three groups of fermented milk, with 2-heptanone and 2-nonanone being the most prominent. Additionally, 2-undecaneone was detected in the LB and SL groups; this substance possesses a fatty, creamy, and cheese-like aroma, contributing to the complexity of the flavor profile. Overall, there was little difference in the types and amounts of ketones among the three groups of fermented milk.

[0211] Acids are the main flavor components of fermented milk, playing a decisive role in the overall flavor profile. A total of eight acids were detected in the three groups of fermented milk. The relative content of acids was 21.61% in the LB group, 26.78% in the SL group, and 13.02% in the Control group. Acetic acid and heptanoic acid were only detected in the LB group. These two acids not only enhanced the sourness and flavor profile of the LB group's fermented milk, but acetic acid also has antibacterial activity and intestinal health benefits, further enhancing the product's functionality and unique flavor. Butyric acid, hexanoic acid, octanoic acid, and decanoic acid were detected in all three groups, while nonanoic acid was not detected in the Control group.

[0212] In summary, the LB group performed best in terms of the variety, composition, and uniqueness of flavor substances, and its metabolite profile was richer. It could give fermented milk a richer fermented aroma, a more harmonious acidity level, and higher functional value, indicating that the addition of Lactobacillus brucellosis PB6 has significant advantages in improving the flavor quality of fermented milk.

[0213] Table 14 Volatile Flavor Compounds in Fermented Milk

[0214]

[0215] Table 15 Other volatile flavor compounds in fermented milk

[0216]

[0217] 5.7 Determination of antioxidant capacity of fermented milk

[0218] The antioxidant capacity of fermented milk was characterized by measuring the DPPH, hydroxyl radical, and superoxide radical scavenging rates of the supernatant. The procedure is described in section 3.3. The LB group of fermented milk exhibited strong antioxidant capacity, such as... Figure 21 As shown in (a), when the fermented milk was undiluted, the DPPH scavenging rate of the LB group, SL group and Control group were close to 100%. After the fermentation liquid was diluted to 25%, the DPPH scavenging rate of the LB group was 34.41%, which was 36.28% higher than that of the SL group and 5.75% higher than that of the CON group. Figure 21 As shown in (b), the LB, SL, and Control groups all exhibited strong hydroxyl radical scavenging capabilities, with scavenging rates exceeding 95% even at a 25% dilution, indicating that the fermented milk matrix itself possesses excellent hydroxyl radical scavenging potential. Figure 21 As shown in (c), when the fermented milk was undiluted, the superoxide anion radical scavenging rate of the LB group was 76.78%, which was 16.21% higher than that of the SL group and 36.57% higher than that of the CON group. The addition of PB6 endowed the fermented milk with superior antioxidant capacity.

[0219] 5.8 Determination of the antibacterial ability of fermented milk

[0220] The inhibitory activity of fermented milk supernatant against Gram-negative bacteria (Escherichia coli ATCC 25922, Salmonella Typhimurium A) and Gram-positive bacteria (Staphylococcus aureus ATCC 12598) was investigated as described in section 3.4. The results are shown in Table 16. Both the LB and SL groups showed some inhibitory activity against common foodborne pathogens. The inhibition zones of the LB group against all three bacteria were slightly larger than those of the SL group. Compared with the SL group, the diameters of the inhibition zones against Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium increased by 21.48%, 14.10%, and 4.04%, respectively. This indicates that fermented milk containing Lactobacillus brucellosis PB6 has antibacterial activity against both Gram-negative and Gram-positive bacteria, and also has antibacterial activity while serving as an auxiliary starter. The CON group, however, showed no antibacterial activity against any of these three bacteria.

[0221] Table 16 Antibacterial activity of fermented milk against two types of bacteria

[0222]

[0223] 5.9 Stability determination of fermented milk during storage

[0224] The pH, titratable acidity, water-holding capacity, and viable cell count of the fermented milk were measured at 0, 7, 14, 21, and 28 days to monitor its stability during storage.

[0225] The changes in pH and titratable acidity of the three groups of fermented milk during a 4-week storage period are as follows: Figure 22 As shown in (a) and (b), the results indicate that the pH of the three groups of fermented milk first decreased and then stabilized during the storage period. The pH of the LB group fermented milk gradually decreased from 4.23 on day 0 to 3.89 on day 14, and then slowly recovered and remained stable over the next two weeks. This suggests that the intracellular enzyme system in the LB group maintained high activity during the first two weeks of low-temperature storage, continuously producing acid. Subsequently, lactic acid and other organic acids were metabolized into other substances, and the pH slowly recovered, eventually reaching 4.02. The pH of the SL group fermented milk was slightly higher than that of the LB group. The pH of the CON group fermented milk was higher than the other two groups, with a final pH of 4.25. The titratable acidity of the LB group fermented milk gradually increased from 95.33 °T to 119.45 °T from day 0 to day 14, reaching a final acidity of 112.60 °T on day 28. The acidity of the fermented milk in group SL was slightly lower than that in group PB6, gradually increasing from 85.64 °T to 104.60 °T from 0 to 14 days, reaching a final acidity of 104.41 °T at 28 days. The acidity of the fermented milk in group Control was lower than the other two groups, with a final acidity of 88.29 °T at 28 days. This indicates that the pH and acidity of the fermented milk remained stable at a good level during storage, meeting the requirement of ≥60 °T for acidity in GB19302-2025 "National Food Safety Standard for Fermented Milk".

[0226] The changes in viable bacterial count and water-holding capacity of the three groups of fermented milk during a 4-week storage period are as follows: Figure 22 As shown in (c) and (d), the water-holding capacity of the three fermented milk groups remained essentially unchanged during the storage period, fluctuating around 80%. This indicates that the protein gel network structure formed by the three fermented milk groups was dense, uniform, and stable, effectively locking in water and preventing whey separation. The viable count of the LB group fermented milk was 2.47 × 10⁻⁶ after 12 hours following the fermentation endpoint. 9 The CFU / mL count meets the national standard requirements for live bacteria count in fermented milk. During the storage period, the live bacteria count remained relatively stable at 2.4 × 10⁻⁶ for the first three weeks. 9 Around CFU / mL, it decreased to 2.19×10⁻⁶ in the fourth week. 9 CFU / mL, meaning the number of live bacteria in fermented milk remains at a high level during storage, meeting the requirement of ≥1×10⁻⁶ lactic acid bacteria count in GB19302-2025 "National Food Safety Standard for Fermented Milk". 6 The required CFU / mL concentration was 1.75 × 10⁻⁶ after 12 hours following the reaching the fermentation endpoint in the SL group of fermented milk. 9 The CFU / mL level remained relatively stable for the first three weeks, then decreased to 1.4 × 10⁻⁶ in the fourth week. 9 The CFU / mL count was lower than that of the LB group. The viable count in the Control group was significantly higher than the previous two groups, with a final viable count of 8.4 × 10⁻⁶. 9 CFU / mL.

[0227] Example 5 demonstrates that fermented milk formulated with PB6 and traditional starter cultures exhibits significant hypoglycemic activity, and the addition of PB6 helps improve the rheological properties of the fermented milk. PB6 fermentation produces a variety of organic acids. The LB group showed the highest total amount of free amino acids. A total of 34 volatile flavor compounds were detected in the LB group, representing the richest variety, and it also exhibited strong antioxidant and antibacterial capabilities.

Claims

1. A type of Lactobacillus brucellosis PB6 with hypoglycemic activity, characterized in that, The strain is Lentilactobacillus buchneri PB6, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2025, with accession number GDMCC No: 66352.

2. The use of Lactobacillus brucellosis PB6 as a starter in fermented milk as described in claim 1, or the use of Lactobacillus brucellosis PB6 in the preparation of drugs for the prevention and treatment of diabetes.

3. The application according to claim 2, characterized in that, The starter culture is a mixture of Lactobacillus brucellosis PB6 and a traditional starter culture.

4. The application according to claim 3, characterized in that, The traditional starter culture is Streptococcus thermophilus DMST-H2 and Lactobacillus delbrueckii DMLD-H1.

5. The application according to claim 4, characterized in that, The mass ratio of Lactobacillus bruneri PB6 to the traditional starter culture is 1:4-3:2; the mass ratio of Streptococcus thermophilus DMST-H2 to Lactobacillus delbrueckii DMLD-H1 is 1:(0.5-2.0).

6. The application according to claim 2, 3, 4, or 5, characterized in that, The method for preparing the fermented milk is as follows: by weight, 2-3 parts of erythritol, 0.5-1.5 parts of xylitol, and 0.01-0.02 parts of steviol glycosides are added and the volume is adjusted to 100 parts with whole milk. The mixture is heated at 90-95℃ for 5±3 minutes, cooled, and then fermented with a starter culture to obtain sugar-free fermented milk.

7. The application according to claim 6, characterized in that, Fermentation conditions: ferment at 43±5℃ for 6~8 h, then place at 4℃ for 12~24 h of post-fermentation.

8. A compound fermentation agent, characterized in that, It includes Lactobacillus brucellosis PB6 as described in claim 1 and conventional starter cultures.

9. The compound fermentation agent according to claim 8, characterized in that, The traditional starter culture is Streptococcus thermophilus DMST-H2 and Lactobacillus delbrueckii DMLD-H1.

10. The compound fermentation agent according to claim 9, characterized in that, The mass ratio of Lactobacillus bruneri PB6 to the traditional starter culture is 1:4-3:2; the mass ratio of Streptococcus thermophilus DMST-H2 to Lactobacillus delbrueckii DMLD-H1 is 1:(0.5-2.0).

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