Bifidobacterium bifidum bglp1 capable of regulating sugar digestion and high yield of glp-1 and application thereof

Abstract

The present application relates to the technical field of microorganisms, and particularly relates to a bifidobacterium BGLP1 capable of regulating sugar digestion and high GLP-1 production and application thereof. Bifidobacterium bifidum The bifidobacterium BGLP1 (CGMCC No. 35718) has the abilities of inhibiting alpha-glucosidase and alpha-amylase, stimulating STC-1 cells to secrete GLP-1 and activating the intestinal glucose-lowering pathway, significantly inhibiting pancreatic lipase activity and effectively reducing dietary fat absorption, converting excess sucrose into extracellular polysaccharides to increase the probiotic function, promoting the conversion of white fat into brown fat, and activating macrophages and improving the immune function of the body. In addition, the bifidobacterium BGLP1 has strong intestinal colonization ability, has good tolerance in artificial gastric juice and artificial intestinal juice, and can successfully reach the human intestinal tract.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bifidobacterium bifidum BGLP1 that regulates sugar digestion and produces high levels of GLP-1, and its applications. Background Technology

[0002] The rising incidence of diabetes, obesity, and related metabolic syndromes has become a major global public health challenge. Current clinical treatments primarily rely on chemically synthesized drugs, such as the alpha-glucosidase inhibitor acarbose and the pancreatic lipase inhibitor orlistat, for blood glucose and lipid regulation. However, long-term use often results in gastrointestinal problems such as bloating, diarrhea, and diminishing efficacy. Furthermore, traditional drugs often target single metabolic pathways (e.g., inhibiting only glucose absorption or lipolysis), making it difficult to synergistically improve glucose and lipid metabolism disorders. In addition, insufficient secretion of glucagon-like peptide-1 (GLP-1) fails to repair intestinal endocrine function, limiting the effectiveness of comprehensive interventions.

[0003] In recent years, probiotics have been regarded as a potential alternative for the management of metabolic diseases due to their high safety and good host compatibility. However, most of the probiotic strains reported so far have limitations in terms of single function or insufficient efficacy. Although some strains show certain α-glucosidase inhibitory activity, their inhibitory ability against α-amylase or pancreatic lipase is weak, making it difficult to effectively block the simultaneous absorption of polysaccharides and fats. At the same time, few strains can significantly activate the secretion of GLP-1 by intestinal L cells, and GLP-1, as a core incretin, is crucial for blood glucose homeostasis and appetite regulation. In addition, multifunctional probiotics that have the triple effects of "lowering blood sugar, lowering blood lipids, and regulating intestinal endocrine function" and can convert dietary sugars into beneficial metabolites (such as extracellular polysaccharides) are still scarce.

[0004] Therefore, how to provide a probiotic with multiple synergistic metabolic regulatory functions is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum BGLP1, with accession number CGMCC No.35718, is deposited at the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 22, 2025.

[0006] The present invention also provides a composition comprising Bifidobacterium bifidum BGLP1 as described above.

[0007] Furthermore, the composition comprises one or more combinations of Bifidobacterium bifidum BGLP1 non-inactivated bacteria, Bifidobacterium bifidum BGLP1 metabolites, and lyophilized Bifidobacterium bifidum BGLP1.

[0008] The present invention also provides a bacterial agent whose components include Bifidobacterium bifidum BGLP1 as described above.

[0009] The present invention also provides a fermentation broth, which is obtained by fermentation of Bifidobacterium bifidum BGLP1 as described above.

[0010] The present invention also provides a probiotic product, the components of which include Bifidobacterium bifidum BGLP1 as described above.

[0011] The present invention also provides a freeze-dried product, the components of which include Bifidobacterium bifidum BGLP1 as described above.

[0012] This invention also provides the use of Bifidobacterium bifidum BGLP1 as described above in the preparation of any of the following functional products,

[0013] (1) Functional products used to inhibit α-glucosidase;

[0014] (2) Functional products used to inhibit α-amylase;

[0015] (3) Functional products used to inhibit lipase;

[0016] (4) Functional products used to stimulate STC-1 cells to secrete GLP-1 and activate the intestinal glucose-lowering pathway;

[0017] (5) Functional products used to convert sucrose into extracellular polysaccharides;

[0018] (6) Functional products used to promote the conversion of white fat into brown fat;

[0019] (7) Functional products used to activate macrophages and enhance the body's immune function.

[0020] The present invention also provides the application of Bifidobacterium bifidum BGLP1 as described above in the preparation of weight loss products.

[0021] Furthermore, the weight loss product includes one or more of the following: weight loss capsules, weight loss probiotic powder, and weight loss enzymes.

[0022] In summary, compared with the prior art, the Bifidobacterium bifidum BGLP1 provided by the present invention has the following beneficial effects:

[0023] The Bifidobacterium bifidum BGLP1 provided by this invention has strong acid and bile salt resistance, good tolerance in artificial gastric juice and artificial intestinal juice, and can successfully reach the human intestine.

[0024] The Bifidobacterium bifidum BGLP1 provided by this invention significantly improves glucose metabolism through three physiological pathways: ① highly efficient inhibition of α-glucosidase; ② moderate inhibition of α-amylase; ③ strong promotion of GLP-1 secretion. Through the synergistic effect of these three pathways, glucose homeostasis is reshaped from multiple dimensions of "substrate blockade - delayed digestion - intestinal hormone regulation".

[0025] In this invention, Bifidobacterium bifidum BGLP1 significantly inhibits pancreatic lipase activity, effectively reducing dietary fat absorption; its lipid-lowering mechanism is compatible with its hypoglycemic function, avoiding the risk of gastrointestinal adverse reactions when used in combination with chemical drugs; it simultaneously utilizes excess sucrose to synthesize extracellular polysaccharides, which not only reduces free sugar toxicity but also generates prebiotic substances, providing a protective carbon source for the intestinal flora, thus avoiding the "steatorrhea" and flora imbalance side effects caused by traditional lipid-lowering drugs from the source;

[0026] In this invention, Bifidobacterium bifidum BGLP1 integrates four functions: "lowering blood sugar, lowering blood lipids, promoting GLP-1, and producing extracellular polysaccharides." This breaks through the limitation of existing probiotics having only a single function, simplifies the development of compound preparations, and can be widely used in weight loss products. Attached Figure Description

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

[0028] Figure 1 The colony morphology of Bifidobacterium bifidum BGLP1 provided by this invention;

[0029] Figure 2 The microscopic morphology of Bifidobacterium bifidum BGLP1 provided by this invention;

[0030] Figure 3 The standard curve for GLP-1 content provided by this invention;

[0031] Figure 4 The extracellular polysaccharide standard curve provided for this invention;

[0032] Figure 5 Determination of extracellular polysaccharide production of Bifidobacterium bifidum BGLP1 in different culture media provided by the present invention;

[0033] Figure 6 The conversion rate of sucrose by Bifidobacterium bifidum BGLP1 provided by this invention;

[0034] Figure 7The results of the expression assay of various immune factors by Bifidobacterium bifidum BGLP1 provided by the present invention;

[0035] Figure 8 The hemolytic test results of Bifidobacterium bifidum BGLP1 provided by the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The present invention provides the following operation examples and embodiments:

[0038] This invention provides a Bifidobacterium bifidum ( Bifidobacterium bifidum BGLP1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35718, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 22, 2025.

[0039] Source of raw materials: The Bifidobacterium bifidum was isolated from the intestines of healthy infants in Xiamen, Fujian Province. After sequencing analysis and Blast sequence alignment, this strain was found to be highly homologous to Bifidobacterium bifidum, and was named Bifidobacterium bifidum BGLP1.

[0040] Colony morphology: In MRS+cysteine ​​solid medium, the colonies are white, round, and have smooth and neat edges.

[0041] Example 1: Isolation and identification of Bifidobacterium bifidum BGLP1

[0042] 1. Separation:

[0043] The plate coating method was used. 5 g of sample was placed in a sterile homogenizing bag, and 45 mL of 0.85% physiological saline was added. The mixture was then homogenized to obtain the sample dilution. After thorough mixing, 100 μL of sample was sequentially diluted 10-fold. A dilution factor of 10 was selected. -3 10 -4 10 -5 The sample dilution solution was pipetted into a 200 μL container and spread onto a solid medium containing MRS and cysteine. The container was then incubated anaerobically at 37°C for 48 h.

[0044] Different strains were initially selected based on their morphology and color. They were repeatedly isolated and purified using the streak plate method until all colonies on the MRS+cysteine ​​solid medium maintained a single morphology. Single colonies were then picked and cultured in MRS+cysteine ​​liquid medium at 37°C for 12 h. The fermentation broth of the strains was then mixed with 50% glycerol in equal proportions and stored in glycerol tubes, and preserved in a bacterial bank at -80°C.

[0045] The colony morphology of the isolated and purified Bifidobacterium bifidum BGLP1 was as follows: in MRS + cysteine ​​solid medium, the colonies were white, round, with smooth and regular edges, as shown in the figure. Figure 1 As shown.

[0046] The formula for MRS liquid medium with cysteine ​​is as follows: 10.0 g beef meal, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 0.5 g cysteine, 1.0 L deionized water, pH 6.5 (adding 2% agar makes it MRS solid medium).

[0047] 2. Strain identification:

[0048] The screened and purified strains were Gram-stained to check whether their morphology was uniform. Figure 2 As shown.

[0049] The bacterial genome was extracted according to the instructions of the bacterial DNA extraction kit, and 16S rDNA was amplified. The amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing, and the isolated strain was identified as Bifidobacterium bifidum. Bifidobacterium bifidum BGLP1. Its gene sequence is shown in SEQ ID NO.1.

[0050] The 16S rDNA gene sequence determination results are as follows:

[0051]

[0052] Example 2: Experimental determination of the intestinal colonization ability of the strain

[0053] 1. Hydrophobicity test

[0054] After activating the bacterial strain for 2-3 generations, the cells were washed 2-3 times with PBS (Phosphate Buffered Saline) and resuspended. The absorbance A0 at 600 nm was measured. 3 mL of the bacterial suspension was added to 1 mL of xylene, pre-cultured at room temperature for 10 min, then rapidly vortexed for 2 min, and allowed to stand at room temperature for 15 min. After the solution separated into layers, the absorbance A of the lower aqueous phase at 600 nm was measured. Each sample was tested in triplicate. The hydrophobicity of the bacterial cell surface was calculated according to equation (1).

[0055] Among them, the hydrophobicity of Bifidobacterium bifidum BGLP1 was (30.76±0.15)%, which is moderately hydrophobic, indicating that this strain has a strong ability to colonize the intestine.

[0056] Equation (1);

[0057] 2. Cell adhesion rate determination: the ability of the strain to adhere to HT-29 cells.

[0058] HT-29 cells were seeded into 6-well cell culture plates and cultured until a monolayer was formed. The cells were washed twice with sterile PBS, and 0.3 mL of 10⁻⁶ HCl was added. 5 CFU / mL Bifidobacterium bifidum BGLP1 was cultured at 37℃ for 2 h. After washing three times with sterile PBS to remove unattached strains, 150 μL of 0.25% trypsin digestion solution was added, and the cells were digested in a 37℃, 5% CO2 incubator for 3-5 min until the cells were completely detached. Then, 350 μL of MEM complete medium was added to stop the digestion. The bacterial suspension was collected, diluted, and spread. The viable number of Lactobacillus was determined by plate count.

[0059] The adhesion rate of Bifidobacterium bifidum BGLP1 to HT-29 cells was calculated to be (52.22±0.02)%.

[0060] Example 3: Determination of the ability of Bifidobacterium bifidum BGLP1 to inhibit α-glucosidase

[0061] After activating the lactic acid bacteria frozen in glycerol tubes for 2-3 generations, they were inoculated into MRS+cysteine ​​liquid medium at a 3% inoculum and cultured at 37℃ for 24 h. The fermentation broth was centrifuged at 6000 r / min and 4℃ for 10 min, and the supernatant was retained for later use.

[0062] 50 μL of α-glucosidase (0.5 U / mL), 50 μL of sodium dihydrogen phosphate buffer (0.2 M, pH 6.8) and 50 μL of Bifidobacterium bifidum BGLP1 fermentation supernatant were mixed and pre-incubated at 37℃ for 15 min. 100 μL of 4 mM PNPG substrate was added, and the mixture was incubated at 37℃ for 20 min. Then, 100 μL of Na2CO3 (0.1 M) was added to terminate the reaction. The absorbance was measured at 405 nm and recorded as sample A. The group without enzyme was used as the blank control group of the sample group, and ultrapure water was used instead of the sample and recorded as control A. The group without enzyme and the sample group were used as the blank control group. The inhibition rate of the sample against α-glucosidase was calculated according to formula (2).

[0063] The results showed that the inhibition rate of α-glucosidase by Bifidobacterium bifidum BGLP1 was (89.89±0.05)%.

[0064] Equation (2)

[0065] Example 4: Determination of the ability of Bifidobacterium bifidum BGLP1 to inhibit α-amylase

[0066] After activating the lactic acid bacteria frozen in glycerol tubes for 2-3 generations, they were inoculated into MRS+cysteine ​​liquid medium at a 3% inoculum and cultured at 37°C for 24 h. The fermentation broth was then centrifuged at 6000 r / min and 4°C for 10 min, and the supernatant was retained for later use.

[0067] 125 μL of sample supernatant was mixed with an equal volume of 1 mg / mL α-amylase solution and incubated at 37 °C for 10 min. The mixture was then added to 250 μL of 1.5% soluble starch solution at 37 °C and reacted at 37 °C for 15 min. Next, 500 μL of DNS solution was added, and the mixture was reacted in a boiling water bath for 5 min, then rapidly cooled to room temperature. After diluting 20-fold, the mixture was allowed to stand for 30 min, and the absorbance was measured at 540 nm. PBS solution (0.1 mol / L, pH=6.8) was used as a blank control for both the α-amylase solution and the test sample. Three replicates were set up for each group.

[0068] The results showed that the inhibition rate of α-amylase by the fermentation supernatant of Bifidobacterium bifidum BGLP1 was (32.50±0.23)%.

[0069] Equation (3)

[0070] In the formula: A is the sample group, containing the sample solution and α-amylase solution; B is the blank group, containing the sample solution but not the α-amylase solution; C is the control group, containing the α-amylase solution but not the sample solution; D is the blank group, containing neither the sample solution nor the α-amylase solution.

[0071] Example 5: Determination of the ability of Bifidobacterium bifidum BGLP1 to inhibit pancreatic lipase.

[0072] Mix 200 μL of Bifidobacterium bifidum BGLP1 fermentation broth and 200 μL of 2 U / mL pancreatic lipase solution thoroughly, incubate at 37°C for 15 min, then immediately add 400 μL of 10 mmol / L pNPP solution and mix well. Incubate again at 37°C for 15 min. After the reaction is complete, quickly place the mixture in a 100°C water bath for 5 min to terminate the reaction. Centrifuge the terminated reaction solution at 6000 r / min for 5 min, collect the supernatant, and measure the absorbance at 405 nm. The enzyme activity inhibition rate is calculated using the following formula.

[0073] The results showed that the inhibition rate of pancreatic lipase by the fermentation supernatant of Bifidobacterium bifidum BGLP1 was (47.04±0.18)%.

[0074] Equation (4)

[0075] In the formula: A s The absorbance of the sample group includes the sample solution and pancreatic lipase solution; A n The absorbance of the sample control group includes the sample solution but does not include pancreatic lipase solution; A c A1 represents the absorbance of the blank control group, which contains pancreatic lipase solution but no sample solution; A2 represents the absorbance of the blank control group, which contains neither sample solution nor pancreatic lipase solution.

[0076] Example 6: Assay of Bifidobacterium bifidum BGLP1 promoting GLP-1 secretion in enteroendocrine cells

[0077] Cell viability was assessed using the CCK-8 assay. Well-grown STC-1 cells were seeded in 96-well plates at a density of 10,000 cells / well and cultured at 37°C with 5% CO2 for 24 h. After cell attachment, cells were cultured in medium containing 2% serum. Separate solutions containing 0% (control group), 1%, 3%, 6%, and 9% of the test sample were added. After 24 h of intervention, the absorbance at 450 nm was measured using the CCK-8 assay. The cell viability results are shown in Table 1. The results were based on a 1% concentration.

[0078] Table 1. Results of STC-1 cell viability assay using fermentation broth supernatant at different concentrations.

[0079]

[0080] Assay for GLP-1 secretion by STC-1 cells: STC-1 cells were seeded into 96-well plates and cultured overnight. Cells were washed three times with PBS buffer (pH 7.4), and then co-incubated with 1% filtered sterile Bifidobacterium bifidum BGLP1 fermentation supernatant for 24 h. The cell supernatant was collected, and the secretion of GLP-1 was measured using an ELISA kit. The measured OD values ​​were substituted into the standard curve (as shown in Table 2), and the calculated secretion level of GLP-1 was 1.951 pmol / L. The standard curve for GLP-1 content is shown in Table 2. Figure 3 As shown.

[0081] Table 2. Results of GLP-1 secretion from fermentation supernatant of Bifidobacterium bifidum BGLP1

[0082]

[0083] Example 7: Study on the characteristics of Bifidobacterium bifidum BGLP1 in converting sucrose into extracellular polysaccharides (EPS).

[0084] 1. Activation: Inoculate the frozen bacterial strain onto MRS+cysteine ​​agar plates and anaerobic culture at 37°C for 24-48 hours.

[0085] 2. Pre-culture: Select a single colony and inoculate it into MRS liquid medium containing sucrose, and incubate at 37°C for 18 hours (seed culture). Inoculate each group of culture medium with the seed culture at a 2% inoculation rate, and incubate at 37°C for 24 hours.

[0086] 3. Heat the fermentation broth in a water bath at 90℃ for 15 min to inactivate bacterial endogenous enzymes. Add 80% trichloroacetic acid solution (to a final trichloroacetic acid concentration of 4 g / L), and let it stand at 4℃ for 12 h to precipitate proteins in the fermentation broth. Centrifuge at 8000 rpm for 10 min to remove proteins and bacterial cells from the fermentation broth. Add 3 times the volume of anhydrous ethanol to the supernatant after centrifugation, and let it stand at 4℃ for 12 h to precipitate extracellular polysaccharides in the fermentation broth. Centrifuge at 8000 rpm for 10 min and collect the extracellular polysaccharide precipitate. Dissolve the precipitate with a small amount of deionized water, transfer it to a dialysis bag (molecular weight cutoff 3500-4000 Da), and dialyze it in 4 L of deionized water, changing the water every 6 h for a total of 48 h. After dialysis, freeze-dry the dialysate for 24 h to obtain crude extracellular polysaccharides.

[0087] Glucose was used as a standard to plot a standard curve and calculate the linear regression equation. 50 mg of glucose was weighed into a 500 mL volumetric flask, and water was added to the mark. 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mL of each flask were pipetted in, and each was brought to 2.0 mL with distilled water. Then, 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid were added, and the mixture was shaken well and allowed to stand for 30 min. The absorbance was measured at 490 nm. A blank was prepared using 2.0 mL of water and the same colorimetric procedure. The standard curve was plotted with polysaccharide content on the x-axis and the measured absorbance values ​​on the y-axis, as shown below. Figure 4 As shown.

[0088] Dissolve the lyophilized crude polysaccharide sample in 1 mL of water, add 1.0 mL of distilled water and 1.0 mL of 6% phenol, then quickly add 5.0 mL of concentrated sulfuric acid, mix thoroughly, let stand for 30 min, and measure the absorbance at 490 nm. Substitute the measured absorbance value into the standard curve to obtain the concentration of extracellular polysaccharides in the sample.

[0089] The measured standard curve is: y = 0.0029x + 0.0182, R0 2 =0.9938.

[0090] Test the extracellular polysaccharide yield in each culture medium, such as Figure 5 As shown, the production of extracellular polysaccharides in Bifidobacterium bifidum BGLP1 increases with the increase of sucrose content, indicating that this strain can convert excess ingested sucrose into probiotics—extracellular polysaccharides.

[0091] The culture medium used in Example 7 is as follows:

[0092] Complete culture medium: MRS broth medium;

[0093] Basic culture medium: MRS broth medium with glucose removed;

[0094] Experimental culture medium: basal medium + 2%, 4%, 6% sucrose;

[0095] Control medium: MRS medium + 2% sucrose.

[0096] Example 8: Determination of sucrose conversion ability of Bifidobacterium bifidum BGLP1

[0097] Fermentation broth was inoculated at a ratio of 2% into experimental media containing 2%, 4%, 6%, and 8% sucrose, respectively, and cultured anaerobically for 48 hours. Uninoculated blank media served as a blank control. The sucrose content in each culture medium was tested according to Method II of the national standard GB / T5009.8-2008. Samples were treated with acid hydrolysis, and the concentration was determined by alkaline tartaric acid solution standardization, sample solution concentration prediction, and sample solution measurement. The results showed that after 48 hours of culture with 2% sucrose, the sucrose conversion rate reached 81.19%. (The results are as follows...) Figure 6 As shown.

[0098] The basal medium was MRS broth medium with glucose removed; the experimental media were basal medium supplemented with 2%, 4%, 6%, and 8% sucrose, respectively.

[0099] Example 9: Assay of the ability to overexpress interleukin-12 (IL-12) and interleukin-27 (IL-27)

[0100] Group setup: Positive control: LPS (induces IL-12, induces IL-27); Negative control: non-irritating culture medium; Sample group: Probiotic group: incubated for 24 h.

[0101] CCK8 cell proliferation: Macrophages in good growth condition were diluted and transferred into 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, cells were cultured in medium containing 2% serum, and then 0% (control group) and 10% (control group) serum were added respectively. -3 10 -4 The test samples were cultured in 2% serum. After 24 h of intervention, the absorbance at 450 nm was measured using the CCK-8 assay to calculate cell viability. The results are shown in Table 3. 10-1 of the test results were selected. -3 At certain concentrations, it can promote the proliferation of macrophages.

[0102] IL-12 / IL-27 secretion and mRNA expression levels in macrophages treated with probiotics: Macrophages in good growth condition were diluted and transferred into 6-well plates and cultured at 37°C with 5% CO2 for 24 h. After cell adhesion, 0 (control group), LPS, and 10% probiotics were added respectively. -3 After 24 h of intervention with 2% serum culture medium, the RNA of the cells was extracted, reverse transcribed, and qPCR was performed to test the relative expression levels of IL-12 and IL-27 mRNA. The test results are shown in Table 4. The results show that Bifidobacterium bifidum BGLP1 can upregulate the expression of IL-12 gene.

[0103] Table 3: Results of macrophage survival rate determination using fermentation broth supernatant at different concentrations

[0104]

[0105] Table 4: Results of determination of relative expression levels of IL-12 and IL-27 mRNA in the supernatant of Bifidobacterium bifidum BGLP1 fermentation broth

[0106]

[0107] Example 10: Study on the immune-enhancing properties of Bifidobacterium bifidum

[0108] 1. Macrophage phagocytic activity

[0109] Macrophages were seeded into 96-well plates and cultured overnight. The fermentation supernatant from the sonicated disruption of Bifidobacterium bifidum BGLP1 was added, and the plates were cultured for another 24 h at 37°C in a 5% CO2 incubator. The supernatant was discarded, and 200 μL of 0.075% neutral red solution was added to each well. The plates were cultured for another 2 h. The staining solution was discarded, and the plates were washed three times with sterile PBS buffer to remove any unphagocytosed neutral red staining. 200 μL of cell lysis buffer (glacial acetic acid: anhydrous ethanol = 1:1, v / v) was added to each well, and the plates were cultured for another 2 h at 37°C. After thorough lysis and mixing, the plates were placed in a microplate reader to measure the optical density at 540 nm.

[0110] The results showed that Bifidobacterium bifidum BGLP1 could increase the phagocytic capacity of macrophages by 33%.

[0111] 2. Macrophages release NO levels

[0112] Macrophages were seeded into 96-well plates and cultured overnight. The fermentation supernatant from *Bifidobacterium bifidum* BGLP1, after ultrasonic disruption, was added, and the plates were cultured for another 24 h at 37°C with 5% CO2. Then, 75 μL of the supernatant was transferred to a new 96-well plate, Griess solution was added, the mixture was stirred, and the plates were allowed to stand. The absorbance was then measured, as shown in Table 5. The results indicate that *Bifidobacterium bifidum* BGLP1 can increase the amount of NO released by macrophages, and compared to LPS production, the increase is more moderate. This approach not only promotes immunity and has anti-inflammatory effects but also avoids the oxidative damage to tissues and DNA caused by high NO levels, as well as the cartilage-damaging side effects (arthritis) in chronic inflammation.

[0113] Table 5: Results of NO release from macrophages by Bifidobacterium bifidum BGLP1

[0114]

[0115] 3. Measurement of macrophage immune factor gene expression levels

[0116] Macrophages were seeded into 6-well plates and cultured overnight. The fermentation supernatant from *Bifidobacterium bifidum* BGLP1 was added, and the plates were cultured for another 24 h at 37°C in a 5% CO2 incubator. RNA was extracted from the cells, reverse transcribed, and qPCR was performed to test the gene expression levels of IL-6, IL-1β, TNF-α, and COX-2.

[0117] The results are as follows Figure 7 As shown, Bifidobacterium bifidum BGLP1 promotes the expression of IL-6, TNF-α, and COX-2 immune factors.

[0118] Example 11 Study on the acid and bile salt resistance of Bifidobacterium bifidum BGLP1

[0119] 1. Tolerance in artificial gastric fluid

[0120] Activated Bifidobacterium bifidum BGLP1 was inoculated into MRS + cysteine ​​liquid medium and cultured at 37℃ for 24 h to obtain fermentation broth. A 10% inoculum was then inoculated into MRS + cysteine ​​medium at pH 3 and placed in a 37℃ incubator. At 0 h, 1 h, and 2 h, 1 mL of the bacterial culture was taken and diluted to a suitable concentration with sterile physiological saline and spread onto MRS + cysteine ​​solid plates. After anaerobic incubation at 37℃ for 36-48 h, plate counts were performed to calculate the viable count and survival rate of Bifidobacterium bifidum BGLP1.

[0121] The results showed that the survival rate of Bifidobacterium bifidum BGLP1 was high at pH 3 for 1 h and 2 h, respectively, which was 97.38% and 93.36%, indicating that Bifidobacterium bifidum BGLP1 had strong tolerance to gastric juice.

[0122] 2. Tolerance in artificial pancreatic juice

[0123] Pancreatic enzyme suspension was prepared in MRS+cysteine ​​liquid medium to form 0.2% simulated artificial pancreatic juice. Bifidobacterium bifidum BGLP1 fermentation broth was inoculated into 0.2% simulated artificial pancreatic juice at an inoculum volume of 10%, and incubated at 37℃ for 1 h. After dilution and viable count, the survival rate of Bifidobacterium bifidum BGLP1 was calculated to be 83.98%, indicating that Bifidobacterium bifidum BGLP1 has strong tolerance to simulated pancreatic juice.

[0124] Example 12: Antibiotic resistance analysis of Bifidobacterium bifidum BGLP1

[0125] Bifidobacterium bifidum BGLP1 was activated for two generations and cultured in MRS + cysteine ​​liquid medium at 37°C until McFarland turbidity reached 0.5. 200 μL of bacterial culture was added dropwise to the surface of MRS + cysteine ​​solid medium and evenly spread using a spreader. Within 15 min after inoculation, Liofilchem ​​antimicrobial susceptibility testing discs were laid flat on the medium surface, air-dried, and then incubated upside down at 37°C for 8–24 h. The diameter of the complete inhibition zone was measured to assess the antibiotic susceptibility of the strain. The results are summarized in Table 6.

[0126] The specifications of the Liofilchem ​​drug sensitivity test strips are as follows: tetracycline (TE, 30 μg / tablet), amoxicillin (AML, 10 μg / tablet), and azithromycin (AZM, 15 μg / tablet).

[0127] According to the interpretation criteria of the inhibition range of the paper disc method for antibiotic susceptibility testing, tetracycline (TE, 30 μg / tablet) is considered sensitive (S) with an inhibition zone diameter ≥19 mm; amoxicillin (AML, 10 μg / tablet) is considered sensitive (S) with an inhibition zone diameter ≥18 mm; and azithromycin (AZM, 15 μg / tablet) is considered sensitive (S) with an inhibition zone diameter ≥17 mm.

[0128] Table 6: Results of resistance analysis of Bifidobacterium bifidum BGLP1 to common antibiotics

[0129]

[0130] Example 13 Hemolytic activity test of Bifidobacterium bifidum BGLP1

[0131] Take Bifidobacterium bifidum BGLP1 glycerol-preserved bacteria from the bacterial bank and activate it for two generations. Add 20% sterile defibrinated sheep blood to the MRS+cysteine ​​solid medium preparation solution at 50-55℃, mix well, pour into a petri dish, and obtain blood agar medium after solidification. Pick a single colony of activated Bifidobacterium bifidum BGLP1 and streak it on the blood agar medium. Incubate at 37℃ for 24 h. Observe whether there is a hemolytic clear zone around the colony. If there is, it is a positive hemolysis test.

[0132] After 24 hours of blood agar incubation, the experimental results are as follows: Figure 8 As shown, no hemolytic zone was observed, indicating that this strain does not exhibit hemolysis and is safe for use in food.

[0133] Example 14 Fermentation broth and inoculum of Bifidobacterium bifidum BGLP1

[0134] This invention provides a Bifidobacterium bifidum BGLP1 inoculum, prepared according to the following steps:

[0135] Bifidobacterium bifidum BGLP1 was activated and inoculated at a rate of 3% (v / v) into MRS+cysteine ​​liquid medium sterilized at 121°C for 15 min. 12% skim milk, 2.0% glucose, 1.5% peptone, 0.6% yeast extract, and the balance water were added to the total mass of the medium, and the pH was adjusted to 6.8.

[0136] The bacteria were anaerobically cultured at 37℃ for 48 h, centrifuged at 4℃ and 6000 rpm for 20 min, the supernatant was discarded, and the bacteria were washed 2 to 4 times with phosphate buffer (pH=7.2) to obtain bacterial sludge. The bacterial sludge was resuspended in physiological saline or PBS buffer to prepare fermentation broth containing Bifidobacterium bifidum BGLP1.

[0137] Optionally, the bacterial sludge and the protective agent are mixed and emulsified in a 1:1 mass ratio; wherein the protective agent comprises 150 g / L crystalline trehalose, 40 mL / L glycerol, 30 g / L isomaltooligosaccharide, 25 g / L fructooligosaccharide and 10 g / L L-sodium glutamate.

[0138] The emulsion was pre-frozen at -40°C for 2 hours and then freeze-dried to obtain the Bifidobacterium bifidum BGLP1 bacterial agent.

[0139] Example 15: Bifidobacterium bifidum BGLP1 probiotic product

[0140] The probiotic product is a composition of Bifidobacterium bifidum BGLP1 and other ingredients. The ingredients include one or more of the following: freeze-drying protectants, dietary fiber carriers, flavor modifiers, and synergists.

[0141] The dosage form of the probiotic product can be compressed candy, solid beverage, beverage, granules, capsules, tablets, pills, or oral liquid, etc.

[0142] More specifically, this embodiment provides a functional solid beverage with Bifidobacterium bifidum BGLP1 as its core. Each 100g of the finished product contains: 10.0g of active bacterial powder (obtained by high-density fermentation of the strain, centrifugation to collect bacterial sludge, mixed with a freeze-drying protectant [trehalose:skim milk = 1:1] and vacuum freeze-dried); dietary fiber carrier: inulin (80.0g, accounting for 80.0%); flavor modifiers: erythritol (8.0g) and citric acid (1.5g); and functional synergist: green tea extract (EGCG ≥ 50%, 0.5g).

[0143] Preparation process: The above components are dry-mixed evenly under conditions of humidity <10% and temperature 25℃, and packaged into 2g / bags. One bag per day can be brewed and consumed to simultaneously ingest sufficient active BGLP1 cells, which, together with inulin, enhance the secretion efficacy of GLP-1, and EGCG (epigallocatechin gallate) helps to improve the lipase inhibition rate.

[0144] Based on the results of the above embodiments, the Bifidobacterium bifidum BGLP1 provided by the present invention has the following properties and effects:

[0145] 1. It has strong resistance to acid and bile salts, and is well tolerated in artificial gastric juice and artificial intestinal juice, and can smoothly reach the human intestine;

[0146] 2. It has the ability to inhibit α-glucosidase, α-amylase, and lipase;

[0147] 3. It can strongly stimulate STC-1 cells to secrete GLP-1. GLP-1 can activate the mitochondrial generation pathway of adipocytes and the intestinal glucose-lowering pathway, promote the synthesis of UCP1, a marker protein of brown fat, and thus promote the conversion of white fat into brown fat, which is a more scientific way to reduce fat.

[0148] 4. It can convert excess sucrose into extracellular polysaccharides, which not only increases the probiotic effect but also helps with weight loss.

[0149] 5. It can activate macrophages and make the release of NO moderate, avoiding excessive inflammation and improving the body's immunity;

[0150] 6. Strong self-aggregation ability and hydrophobicity indicate that this strain has good intestinal adhesion and colonization ability.

[0151] In summary, compared with the prior art, the Bifidobacterium bifidum BGLP1 provided by the present invention has the following beneficial effects:

[0152] The Bifidobacterium bifidum BGLP1 provided by this invention has strong acid and bile salt resistance, good tolerance in artificial gastric juice and artificial intestinal juice, and can successfully reach the human intestine.

[0153] The Bifidobacterium bifidum BGLP1 provided by this invention significantly improves glucose metabolism through three physiological pathways: ① highly efficient inhibition of α-glucosidase; ② moderate inhibition of α-amylase; ③ strong promotion of GLP-1 secretion. Through the synergistic effect of these three pathways, glucose homeostasis is reshaped from multiple dimensions of "substrate blockade - delayed digestion - intestinal hormone regulation".

[0154] In this invention, Bifidobacterium bifidum BGLP1 significantly inhibits pancreatic lipase activity, effectively reducing dietary fat absorption; its lipid-lowering mechanism is compatible with its hypoglycemic function, avoiding the risk of gastrointestinal adverse reactions when used in combination with chemical drugs; it simultaneously utilizes excess sucrose to synthesize extracellular polysaccharides, which not only reduces free sugar toxicity but also generates prebiotic substances, providing a protective carbon source for the intestinal flora, thus avoiding the "steatorrhea" and flora imbalance side effects caused by traditional lipid-lowering drugs from the source;

[0155] In this invention, Bifidobacterium bifidum BGLP1 integrates four functions: "lowering blood sugar, lowering blood lipids, promoting GLP-1, and producing extracellular polysaccharides." This breaks through the limitation of existing probiotics having only a single function, simplifies the development of compound preparations, and can be widely used in weight loss products.

[0156] It should be noted that:

[0157] (1) Definition:

[0158] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0159] (2) The relevant prior art means or prior art terms involved in this application:

[0160] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. 625 "OD" is the optical density value measured when the wavelength is set to 625nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0161] Application of strains:

[0162] The example illustrates that Bifidobacterium bifidum BGLP1 can be applied to weight loss products. Based on the above design concept, this strain can be applied to weight loss capsules, weight loss probiotic powders, etc.

[0163] Although this document frequently uses terms such as Bifidobacterium bifidum, α-glucosidase, α-amylase, and pancreatic lipase, the possibility of using other terms is not excluded. These terms are used merely for the convenience of describing and explaining the essence of the invention; interpreting them as any additional limitation would be contrary to the spirit of the invention.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Bifidobacterium ( Bifidobacterium bifidum BGLP1, characterized in that: Its accession number is CGMCC No.35718.

2. A composition, characterized in that: Including Bifidobacterium bifidum BGLP1 as described in claim 1.

3. The composition according to claim 2, characterized in that: The composition contains one or more combinations of Bifidobacterium bifidum BGLP1 non-inactivated bacteria and Bifidobacterium bifidum BGLP1 lyophilized bacteria.

4. A microbial agent, characterized in that: Its components include Bifidobacterium bifidum BGLP1 as described in claim 1.

5. A fermentation broth, characterized in that: It is obtained by fermentation of Bifidobacterium bifidum BGLP1 as described in claim 1.

6. A probiotic product, characterized in that: Its components include Bifidobacterium bifidum BGLP1 as described in claim 1.

7. A freeze-dried product, characterized in that: Its components include Bifidobacterium bifidum BGLP1 as described in claim 1.

8. The application of Bifidobacterium bifidum BGLP1 as described in claim 1 in the preparation of weight loss products.

9. The application of Bifidobacterium bifidum BGLP1 according to claim 8 in the preparation of weight loss products, characterized in that: The weight loss products include one or more of the following: weight loss capsules, weight loss probiotic powder, and weight loss enzymes.

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