Lactobacillus mucilaginosus capable of regulating blood sugar and blood fat level and application thereof

Abstract

The invention discloses a lactobacillus mucilaginosus for regulating blood sugar and blood fat level and application thereof, the lactobacillus mucilaginosus is preserved in Guangdong Microbial Culture Collection Center on February 18, 2025, and the preservation number is GDMCCNO: 65910. The compound not only shows a remarkable inhibition effect on alpha-glucosidase and DPP-IV in vitro, but also shows the capabilities of improving insulin sensitivity, promoting glucose uptake and reducing the hyperglycemia level in cell and zebra fish models; in addition, deposition of liver lipid droplets in a zebra fish high-fat model can be remarkably reduced, and a good blood fat reducing effect is shown. Compared with an existing standard strain and a traditional medicine, the SHY0007 shows a better effect in the aspects of reducing blood glucose and blood lipid.

Description

Technical Field

[0001] The invention relates to the field of probiotics, and more specifically to a fermented mucus lactobacillus capable of regulating blood sugar and blood lipid levels and application thereof. Background Art

[0002] In recent years, the explosive growth in the incidence of diabetes has become one of the most serious public health problems in the world in the 21st century. Diabetes is a disease associated with various metabolic disorders caused by genetic and environmental factors, and its main feature is chronic hyperglycemia caused by insufficient insulin action. The hazards of diabetes are mainly complex complications, including neuropathy, nephropathy and retinopathy. In addition, diabetic patients are often accompanied by dyslipidemia, manifested as elevated levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), and decreased levels of high-density lipoprotein cholesterol (HDL-C), further increasing the risk of cardiovascular disease and stroke. However, there is currently no cure for diabetes, and most patients achieve the effect of controlling blood sugar through long-term oral medication.

[0003] Most common hypoglycemic drugs have certain side effects. For example, thiazolidinediones may cause abnormal liver function; α-glucosidase inhibitors may affect normal intestinal function, causing abdominal distension, increased flatulence, constipation or diarrhea; dipeptidyl peptidase IV (DPP-IV) inhibitors may increase the risk of upper respiratory tract infection during use; sodium-glucose cotransporter 2 (SGLT2) inhibitors increase the risk of genitourinary system infection; sulfonylurea hypoglycemic drugs may increase the burden on the liver and kidneys, leading to liver and kidney dysfunction. Long-term use of a hypoglycemic drug is prone to tolerance, resulting in reduced efficacy; in addition, some patients' poor diet control, lack of exercise, and improper use of drugs will also accelerate the reduction of drug efficacy. This will affect the effect of blood sugar management. Therefore, more and more research is focused on finding treatments that can effectively relieve diabetes with fewer side effects.

[0004] Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and regulating the structure of the host's specific bacterial flora. This type of microorganism has been widely used in many fields such as food fermentation, health products and medicine. Limosilactobacillus fermentum is a lactic acid bacterium with multiple probiotic functions. It is widely used in food fermentation, probiotic supplements and health products. It has the effects of promoting body growth, improving immunity and regulating intestinal flora.

[0005] The Limosilactobacillus fermentum CECT5716 isolated from healthy breast milk has been recognized as GRAS (Generally Recognized as Safe) by the US Food and Drug Administration and can be used in infant formula. Infants using the second-generation formula containing Limosilactobacillus fermentum CECT5716 can help prevent infectious gastrointestinal diseases and upper respiratory tract infections; Limosilactobacillus fermentum HCS08005 has the ability to anti-allergy and regulate immunity. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide a Limosilactobacillus fermentum with the ability to regulate blood glucose and lipid levels and its applications.

[0007] The first object of the present invention is to provide a Limosilactobacillus fermentum.

[0008] The second object of the present invention is to provide the fermentation supernatant of the Limosilactobacillus fermentum.

[0009] The third object of the present invention is to provide the supernatant of the cell lysate of the Limosilactobacillus fermentum.

[0010] The fourth object of the present invention is to provide the cell-containing culture of the Limosilactobacillus fermentum.

[0011] The fifth object of the present invention is to provide the application of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate or the cell-containing culture according to claim 4 in the preparation of an α-glucosidase inhibitor.

[0012] The sixth object of the present invention is to provide the application of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate or the cell-containing culture in the preparation of a dipeptidyl peptidase 4 inhibitor.

[0013] The seventh object of the present invention is to provide the application of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate or the cell-containing culture in the preparation of a drug for treating diabetes.

[0014] The eighth object of the present invention is to provide the application of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate or the cell-containing culture according to claim 4 in the preparation of a drug for treating insulin resistance.

[0015] The ninth object of the present invention is to provide the application of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate or the cell-containing culture in the preparation of a drug for treating hyperlipidemia.

[0016] The tenth object of the present invention is to provide a preparation.

[0017] To achieve the above object, the present invention is realized by the following technical solutions:

[0018] The present invention claims the following:

[0019] A strain of Limosilactobacillus fermentum SHY0007, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on February 18, 2025, with the deposit number GDMCC NO: 65910.

[0020] The fermentation supernatant of the Limosilactobacillus fermentum.

[0021] The supernatant of the cell lysate of the Limosilactobacillus fermentum.

[0022] The cell-containing culture of the Limosilactobacillus fermentum.

[0023] And the following applications:

[0024] Use of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture in the preparation of an α-glucosidase inhibitor.

[0025] Use of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture in the preparation of a dipeptidyl peptidase 4 inhibitor.

[0026] Use of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture in the preparation of a drug for treating diabetes.

[0027] Use of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture in the preparation of a drug for treating insulin resistance.

[0028] Use of one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture in the preparation of a drug for treating hyperlipidemia.

[0029] And a preparation containing one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture, or prepared from one or more of the Limosilactobacillus fermentum, the fermentation supernatant, the supernatant of the cell lysate, or the cell-containing culture.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention has obtained a fermented Lactobacillus mucosae SHY0007 with dual effects of reducing blood sugar and blood lipid. This strain has good inhibitory effects on both α-glucosidase and DPP-IV. In addition, it also shows a significant ability to promote glucose uptake and regulate blood sugar levels in insulin-resistant mouse myoblast models and zebrafish models, showing an insulin-sensitizing effect. At the same time, SHY0007 can also significantly reduce the deposition of liver lipid droplets in the zebrafish high-fat model, showing excellent blood lipid-lowering efficacy. Through the above experiments, it shows that SHY0007 has superior effects in both blood sugar reduction and blood lipid reduction. Therefore, the fermented Lactobacillus mucosae SHY0007 can be applied to the preparation of foods, drugs, and health products for assisting in reducing blood sugar. Description of the Drawings

[0032] Figure 1 Inhibition rate of the fermentation supernatant and the supernatant of broken cells of Lactobacillus mucosae SHY0007 on α-glucosidase (**P<0.01, vs positive control group).

[0033] Figure 2 Inhibition rate of the fermentation supernatant and the supernatant of broken cells of Lactobacillus mucosae SHY0007 on DPP-IV.

[0034] Figure 3 Microscopic images of normal C2C12 myotubes and insulin-resistant C2C12 myotubes after differentiation and modeling are shown.

[0035] Figure 4 Concentrate of the fermentation supernatant of Lactobacillus mucosae SHY0007 improves the ability of 2-NBDG cells to uptake glucose (**P<0.01 vs model group, P<0.001 vs control group).

[0036] Figure 5 Bacterial suspension of Lactobacillus mucosae SHY0007 can reduce the glucose content in the zebrafish hyperglycemic model (***P<0.001 vs normal group, P<0.001 vs model group).

[0037] Figure 6 Effect of Lactobacillus mucosae SHY0007 on liver lipid droplets in the zebrafish high-fat model (***P<0.001 vs normal group, P<0.001 vs model group). Detailed Embodiments

[0038] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0039] Depsipeptide A: Shanghai Yuanye Bio-Technology Co., Ltd., product number S26288;

[0040] E3 zebrafish embryo incubation solution: 17.4 g / L sodium chloride, 0.8 g / L potassium chloride, 2.9 g / L CaCl2·2H2O and 4.89 g / L MgCl2·6H2O, pH 7.2.

[0041] Example 1 Isolation and Identification of Lactobacillus mucosae SHY0007

[0042] I. Experimental Method

[0043] The Baitang sample from Kaili City, Qiandongnan Prefecture, Guizhou Province was vortexed and mixed evenly, and then 10-fold gradient diluted with sterile normal saline (10 6 、10 7 、10 8 times) in a laminar flow cabinet. 100 μL of the bacterial solution at each gradient was taken and spread on MRS agar medium, and cultured at 37 °C for 48 h.

[0044] After the culture, petri dishes with 50 - 150 colonies were selected, and typical colonies were selected through morphological observation and streaked and purified 2 - 3 times on MRS agar plates until the colony morphology on the entire plate was consistent. Single colonies were picked and amplified in MRS liquid medium. The cultured bacterial solution was mixed with 50% glycerol in equal proportion and stored frozen at -80 °C.

[0045] II. Experimental Results

[0046] Strain SHY0007 was Gram-positive and negative in the catalase test. After culturing on MRS agar medium for 48 h, the colony diameter was between 0.3 - 1.5 mm, round, with neat edges, white, and the surface was moist and smooth.

[0047] Sequencing showed that its 16S rDNA was as shown in SEQ ID NO: 1,

[0048] SEQ ID NO: 1:

[0049] TCAGGATGAACGCTGGCGGTGTGCCTAATACATGCAAGTCGAACGCGTTGG

[0050] CCCAATTGATTGACGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGT

[0051] GGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACA

[0052] ACATTTGGAAACAGATGCTAATACCGCATAACAGCGTTGTTCGCATGAACA

[0053] ACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCAT

[0054] TAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGT

[0055] TGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACG

[0056] GGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCA

[0057] ACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAA

[0058] GAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGA

[0059] AAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAA

[0060] GCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGT

[0061] CTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATA

[0062] ACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGC

[0063] GTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAAC

[0064] TGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGT

[0065] AGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTC

[0066] AGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAA

[0067] GGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG

[0068] TGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGC

[0069] GCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGT

[0070] GCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC

[0071] GAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGA

[0072] GACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCAT

[0073] GCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGT

[0074] CGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGGTCTCAGTTCGGA

[0075] CTGCAGGCTGCAACTCGTCTGCACGAAGTCGGAATCGCTAGTAATCGCGGA

[0076] TCAGCATGCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGAGAG。

[0077] After the strain SHY0007 was isolated and amplified, the bacterial liquid was sent to BGI-Shenzhen Co., Ltd. for 16S rDNA detection. The sequencing results were compared by NCBI-BLAST. The results showed that the strain SHY0007 was Limosilactobacillus fermentum. It was deposited in the Guangdong Provincial Culture Collection of Microorganisms on February 18, 2025, with the deposit number GDMCC NO: 65910. Its taxonomic name is Limosilactobacillus fermentum, hereinafter referred to as Limosilactobacillus fermentum SHY0007. Example 2 Inhibition of α-glucosidase by the fermentation supernatant and cell lysate of Limosilactobacillus fermentum SHY0007

[0078] I. Experimental method

[0079] 1. Preparation of the fermentation supernatant and cell lysate of Limosilactobacillus fermentum SHY0007

[0080] (1) Preparation of the fermentation broth of Limosilactobacillus fermentum SHY0007 strain

[0081] The Limosilactobacillus fermentum SHY0007 strain stored in glycerol at -80°C was inoculated into MRS liquid medium at an inoculation amount of 2% (volume fraction) and cultured at 37°C for 12 h, with two consecutive activations. Then, it was inoculated into lactose-free MRS liquid medium containing 20 g / L lactose at the same inoculation amount and cultured for 18 h to obtain the fermentation broth of Limosilactobacillus fermentum SHY0007 strain.

[0082] (2) Preparation of the fermentation supernatant and supernatant of cell lysate of Limosilactobacillus fermentum SHY0007

[0083] The fermentation broth of Limosilactobacillus fermentum SHY0007 strain was centrifuged at 4°C and 8000×g for 10 min, and the supernatant and cells were collected separately. The supernatant was passed through a 0.22 μm hydrophilic microfiltration membrane to obtain the SHY0007 fermentation supernatant.

[0084] After the cells were washed twice, the bacteria were resuspended in PBS (pH 6.8), and the concentration of the bacterial suspension was adjusted to 1×10 9 CFU / mL. Ultrasonic treatment was performed in an ice bath with a pulse of 3 - 5 seconds for 15 min. The ultrasonicated lysate was centrifuged at 4°C and 12000×g for 15 min, and the supernatant was filtered through a 0.22 μm hydrophilic microfiltration membrane to obtain the SHY0007 cell lysate supernatant.

[0085] Both the fermentation supernatant and the supernatant of cell lysate were stored at -80 °C.

[0086] 2. Detection of α-glucosidase inhibitory rate

[0087] PNPG (4-Nitrophenyl-β-D-glucopyranoside) is a chromogenic β-D-glucosidase substrate that produces a yellow solution after cleavage.

[0088] Group A: Take a 96-well plate, add 25 μL of 2.5 mmol / L PNPG and 25 μL of the treatment to the reaction wells, incubate at 37 °C for 10 min; then add 50 μL of 0.2 U / mL α-glucosidase and react at 37 °C for 15 min; finally, add 100 μL of 0.2 mol / L Na2CO3 solution to terminate the reaction; use a microplate reader to detect the absorbance of the reaction solution at a wavelength of 405 nm, and make three parallels for each sample.

[0089] The treatments were: the fermentation supernatant of SHY0007, the supernatant of cell lysate of SHY0007, 1 mg / mL acarbose (positive control), the fermentation supernatant of Lactobacillus rhamnosus ATCC53103, the supernatant of cell lysate of Lactobacillus rhamnosus ATCC53103, or MRS lactose medium (negative control).

[0090] Group B: The difference from Group A is that α-glucosidase was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L.

[0091] Group C: The difference from Group A is that the treatment was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L.

[0092] Group D: The difference from Group A is that α-glucosidase was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L, and the treatment was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L.

[0093] α-glucosidase activity inhibition rate = [1 - (A - B) / (C - D)] × 100%. Where A, B, C, and D are the absorbance values measured for each group

[0094] II. Experimental results

[0095] The results are as Figure 1As shown in Table 1, the inhibition rates of the fermentation supernatant and the cell lysate supernatant of Lactobacillus mucosae SHY0007 against α-glucosidase were significantly better than those of the standard strain Lactobacillus rhamnosus ATCC53103. In addition, the inhibitory effect of the fermentation supernatant of this strain against α-glucosidase was also significantly better than that of acarbose at 1.6 mg / mL (inhibition rate 37.16 ± 4.81%), indicating that Lactobacillus mucosae SHY0007 has excellent α-glucosidase inhibitory ability and hypoglycemic effect.

[0096] Table 1: Inhibition rates of the fermentation supernatant and the cell lysate supernatant of the strain against α-glucosidase

[0097] Treatment substance Inhibition rate of α-glucosidase (%) Fermentation supernatant of SHY0007 77.69±9.18* Supernatant of disrupted cells of SHY0007 6.67±0.59* Fermentation supernatant of ATCC53103 12.89±0.24 Supernatant of disrupted cells of ATCC53103 0.95±0.24 Acarbose 37.16±4.81 MRS lactose medium ND

[0098] * indicates significant difference compared with the same sample of ATCC53103, P < 0.05, ND indicates that the enzyme inhibition rate of the sample is 0. Example 3 Inhibition of dipeptidyl peptidase by the fermentation supernatant and cell lysate of Lactobacillus mucosae SHY0007

[0099] I. Experimental method

[0100] 1. Preparation of the fermentation supernatant and cell lysate of Lactobacillus mucosae SHY0007

[0101] (1) Preparation of the fermentation broth of Lactobacillus mucosae SHY0007 strain

[0102] The Lactobacillus mucosae SHY0007 strain stored in glycerol at -80°C was inoculated into MRS liquid medium at an inoculum size of 2% (v / v), cultured at 37°C for 12 h, and then inoculated into MRS liquid medium after continuous activation twice.

[0103] (2) Preparation of the fermentation supernatant and cell lysate supernatant of Lactobacillus mucosae SHY0007

[0104] Same as Example 4

[0105] 2. Detection of dipeptidyl peptidase inhibition rate

[0106] Glycyl-prolyl-p-nitroanilide hydrochloride (Gly-Pro-PNA) is the substrate of dipeptidyl peptidase IV (DPP-IV).

[0107] Group A: The reaction was carried out in a 96-well plate. 25 μL of 0.4 mmol / L Gly-Pro-PNA and the treatment were added to the reaction wells and incubated at 37 °C for 10 min. Then, 50 μL of 0.1 U / L DPP-IV was added and reacted at 37 °C for 60 min. Finally, 100 μL of 1 mol / L sodium acetate buffer solution (pH 4.0) was added to terminate the reaction. The absorbance of the reaction solution was measured at a wavelength of 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0108] The treatments were: the fermentation supernatant of SHY0007, the supernatant of the disrupted cells of SHY0007, 6.36 μmol / L dipeptidyl peptidase inhibitor A (positive control), the fermentation supernatant of Lactobacillus rhamnosus ATCC53103, the supernatant of the disrupted cells of Lactobacillus rhamnosus ATCC53103, or MRS lactose medium (negative control).

[0109] Group B: The difference from Group A was that DPP-IV was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L.

[0110] Group C: The difference from Group A was that the treatment was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L.

[0111] Group D: The difference from Group A was that DPP-IV was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L, and the treatment was replaced with an equal volume of PBS solution with a pH of 6.8 and a concentration of 0.1 mol / L.

[0112] The inhibition rate of DPP-IV enzyme activity = [1 - (A - B) / (C - D)] × 100%. Among them, A, B, C, and D were the absorbance values measured in each group.

[0113] II. Experimental Results

[0114] The results were as Figure 2 shown in Table 2. The inhibition rates of the fermentation supernatant and the supernatant of the disrupted cells of Lactobacillus mucosae SHY0007 on DPP-IV were significantly better than those of the standard strain Lactobacillus rhamnosus ATCC53103. In addition, the inhibitory effect of the fermentation supernatant of this strain on DPP-IV was also similar to that of diprotin A (inhibition rate 23.51 ± 2.54%), indicating that Lactobacillus mucosae SHY0007 has excellent DPP-IV inhibitory ability and has the effect of reducing blood lipid.

[0115] Table 2 Inhibition rates of the fermentation supernatant and the supernatant of the disrupted cells of strains on DPP-IV

[0116] Treatment substance Inhibition rate of DPP-IV enzyme activity (%) Fermentation supernatant of SHY0007 26.86±2.11** Supernatant of disrupted cells of SHY0007 1.57±0.76 Fermentation supernatant of ATCC53103 18.47±2.13 Supernatant of disrupted cells of ATCC53103 ND Dual protein A 23.51±2.54 MRS medium ND

[0117] **Indicates a significant difference compared with the fermentation supernatant of ATCC53103, P<0.01, ND indicates that the enzyme inhibition rate of the sample is 0.

[0118] Example 4 Fermented Lactobacillus mucosae SHY0007 Improves Cellular Insulin Resistance

[0119] I. Experimental Methods

[0120] 1. Sample Preparation

[0121] Inoculate the cryopreserved Lactobacillus mucosae SHY0007 stored in glycerol at -80°C into MRS broth medium at an inoculation amount of 2% (volume fraction), and culture it at 37°C for 12 h, and activate it continuously for 2 times.

[0122] Inoculate the seed liquid at an inoculation amount of 2% into 50 mL of liquid medium and culture it at 37°C for 15 h; then, centrifuge at 8000 rpm for 10 min at 4°C, collect the supernatant, and filter it with a 0.22 μm aqueous filter membrane; take 30 mL of the filtered supernatant into a 50 mL centrifuge tube, adjust the pH of the fermentation broth to 7.0±0.1 with 2M sodium hydroxide solution, and store it at -80°C until use.

[0123] Freeze-dry the fermentation supernatant with a vacuum freeze dryer, weigh it after freeze-drying and make it up to 12 mL to prepare a fermentation supernatant mother liquor concentrated 4 times. After filtering with a 0.22 μm aqueous filter membrane, store it at -80°C for standby.

[0124] 2. Improvement Effect of Concentrated Fermentation Supernatant of the Strain on Insulin Resistance of Mouse Myoblasts

[0125] 2-NBDG (2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) is a fluorescently labeled deoxyglucose analog that can be used to monitor glucose uptake by cells.

[0126] Mouse myoblast C2C12 cells were seeded at 2.5×10 5 cells / well into a 12-well plate pretreated with polylysine (0.1 mg / mL) and grown in 1 mL of Dulbecco's Modified (DMEM) medium containing 10% fetal bovine serum (FBS) for 12 h until contact inhibition. The cells were divided into four groups in the experiment. The following are the cell culture and treatment conditions for each group:

[0127] The treatment conditions of normal group cells are as follows: induction of myotubes: the culture medium in each well was replaced with 1 mL of DMEM culture medium containing 2% horse serum (HS), and differentiation was induced for 72 h (the medium was changed every 24 h); intervention with glucose fluorescent analogs: after the differentiation of myotube cells was completed, the cells were washed twice with 1 mL of DPBS, 1 mL of glucose-free DMEM culture medium was added, and the culture medium was discarded after incubation for 10 min, and replaced with glucose-free DMEM culture medium containing 50 uM 2-NBDG. After incubation for 1 h, the supernatant was removed and the cells were washed once with 1 mL of cold DPBS to remove free 2-NBDG.

[0128] The cell treatment conditions of the control group are as follows: induction of myotubes: the same as the normal group; intervention with glucose fluorescent analogs: the difference from the normal group is that the "glucose-free DMEM medium containing 50 μM 2-NBDG" is replaced with "glucose-free DMEM medium containing 50 μM 2-NBDG and 120 nM insulin".

[0129] The cell treatment conditions of the model group are as follows: induction of myotubes and modeling: the cell culture medium was replaced with 1 mL of DMEM medium containing 2% HS and 100 nM insulin, and differentiation was induced for 72 h (the medium was changed every 12 h); intervention with glucose fluorescent analogs: the same as the control group.

[0130] The cell treatment conditions of the intervention group are as follows: induction of myotubes and modeling: the cell culture medium was replaced with 1 mL of DMEM culture medium containing 2% HS and 100 nM insulin, and differentiation was induced for 48 h (the medium was changed every 12 h), and then 5% (v / v) of 4-fold concentrated fermentation supernatant mother solution was added to the culture medium (the 4-fold concentration of fermentation supernatant was the maximum concentration without damaging the viability of C2C12 cells), and then differentiation was induced for 24 h (the medium was changed every 12 h); intervention with glucose fluorescent analogs: the same as the control group.

[0131] After the treatment of each group of cells was completed, 300 μL of trypsin (without EDTA) was added to each well. After digestion for 8 minutes, 600 μL of DPBS was added to terminate the digestion. After aspiration until there was basically no cell adhesion, the cells were transferred to a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 6 minutes. The supernatant was removed and suspended with 1 mL of DPBS to prepare a single cell suspension. The prepared suspension was detected using a BD Accuri C6 Plus flow cytometer, and the FITC-A channel was selected.

[0132] 2. Experimental Results

[0133] C2C12 cell differentiation and modeling results Figure 3 As shown in the figure, after 72 hours of induction of differentiation in the normal group and the control group, more than 95% of the cells began to fuse and form myotubes. The myotubes were densely arranged and mostly in the shape of thin and long strips, indicating that the differentiation was complete.Figure 3 (Left) as shown; in the model group and the intervention-induced differentiation and modeling for 72 h, more than 95% of the cells began to fuse and form myotubes, and compared with normal differentiation, the myotubes were arranged more closely and mostly showed short and thick rod shapes, indicating successful insulin resistance modeling. Most of the cells were differentiated and had insulin resistance symptoms, such as Figure 3 (Right) as shown.

[0134] The experimental results are as Figure 4 shown. The proportion of cells that absorbed 2-NBDG in the control group was significantly higher than that in the normal group, proving that insulin played an effect of promoting cell glucose absorption. The proportion of cells that absorbed 2-NBDG in the model group was significantly lower than that in the control group, proving that the insulin resistance modeling was successful.

[0135] Compared with the model group, the fermentation supernatant concentrate of Lactobacillus mucosae SHY0007 significantly increased the proportion of cells that absorbed 2-NBDG, proving that this strain can improve cell insulin resistance.

[0136] Hypoglycemic effect of Lactobacillus mucosae SHY0007 in Example 5

[0137] I. Experimental method

[0138] 1. Sample preparation

[0139] Take the seed liquid of Lactobacillus mucosae SHY0007 in the logarithmic phase, inoculate it into 5 mL of MRS liquid medium at an inoculation amount of 2%, and culture it in a shaker at 37 °C for 12 h. After incubation, the fermentation broth was centrifuged at 6000 rpm for 10 min at 4 °C, and the supernatant was discarded. The cell precipitate was washed twice with sterile normal saline and then resuspended with 5 mL of sterile normal saline. The cell suspension was counted with a microbial cell counter, and the cell density was adjusted to 10 9 cells / mL and placed in a refrigerator at 4 °C for standby.

[0140] 2. Detection of glucose content in hyperglycemic zebrafish

[0141] In a 6-well plate, 40 zebrafish at 3 dpf (days post fertilization) were placed in each well and treated as follows:

[0142] Blank control group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution;

[0143] Model group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution containing 4% glucose;

[0144] Positive drug group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution containing 4% glucose and 1.6 mg / mL acarbose;

[0145] Treatment group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution containing 4% glucose and 10 6 CFU / mL of fermented Limosilactobacillus mucosae SHY0007;

[0146] For each group, three replicates were set up and incubated in a constant temperature incubator at 28.5 ± 0.5 °C for 48 h, with the liquid changed once during this period.

[0147] After treatment until 5 dpf, all the fish in each group were collected, rinsed 3 - 4 times with E3 zebrafish embryo incubation solution to remove the residual liquid, and centrifuged at low speed 3 - 4 times to fully remove the liquid, and the fish bodies were retained.

[0148] The fish bodies together with 2 μL of E3 zebrafish embryo incubation solution and 3 zirconium beads were placed into a high-throughput cryogenic grinder for grinding (4 °C, 20 seconds, with an interval of 10 seconds, cycling 3 times). After grinding, centrifugation was carried out at 4 °C, 13,000 rpm for 5 minutes. After centrifugation, the supernatant was removed, 2 μL of zebrafish tissue homogenate was aspirated, and the blood glucose content in the zebrafish tissue was detected using a blood glucose meter.

[0149] II. Experimental results

[0150] The results were as Figure 5 shown. The glucose content in the zebrafish of the treatment group was 7.77 ± 0.60 nmol / L, which was significantly different compared with the model group (12.13 ± 0.15 nmol / L) (P < 0.001). In addition, in the positive drug group, the glucose content in the zebrafish was 8.83 ± 0.42 nmol / L, which was significantly different compared with the model group (P < 0.001), consistent with the clinical effect, but slightly higher than that of the treatment group.

[0151] The above results indicate that the fermented Limosilactobacillus mucosae SHY007 suspension can effectively reduce the glucose content in hyperglycemic zebrafish, and its effect is better than acarbose in this experiment.

[0152] Example 6 Hypolipidemic effect of Limosilactobacillus mucosae SHY0007

[0153] I. Experimental method

[0154] 1. Sample preparation

[0155] Take the seed solution of fermented Limosilactobacillus mucosae SHY0007 in the logarithmic phase, inoculate it into 5 mL of MRS liquid medium at an inoculation amount of 2%, and culture it in a shaker at 37 °C for 12 h. After incubation, the fermentation broth was centrifuged at 6000 rpm for 10 min at 4 °C, and the supernatant was discarded. The bacterial cell precipitate was washed 2 times with sterile normal saline and then resuspended with 5 mL of sterile normal saline. The bacterial suspension was counted using a microbial cell counter, and the bacterial density was adjusted to 10 9 CFU / mL and placed in a refrigerator at 4 °C for standby.

[0156] 2. Detection of Fat Deposition in the Liver Tissue of Hyperlipidemic Zebrafish

[0157] The accumulated fat in the liver can be stained by Oil Red O. Oil Red O belongs to azo dyes, which have strong fat solubility and fat-staining property. It has a relatively high solubility in lipids and can combine with triglycerides to form small lipid droplets. When entering the zebrafish body, Oil Red O dissolves in the lipids (lipid droplets) in the tissue, making it appear orange-red. Therefore, the Oil Red O staining technique can be used to identify the fat deposition in the liver tissue.

[0158] In a 6-well plate, 40 zebrafish at 5 dpf (days post fertilization) were placed in each well and treated as follows:

[0159] Blank control group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution.

[0160] Model group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution containing 0.1% egg yolk powder.

[0161] Positive drug group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution containing 0.1% egg yolk powder and 1.5 mg / mL fenofibrate.

[0162] Treatment group: Zebrafish were cultured with 5 mL of E3 zebrafish embryo incubation solution containing 0.1% egg yolk powder and 7 10 CFU / mL of fermented Lactobacillus mucosae SHY0007.

[0163] Three replicates were set for each group and incubated in a constant temperature incubator at 28.5 ± 0.5 °C for 48 h, and the solution was changed once during this period.

[0164] After treatment until 7 dpf, they were fixed with 4% paraformaldehyde, then stained with Oil Red O for 2 h and then eluted. Photographs of zebrafish embryos in each group were taken under a microscope, 40 tails were photographed in each well, the light intensity of the zebrafish liver region was collected using ImageJ, and a significant difference test was performed on the average brightness values of each group.

[0165] II. Experimental Results

[0166] The results were as Figure 6 shown. Compared with the blank control group (mean liver brightness: 58.199 ± 14.848), the mean liver brightness of zebrafish in the model group (115.257 ± 8.431) increased significantly (P < 0.001), indicating that the hyperlipidemic zebrafish model was successfully established in this study.

[0167] The mean liver brightness of zebrafish in the positive drug fenofibrate group was 98.378 ± 15.009, showing a significant difference compared with the model (P < 0.001). Therefore, fenofibrate has a lipid-lowering effect, which is consistent with the clinical results. The mean liver brightness of the treatment group treated with Lactobacillus mucosae SHY0004 was 98.3543 ± 12.15, showing a significant difference compared with the model group (P < 0.001). Therefore, the above results indicate that Lactobacillus mucosae SHY0007 has a lipid-lowering effect, and its lipid-lowering effect is comparable to that of the positive drug fenofibrate in this experiment.

Claims

1. A strain of Limosilactobacillus fermentum, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on February 18, 2025, with the deposit number GDMCCNO:65910.

2. The fermentation supernatant of the fermented mucilaginous lactobacillus according to claim 1.

3. The supernatant of the cell crushed product of the fermented Lactobacillus mucilaginosus according to claim 1.

4. The bacterial culture containing the fermented Lactobacillus mucilaginosus according to claim 1.

5. Use of one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial cell crush according to claim 3, or the bacterial cell-containing culture according to claim 4 in the preparation of an α-glucosidase inhibitor.

6. Use of one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial cell crush according to claim 3 or the bacterial cell-containing culture according to claim 4 in the preparation of a dipeptidyl peptidase 4 inhibitor.

7. Use of one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial cell fragments according to claim 3, or the bacterial cell-containing culture according to claim 4 in the preparation of a drug for treating diabetes.

8. Use of one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial cell fragments according to claim 3, or the bacterial cell-containing culture according to claim 4 in the preparation of a drug for treating insulin resistance.

9. Use of one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial cell crush according to claim 3, or the bacterial cell-containing culture according to claim 4 in the preparation of a drug for treating hyperlipidemia.

10. A preparation, characterized in that The method comprises one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial crushed product according to claim 3, or the bacterial culture containing claim 4, or is prepared by using one or more of the fermented mucus lactobacillus according to claim 1, the fermentation supernatant according to claim 2, the supernatant of the bacterial crushed product according to claim 3, or the bacterial culture containing claim 4.

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