Lactobacillus rhamnosus strain, viable bacteria formulation and preparation method therefor and use thereof

Abstract

The present application relates to the technical field of microorganisms, and in particular to a Lactobacillus rhamnosus strain, a viable bacteria formulation and a preparation method therefor and a use thereof. The preservation number of the Lactobacillus rhamnosus strain is CGMCC No. 25682. The Lactobacillus rhamnosus strain and the viable bacteria formulation thereof have good free radical scavenging, oxidative stress response, anti-oxidation and anti-aging capabilities, have the potential of regulating blood sugar, can adjust the immunity, and are used for maintaining a balance in intestinal flora.

Description

Lactobacillus rhamnosus strain, live bacterial preparation, and preparation method and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 202310744495.8 filed in China on June 21, 2023, Chinese Patent Application No. 202310745739.4 filed in China on June 21, 2023, and Chinese Patent Application No. 202310744266.6 filed in China on June 21, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus rhamnosus strain, a live bacteria preparation, and a preparation method and use thereof. Background Art

[0004] The aging problem is serious in today's world, and delaying aging has become a focus of attention. With the development of science and technology, there is a consensus on the research of aging mechanism. The most studied is the free radical-oxidative stress theory, which believes that under normal circumstances, the body will release free radicals (superoxide anion free radicals (O 2- ), hydroxyl radicals (OH), and other free radicals maintain a balance with the antioxidant system. Once the body is stimulated, free radical content increases, the ability to scavenge free radicals decreases, and antioxidant enzyme levels decrease, damaging the body's cellular components, leading to lipid peroxidation, protein damage, and oxidative stress, ultimately leading to cell apoptosis and aging.

[0005] Diabetes, particularly type 2 diabetes, is a global health concern. It is a metabolic disease characterized by hyperglycemia caused by insufficient insulin secretion. Currently, medications such as α-glucosidase inhibitors and dipeptidyl peptidase 4 inhibitors (DPP-4 inhibitors) can be used to control blood sugar levels in diabetic patients.

[0006] Studies have shown that oxidative damage to pancreatic beta cells is one of the causes of diabetes. Excessive free radicals attack insulin-specific receptors on liver cell membranes, triggering insulin resistance. This significantly reduces the body's antioxidant defense system's ability to scavenge free radicals, leading to a series of oxidative damages.

[0007] Currently, there is insufficient research in the relevant technologies on the effects of Lactobacillus rhamnosus strains in lowering blood sugar, anti-oxidation, anti-aging and improving immunity.

[0008] Summary of the Invention

[0009] To solve the above technical problems, the present invention provides a Lactobacillus rhamnosus strain, its fermentation product, live bacterial preparation and its preparation method and use. The Lactobacillus rhamnosus strain, its fermentation product or live bacterial preparation has excellent performance in anti-oxidation, anti-aging, lowering blood sugar and improving immunity.

[0010] In one aspect, the present invention provides a Lactobacillus rhamnosus strain, the deposit number of which is CGMCC No.25682.

[0011] In one example, the Lactobacillus rhamnosus strain comprises the 16S rRNA gene sequence represented by SEQ ID NO: 1.

[0012] In another aspect, the present invention provides a fermentation product obtained by fermentation using the Lactobacillus rhamnosus strain described above.

[0013] In one example, the fermented product is a fermented dairy product; optionally, the content of active Lactobacillus rhamnosus in the fermented dairy product is higher than 6.0×10 8 CFU / mL.

[0014] In another example, the fermented product is a fermented oat milk product; optionally, the viable count of Lactobacillus rhamnosus in the fermented oat milk product is higher than 5.0×10 8 CFU / mL.

[0015] In another aspect, the present invention provides a live bacterial preparation comprising the Lactobacillus rhamnosus strain described above.

[0016] In one example, the live bacteria preparation contains 3.0×10 11 CFU / g and above.

[0017] In another aspect, the present invention provides a food or health product comprising the live bacteria preparation as described above.

[0018] In one example, the food is selected from one or more of milk powder, solid beverages, fermented dairy products, milk-containing beverages and cheese.

[0019] In one example, the fermented dairy product is selected from fermented cow's milk product and fermented oat milk product. Optionally, the content of active Lactobacillus rhamnosus in the fermented cow's milk product is 6.0×10 8 CFU / mL or more. Optionally, the viable count of Lactobacillus rhamnosus in the fermented oat milk product is 5.0×10 8 CFU / mL or above.

[0020] In another aspect, the present invention provides use of the Lactobacillus rhamnosus strain as described above in the preparation of medicaments for scavenging free radicals, coping with oxidative stress, anti-oxidation, and anti-aging.

[0021] In one example, the Lactobacillus rhamnosus strain is used to increase the levels of superoxide dismutase, glutathione peroxidase, and glutathione, and to reduce the level of malondialdehyde.

[0022] In another example, the Lactobacillus rhamnosus strain is used to scavenge excess free radicals and improve the scavenging rate of hydroxyl radicals and DPPH radicals.

[0023] The present invention also provides use of the Lactobacillus rhamnosus strain described above in the preparation of a drug for producing DPP-4 and α-glucosidase inhibitors and stimulating NCI-H716 cells to produce GLP-1.

[0024] In one example, the Lactobacillus rhamnosus strain is used to assist in lowering blood sugar.

[0025] In another aspect, the present invention provides use of the Lactobacillus rhamnosus strain in preparing a medicament for regulating immunity.

[0026] In one example, the Lactobacillus rhamnosus strain is used to increase the level of cytokines TNF-α and / or IL-6.

[0027] In another aspect, the present invention provides use of the Lactobacillus rhamnosus strain described above for increasing the content of lactic acid or short-chain fatty acids.

[0028] Preferably, the short-chain fatty acid is acetic acid.

[0029] Preferably, the Lactobacillus rhamnosus strain is used to maintain the balance of intestinal flora.

[0030] In another aspect, the present invention provides a method for preparing the live bacteria preparation as described above, comprising the following steps:

[0031] The Lactobacillus rhamnosus strain described above was expanded and cultured in an optimized liquid culture medium;

[0032] Collect bacteria;

[0033] Add protective agent to resuspend, freeze-dry in vacuum, and crush to obtain active bacterial agent. Beneficial effects

[0034] The Lactobacillus rhamnosus strain, its fermentation product or live bacterial preparation of the present invention has good free radical scavenging, oxidative stress response, antioxidant and anti-aging capabilities, can increase the levels of superoxide dismutase, glutathione peroxidase and glutathione, reduce the level of malondialdehyde, and increase the scavenging rate of hydroxyl radicals and DPPH free radicals; can produce DPP-4 and α-glucosidase inhibitors, and stimulate NCI-H716 cells to produce GLP-1, with the potential to regulate blood sugar; can increase the levels of cytokines TNF-α and IL-6, can regulate immunity; and increase the content of lactic acid or short-chain fatty acids, which are used to maintain the balance of intestinal flora. The Lactobacillus rhamnosus of the present invention can also be fermented to produce fermented cow milk products, fermented oat milk products and other fermented products. The production process parameters of the live bacterial preparation of the present invention are simple, easy to control, and short cycle, which ensures the high survival rate of Lactobacillus rhamnosus, and the resulting product can be stored for a long time with stable product quality.

[0035] In addition, the Lactobacillus rhamnosus of the present invention has excellent in vitro prebiotic functions, and the strain performs well in terms of artificial gastrointestinal fluid tolerance, bacteriostasis (inhibiting the proliferation of common pathogenic bacteria), and immune regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 shows a RAPD cluster analysis diagram of Lactobacillus rhamnosus HOM1213 strain and some commercial Lactobacillus rhamnosus strains constructed based on the UPGMA method.

[0037] FIG2 shows the inhibition rate of the Lactobacillus rhamnosus HOM1213 strain of the present invention on DPP-4 and α-glucosidase.

[0038] FIG3 shows the in vitro antioxidant capacity of the Lactobacillus rhamnosus HOM1213 strain of the present invention.

[0039] FIG4 shows the effects of the Lactobacillus rhamnosus HOM1213 strain of the present invention on the antioxidant and anti-aging indicators in vivo of oxidative damage model mice.

[0040] FIG5 shows a process flow chart of the present invention.

[0041] Microorganism Preservation Instructions

[0042] The Lactobacillus rhamnosus HOM1213 strain of the present invention was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on September 9, 2022. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 25682.

[0043] The Lactobacillus rhamnosus HOM1213 strain of the present invention was sent to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0044] The test and identification conclusion is as follows: Under the conditions of our laboratory, based on a comprehensive analysis of the cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, pheS gene sequence, and other experimental data of the tested strain, and with reference to relevant research articles in the "Bergey's Manual of Systematic Bacteriology" and the International Journal of Systematic and Evolutionary Microbiology, the tested strain (strain number: HOM1213) was identified as: Lacticaseibacillus rhamnosus. Synonym: Lactobacillus rhamnosus.

[0045] The cell morphology of the strain is rod-shaped; the physiological and biochemical characteristics are: Gram-positive, catalase-negative (-), oxidase-negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO: 1, and the pheS gene sequence is shown in SEQ ID NO: 9. DETAILED DESCRIPTION

[0046] The technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention are further described below with reference to specific embodiments to further illustrate the technical solution of the present invention, but are not limited thereto. Any modification or equivalent substitution of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. Experimental methods without specific conditions in the following examples were carried out in accordance with conventional methods and conditions in the art.

[0047] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0048] Unless otherwise specified, the reagents and materials used in the present invention were prepared using conventional methods or obtained from commercial channels.

[0049] As identified above, Lactobacillus rhamnosus and Lacticaseibacillus rhamnosus are synonyms, and thus in the present invention, Lactobacillus rhamnosus and Lacticaseibacillus rhamnosus can be used interchangeably.

[0050] The Lactobacillus rhamnosus strain HOM1213 used in the present invention is isolated from the feces of healthy infants. Its physical and chemical properties include an optimal growth temperature of 37°C, rich metabolites, acid and bile salt resistance, antibacterial properties, immunomodulatory properties, blood sugar regulation, and antioxidant and anti-aging activities.

[0051] In order to further understand the present invention, the Lactobacillus rhamnosus and its uses provided by the present invention are described in detail below with reference to the examples. The protection scope of the present invention is not limited by the following examples.

[0052] Example

[0053] Example 1 Isolation and identification of Lactobacillus rhamnosus strain HOM1213

[0054] (1) Preparation of culture medium

[0055] MRS liquid culture medium:

[0056] Mix 1 L of MRS medium (OXOID, CM1163) and double-distilled water, stir well, sterilize at 121°C for 20 min, and set aside.

[0057] Modified MRS solid medium:

[0058] MRS medium (OXOID, CM1163), 0.05 g of bromocresol green (Shanghai Biotech), and 1 L of double-distilled water were mixed and mixed evenly. The pH was adjusted to 5.5, and the mixture was sterilized at 121°C for 20 min.

[0059] (2) Isolation of strains

[0060] Weigh 1 g of healthy infant feces and thoroughly vortex with 9 mL of 0.9% saline. Perform a ten-fold serial dilution of the sample. Select two appropriate dilutions and spread 100 μL onto a modified MRS solid plate. Incubate anaerobically at 37°C for 72 hours. Select a single colony with a smooth, milky white or creamy yellow surface and a yellowing periphery, streak culture, and purification. Observe colony morphology under a microscope. Select a single colony and culture it in MRS liquid medium for pure culture. Store the culture in glycerol stock and designate it as HOM1213.

[0061] (3) Identification of strains

[0062] The strains were inoculated into MRS liquid medium and cultured at 37°C for 24 hours. Total bacterial DNA was extracted from the bacterial solution and 16S rRNA gene amplification was performed. PCR amplification and agarose gel electrophoresis were performed using universal primers 27F and 1492R. The gel was excised and recovered, and sequencing was performed (Shanghai Biotech). Then, using the BLAST tool, the HOM1213 strain was identified as Lactobacillus rhamnosus in the NCBI database. The sequence of the 16S rRNA gene of the HOM1213 strain is shown in SEQ ID NO: 1.

[0063] The sequence of the 16S rRNA gene of the HOM1213 strain was determined as follows:

[0064] (4) Randomly amplified polymorphic DNA (RAPD) strain identification

[0065] Random polymorphic DNA analysis (RAPD) was performed on the isolated Lactobacillus rhamnosus HOM1213. The results showed that HOM1213 was different from some commercial Lactobacillus rhamnosus strains and had specificity.

[0066] DNA was extracted from the preserved strains. Using the strain DNA as a template, PCR was performed to amplify polymorphic DNA fragments using five primers: OPA-02: TGC CGA GCT G; OPA-18: AGG TGA CCG T; OPL-07: AGG CGG GAA C; OPL-16: AGG TTG CAG G; and OPM-05: GGG AAC GTG T. Gel electrophoresis revealed distinct DNA differences, which were analyzed using cluster analysis software. The results are shown in Figure 1. As shown in Figure 1, the RAPD cluster analysis diagram of Lactobacillus rhamnosus HOM1213 is distinct from that of commercial Lactobacillus rhamnosus strains and is unique.

[0067] Table 1

[0068] Example 2 Preparation process of active bacteria powder

[0069] (1) Culture of strains

[0070] The Lactobacillus rhamnosus HOM1213 strain isolated in Example 1 was inoculated into MRS liquid medium at an inoculum rate of 3% of the total medium volume, cultured at 37°C for 24 hours, and activated for the second generation. The viable count of the strain inoculated into MRS medium and cultured at 37°C was 1×10 10 CFU / mL or above.

[0071] (2) Freeze-drying

[0072] The fermentation broth of Lactobacillus rhamnosus HOM1213 was centrifuged to collect bacterial sludge, which was washed with 0.9% sterile physiological saline. The bacterial sludge was mixed with the protective agent (100 g / L skim milk powder) and freeze-dried in a freeze dryer. The bacterial cake was crushed with a fine grinder to obtain the freeze-dried bacterial powder. The number of viable bacteria in the freeze-dried bacterial powder was higher than 3.0×10 11 CFU / g.

[0073] Example 3 Gastrointestinal transit capacity test

[0074] (1) Isolation and activation of strains

[0075] A commercial strain of Lactobacillus rhamnosus GG was selected as a control and purchased from the ATCC (American Type Culture Collection). Each strain (Lactobacillus rhamnosus GG and the Lactobacillus rhamnosus HOM1213 strain isolated in Example 1) was inoculated into MRS liquid medium at an inoculum size of 3% of the total culture volume, cultured at 37°C for 24 hours, activated twice, and then used for standby use.

[0076] (2) Preparation of artificial gastric juice

[0077] Take 16.4 mL of dilute hydrochloric acid and 10 g of pepsin, add about 800 mL of water, shake well, adjust the pH to 3.0, add water to make up to 1 L, filter with a 0.22 μm microporous membrane and set aside.

[0078] (3) Preparation of artificial intestinal fluid

[0079] Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of water and adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution. Dissolve 10 g of pancreatic enzyme and 3 g of oxgall (BD Difco) in appropriate amounts of water. Mix the two solutions and dilute to 1000 mL with water. Filter through a 0.22 μm sterile filter membrane under sterile conditions and set aside.

[0080] (4) Evaluation of the survival ability of strains in simulated artificial gastrointestinal fluid

[0081] Take 1 mL of each bacterial strain, centrifuge, collect the cells, add them to 10 mL of artificial gastric fluid, mix thoroughly, and immediately count the viable cells (T0). Place the cells in a 37°C incubator and incubate for 3 hours before counting the viable cells (T1). Centrifuge the sample, add 10 mL of artificial intestinal fluid, mix thoroughly, and incubate in a 37°C incubator for 3 hours before counting the viable cells (T2). The survival rate is calculated using the following formula:

[0082] T0 is the viable bacterial count (CFU / mL) of the test strain at 0 h before treatment; T1 is the viable bacterial count of the test strain after 3 h of treatment with artificial gastric juice; and T2 is the viable bacterial count of the test strain after 3 h of treatment with artificial gastric juice and artificial intestinal juice.

[0083] As can be seen from Table 2, after 3 hours of treatment with simulated artificial gastric fluid, the survival rate of the Lactobacillus rhamnosus HOM1213 strain can reach more than 98%. After the bacterial solution is further treated with simulated intestinal fluid for 3 hours, its survival rate can still be as high as more than 80%, indicating that the Lactobacillus rhamnosus HOM1213 strain has a high survival rate in the intestine.

[0084] Table 2. Survival rates of strains in simulated gastrointestinal fluid

[0085] Example 4 Antibiotic Sensitivity

[0086] The drug susceptibility test was performed according to the KB agar method recommended by the National Committee for Clinical Standards (NCCLS) of the United States. The method is as follows:

[0087] (1) The commercial strain Lactobacillus rhamnosus GG was selected as the control strain. Lactobacillus rhamnosus HOM1213 and GG were inoculated into MRS liquid culture medium at an inoculum rate of 3% of the total culture medium, cultured at 37°C for 24 hours, activated for three generations, and then used for standby.

[0088] (2) Take 1 mL of bacterial solution (1.5×10 8 CFU / mL), 15 mL of MRS solid culture medium was placed in a sterile culture dish and mixed evenly. After the plate solidified, a standard antibiotic sensitivity paper was placed on it. After incubation at 37 °C for 48 h, the diameter of the inhibition zone was measured and recorded.

[0089] Results were interpreted according to CLSI criteria (see Table 3). Lactobacillus rhamnosus GG strain was used as a commercial control. The results showed that Lactobacillus rhamnosus HOM1213 was resistant to vancomycin, gentamicin, kanamycin, and ampicillin, moderately resistant to streptomycin, and sensitive to the remaining four antibiotics. Lactobacillus rhamnosus exhibited natural resistance to vancomycin.

[0090] Table 3. Results of sensitivity test of Lactobacillus rhamnosus HOM1213 strain to nine antibiotics

[0091] S (susceptible) indicates sensitivity; I (intermediate) indicates intermediate; R (resistance) indicates resistance

[0092] Example 5 Test on the ability to inhibit common pathogenic bacteria

[0093] (1) Activation of pathogenic bacteria

[0094] Five pathogenic bacteria were selected for this experiment: Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, and Listeria monocytogenes ATCC 19111. These pathogenic strains were purchased from the American Type Culture Collection (ATCC).

[0095] The pathogenic strains were inoculated into nutrient agar medium at a 3% inoculum and cultured at 37°C with shaking at 200 rpm for 12 h. The indicator bacteria were adjusted to OD = 0.1 with fresh culture medium for later use.

[0096] (2) Strain activation

[0097] In this experiment, the commercial strain Lactobacillus rhamnosus GG was used as a control strain. The commercial strain GG and the Lactobacillus rhamnosus HOM1213 strain isolated in Example 1 were inoculated into MRS liquid culture medium at a 3% inoculum rate. The culture was incubated at 37°C for 24 hours. After activation twice, the fermentation broth was obtained. The supernatant was centrifuged at 11,000 rpm for 10 minutes, and the antibacterial test was performed using MRS liquid culture medium as a negative control.

[0098] (3) Plate preparation

[0099] Pour the sterilized nutrient agar culture into the culture dish, add 100 μL of indicator bacteria solution into the culture medium, mix well, and let it stand to solidify.

[0100] (4) Antibacterial experiment

[0101] Using sterile tweezers, gently place the Oxford cup on the plate, maintaining a certain distance between the wells. Add 150 μL of fermentation supernatant to each well. After diffusion for 12 hours in a 4°C refrigerator, incubate in a 37°C incubator for 18 hours. Observe and measure the diameter of the inhibition zone. The results are shown in Table 4.

[0102] Table 4. Inhibitory effect of each group on pathogenic bacteria Note: “–” no antibacterial activity, <11mm; “+” 11mm≤inhibition zone<16mm; “++” 16mm≤inhibition zone<23mm; “+++” ≥23mm

[0103] As shown in Table 4, Lactobacillus rhamnosus HOM1213 has an inhibitory effect on all five pathogens. Compared with the commercial strain Lactobacillus rhamnosus GG, Lactobacillus rhamnosus HOM1213 has a stronger antibacterial ability, as reflected in its inhibition of Salmonella and Listeria monocytogenes.

[0104] Example 6 Lactic acid and short-chain fatty acid content

[0105] (1) Preparation of strain supernatant

[0106] The commercial strain Lactobacillus rhamnosus GG was selected as a control strain. The Lactobacillus rhamnosus strain HOM1213 isolated in Example 1 and the commercial strain GG were inoculated into MRS liquid culture medium at a 3% inoculum rate, cultured at 37°C for 24 hours, activated for three generations, and centrifuged at 11,000 rpm and 4°C for subsequent use.

[0107] (2) Determination of lactic acid, acetic acid and formic acid content

[0108] 4 mL of the supernatant was added with 2 drops of phenolphthalein and adjusted with KOH solution until a slight color change occurred. The volume was then made up to 5 mL with deionized water and centrifuged at 11,000 rpm and 4°C for 10 minutes. Samples of varying concentrations of lactic acid, acetic acid, and formic acid were placed in separate 10 mL headspace vials. 2 mL of phosphate buffer solution, 2 mL of a derivatization reagent, and 1 mL of acetone were then added. The vials were sealed and reacted in a 100°C waterbath for 40 minutes. After cooling to room temperature, the derivatized samples were transferred to 2 mL centrifuge tubes and vortex-extracted with an equal volume of isooctane. The isooctane layer was analyzed by gas chromatography (GC). The results are shown in Table 5.

[0109] Table 5. Determination of lactic acid, acetic acid and formic acid content **: p < 0.01 compared with the MRS control group

[0110] As shown in Table 5, both Lactobacillus rhamnosus strains HOM1213 and GG produced high levels of lactic acid and a certain amount of acetic acid. Lactic acid and acetic acid can inhibit the proliferation of pathogenic bacteria and help maintain a healthy intestinal flora.

[0111] Example 7 In vitro cytokine secretion test

[0112] The commercial strain Lactobacillus rhamnosus GG was selected as the control strain. The Lactobacillus rhamnosus strain HOM1213 and the commercial strain GG isolated in Example 1 were inoculated into MRS liquid culture medium at a 3% inoculum volume, cultured at 37°C for 24 hours, and activated for three generations. After centrifugation at 11000 rpm for 10 minutes, the cells were collected and adjusted to a working concentration (about 5.0×10 5CFU / mL). Mouse macrophage RAW264.7 (about 5×10 5 Cells (cells / mL) were added to a 24-well culture plate, with 1 mL per well. After 2 hours of adherence, the culture medium was discarded and 1 mL of culture medium containing bacteria was added to each well. A blank control group was set up, with 1 mL of DMEM medium added. After 24 hours of co-culture, the supernatant of the cells was collected.

[0113] The levels of TNF-α and IL-6 in the cell supernatant were determined by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.

[0114] Table 6. Lactobacillus rhamnosus HOM1213 strain promotes cytokine secretion in mouse macrophages Note: *: compared with the blank control group, p < 0.05; **: compared with the blank control group, p < 0.01

[0115] The results are shown in Table 6. Lactobacillus rhamnosus HOM1213 strain can significantly increase the secretion of TNF-α and IL-6 by RAW264.7 cells. This shows that Lactobacillus rhamnosus HOM1213 strain has potential immunomodulatory ability.

[0116] Example 8 Exploration of in vitro hypoglycemic efficacy and mechanism

[0117] (1) Improved MRS liquid culture medium

[0118] MRS medium (OXOID) was prepared by adding 0.05% L-cysteine ​​hydrochloride to the finished medium, stirring evenly, sterilizing at 121° C. for 20 min, and setting aside.

[0119] (2) Bacterial activation and sample processing

[0120] The commercial strain Lactobacillus rhamnosus GG was selected as a positive control. The Lactobacillus rhamnosus HOM1213 strain isolated in Example 1 and the commercial strain GG were inoculated into fresh modified MRS liquid medium at an inoculum rate of 1% of the total culture medium. The culture was incubated anaerobically at 37°C for 24 hours and activated for three generations. The culture was centrifuged at 11,000 rpm for 10 minutes and washed three times with phosphate buffered saline (PBS). The viable cell count was adjusted to 4 × 10 10 CFU / mL, culture anaerobically for 8 h, centrifuge at 11000 rpm for 10 min, and take the supernatant for later use.

[0121] (3) DPP-4 inhibition and α-glucosidase inhibition assays

[0122] In vitro screening for hypoglycemic activity was performed using a DPP-4 inhibitor screening kit (Abnova #KA1311) and an α-glucosidase inhibitor screening kit (Biovision #K938). The inhibition rate of each sample against these two enzymes was calculated, and the results are shown in Table 7 and Figure 2.

[0123] Table 7. Inhibition rate of DPP-4 and α-glucosidase in each group **: There is a significant difference compared with the Lactobacillus rhamnosus HOM1213 strain group (p<0.01)

[0124] As shown in Table 7 and Figure 2, Lactobacillus rhamnosus HOM1213 has a high inhibition rate against DPP-4 and α-glucosidase. In both indicators, the Lactobacillus rhamnosus HOM1213 group was higher than the Lactobacillus rhamnosus GG group. In summary, the potential of Lactobacillus rhamnosus HOM1213 to regulate blood sugar may be due to its production of higher levels of DPP-4 and α-glucosidase inhibitors.

[0125] Example 9: Lactobacillus rhamnosus HOM1213 stimulates NCI-H716 cells to secrete GLP-1

[0126] (1) Culture of NCI-H716 cells

[0127] NCI-H716 cells (purchased from the Chinese Academy of Sciences Cell Bank) were used in this experiment. NCI-H716 cells were grown in suspension in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (Hyclone) and 1% double antibiotics (penicillin and streptomycin, Hyclone) at 37°C in a 5% CO2 incubator.

[0128] (2) Bacterial activation and sample processing

[0129] The commercial strain Lactobacillus rhamnosus GG was selected as a positive control. The Lactobacillus rhamnosus HOM1213 strain isolated in Example 1 and the commercial strain GG were inoculated into MRS liquid culture medium at a 3% inoculum rate, incubated at 37°C for 24 hours, and activated for three generations. Each bacterial solution was centrifuged at 11000 rpm for 10 minutes and washed three times with Krebs buffer (Sigma). The viable cell count was adjusted to 1×10 10 CFU / mL, set aside.

[0130] (3) GLP-1 endocrine test

[0131] NCI-H716 cells were cultured at 1.5×10 6Cells were seeded at a density of 100 cells / well in a 24-well plate coated with Matrigel (Corning) and incubated in endocrine differentiation medium (DMEM (Gibco)) for 2 days at 37°C in a 5% CO2 incubator for endocrine differentiation experiments. The endocrine differentiation medium consisted of high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum and 1% double-stranded antibody.

[0132] After 2 days, the DMEM medium was replaced with Krebs-Ringer buffer containing 1×10 10 After culturing for 2 hours, the cells were centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. 50 μg / mL of phenylmethylsulfonyl fluoride (Roche) and 10 μg / mL of sitagliptin (Sigma) were added to the supernatant, and GLP-1 concentrations were measured using an ELISA kit (Raybiotech). The results are shown in Table 8.

[0133] Table 8. GLP-1 secretion by NCI-H716 cells stimulated by various strains **: There is a significant difference compared with the Lactobacillus rhamnosus HOM1213 strain group (p<0.01)

[0134] As shown in Table 8, Lactobacillus rhamnosus HOM1213 stimulated NCI-H716 cells to secrete high levels of GLP-1, reaching a concentration of 1781.83±22.22 pg / mL. Compared with the commercial Lactobacillus rhamnosus strain GG, Lactobacillus rhamnosus HOM1213 showed a highly significant difference (p<0.01). These results suggest that Lactobacillus rhamnosus HOM1213 has the potential to lower blood sugar, possibly by stimulating intestinal L cells to produce high levels of GLP-1.

[0135] GLP-1 is an incretin hormone that promotes the secretion of insulin by pancreatic beta cells and inhibits the secretion of glucagon by alpha cells in a glucose concentration-dependent manner. It has a good effect in lowering postprandial blood sugar. When the glucose concentration is lower than normal, it does not stimulate insulin secretion, thereby preventing the occurrence of hypoglycemia.

[0136] Example 10 In vitro antioxidant capacity test

[0137] (1) Bacteria activation

[0138] The rhamnosus Lactobacillus HOM1213 and GG strains isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum volume, cultured at 37°C for 24 h, activated three times, centrifuged at 11000 rpm for 10 min, washed with PBS buffer, resuspended and centrifuged, and repeated three times. The bacterial count was adjusted to 2×10 10 CFU / ml is reserved.

[0139] (2) Detection of antioxidant indicators

[0140] Indicators include total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), hydroxyl radical scavenging (·OH), and DPPH free radical scavenging. The DPPH free radical scavenging test uses a colorimetric method. Its principle is that the free radical scavenger donates an electron to pair with the lone pair of electrons of the DPPH free radical, causing its own purple color to turn yellow and the absorbance at a wavelength of 517nm to decrease. The degree of change is linearly related to the degree of free radical scavenging: that is, the stronger the scavenging ability of the free radical scavenger, the lower the absorbance. Other indicators were measured using kits purchased from Nanjing Jiancheng Company according to the operating instructions. Using 0.05% vitamin C as a positive control, the various antioxidant indicators of bacterial suspensions of Lactobacillus rhamnosus HOM1213 and GG strains were measured. The results are shown in Table 9 and Figure 3.

[0141] Table 9. In vitro antioxidant capacity test Note: ND: not detected; **: extremely significant difference compared with HOM1213 group (p<0.01)

[0142] As shown in Table 9 and Figure 3, Lactobacillus rhamnosus HOM1213 has high T-AOC, SOD, and GSH-Px activities, and high hydroxyl and DPPH free radical scavenging rates, with particular strength in SOD enzyme activity, hydroxyl radical scavenging, and DPPH free radical scavenging. This suggests that Lactobacillus rhamnosus HOM1213 has excellent antioxidant activity.

[0143] Example 11 Improving the Antioxidant Level and Delaying Aging in Mice

[0144] (1) Mouse origin

[0145] Thirty healthy SPF female Kunming mice weighing 22-26 g were bred by Beijing Huafukang Biotechnology Co., Ltd. The experimental animals were housed in the SPF animal room of the Health Food Functional Testing Center of the College of Applied Arts and Sciences, Beijing Union University, and maintained on feed produced by Beijing Huafukang Biotechnology Co., Ltd.

[0146] (2) D-galactose oxidative damage model

[0147] Thirty mice were divided into three groups of 10 mice each. Two groups of mice were treated with D-galactose (300 mg / kg body weight) diluted in saline by subcutaneous injection into the nape of the neck for six consecutive weeks. Blood samples were collected for malondialdehyde (MDA) levels. Mice were randomly divided into two groups based on MDA levels: a model control group and a HOM1213 group. The other group served as a blank control group, which received an equal volume of saline injected subcutaneously into the nape of the neck daily during modeling.

[0148] (3) Dosage design

[0149] After successful modeling, the samples were orally administered once a day, with a gavage volume of 10 mL / kg BW. The Lactobacillus rhamnosus HOM1213 group was given 1×10 9 The blank and model control groups were administered with sterile water instead of sample, and the daily gavage volume was the same as that of the sample group. All groups were given a maintenance diet. Various indices were measured after 45 days of continuous gavage. Simultaneously with the sample administration, the Lactobacillus rhamnosus HOM1213 and model control groups were continuously injected with the same dose of D-galactose, while the blank control group was injected with an equal volume of normal saline.

[0150] The specific experimental methods were based on the Technical Specifications for Inspection and Evaluation of Health Food (2012 edition), and the experimental results were statistically analyzed using SPSS software.

[0151] (4) Index determination

[0152] All the test kits required for the experiment were purchased from Nanjing Jiancheng Bioengineering Institute.

[0153] A. Lipid Peroxide-Determination of Malondialdehyde (MDA) Content in Serum

[0154] 0.5 mL of blood was collected from the medial canthal venous plexus of the mouse eye, and the blood was centrifuged at 3000 r / min for 10 min. The malondialdehyde content in the serum was determined using a kit. The results are shown in Table 12.

[0155] B. Determination of protein carbonyl content in liver tissue protein oxidation products

[0156] Take a certain amount of liver tissue, rinse it in ice-cold physiological saline to remove blood stains on the surface, and wipe it dry with filter paper. Use a protein carbonyl kit (A087-1-2, Nanjing Jiancheng Bioengineering) to determine the protein carbonyl content. Specific method: Add reagent one at a weight-to-volume ratio of 1:9, mechanically homogenize under ice bath conditions, centrifuge the homogenate at 3000r / min for 10 minutes, and take the supernatant, i.e., 10% liver tissue homogenate, for measurement. Take 450μL of 10% liver tissue homogenate, add 50μL of reagent two, leave it at room temperature for 10 minutes, centrifuge it at 11000r / min for 10 minutes, and take the supernatant for measurement. The results are shown in Table 13.

[0157] C. Antioxidant enzymes

[0158] 0.5 mL of blood was collected from the medial canthal venous plexus of aged mice and centrifuged at 3000 r / min for 10 min. The superoxide dismutase (SOD) activity in the serum was determined using a superoxide dismutase activity kit. The results are shown in Table 14.

[0159] 10 μL of blood was collected from the medial canthal venous plexus of aged mice and distilled water was added to 1 mL to prepare a 1:99 hemolyzed solution. Glutathione peroxidase (GSH-Px) activity was measured using a glutathione peroxidase kit. The results are shown in Table 15.

[0160] D. Determination of antioxidant substance - reduced glutathione (GSH) content

[0161] A certain amount of liver was removed, rinsed with saline, wiped dry, weighed, and minced. The mixture was homogenized in a glass homogenizer with cold saline to prepare a 10% tissue homogenate. The mixture was centrifuged at 2500 rpm for 10 minutes. The supernatant was collected and GSH content was determined using a GSH kit. The results are shown in Table 16.

[0162] Table 10. Serum malondialdehyde content of mice after modeling **: P<0.01 compared with the model control group and the blank control group

[0163] As shown in Table 10, 6 weeks after the mice were subcutaneously injected with D-galactose to establish the model, the model group was randomly divided into a model control group and a sample group HOM1213 group according to the serum malondialdehyde content of the mice. There was no significant difference between the HOM1213 group and the model control group (p>0.05), indicating that the serum malondialdehyde content of the mice was relatively balanced between the model groups; compared with the blank control group, the serum malondialdehyde content of the model control group was increased, with a significant difference (p<0.01), indicating that the D-galactose oxidative damage model was successfully established.

[0164] Table 11. Changes in mouse body weight before and after sample administration

[0165] As shown in Table 11, there was no significant difference in the body weight of the mice before and 45 days after oral administration of the sample between the model control group and the blank control group (p>0.05), and there was no significant difference between the sample group and the model control group (p>0.05). In other words, the sample had no adverse effect on the body weight of the mice.

[0166] Table 12. Effects of Lactobacillus rhamnosus HOM1213 strain group on malondialdehyde in mouse serum **: P<0.01 compared with the sample group and the model control group

[0167] As shown in Table 12, 45 days after oral administration of the mouse samples, the serum malondialdehyde level in the Lactobacillus rhamnosus HOM1213 strain group was significantly lower than that in the model control group (p < 0.01). This shows that the Lactobacillus rhamnosus HOM1213 strain group can significantly reduce the serum malondialdehyde level in mice with D-galactose oxidative damage. Malondialdehyde is the main product of polyunsaturated fatty acid peroxidation. Malondialdehyde content has been widely used to measure the level of oxidative damage in the body.

[0168] Table 13. Effects of Lactobacillus rhamnosus HOM1213 strain group on protein carbonyl content in mouse liver tissue

[0169] Protein carbonylation is a type of protein oxidative damage, occurring when amino acid side chains are attacked by oxygen free radicals and converted into carbonyl products. Carbonyl modification can alter protein structure, leading to cellular and tissue pathology and contributing to aging and apoptosis. As shown in Table 13, after 45 days of oral administration to mice, the carbonyl content of liver proteins in the Lactobacillus rhamnosus HOM1213 group was reduced compared to the model control group, but the differences were not statistically significant (p>0.05).

[0170] Table 14. Effects of the Lactobacillus rhamnosus HOM1213 strain group on the activity of superoxide dismutase (SOD) in mouse serum *: P<0.05 compared with the sample group and the model control group

[0171] As shown in Table 14, after 45 days of oral administration of the sample to mice, the serum SOD activity in the Lactobacillus rhamnosus HOM1213 group was significantly higher than that in the model control group (p < 0.05). Lactobacillus rhamnosus HOM1213 can increase the body's SOD level to resist oxidative stress.

[0172] Table 15. Effects of Lactobacillus rhamnosus HOM1213 strain group on glutathione peroxidase activity in whole blood of mice **: Comparison between sample group and model control group p<0.01

[0173] As can be seen from Table 15, 45 days after oral administration of the mouse sample, the whole blood glutathione peroxidase activity in the Lactobacillus rhamnosus HOM1213 strain group increased, with a very significant difference (p < 0.01), that is, the Lactobacillus rhamnosus HOM1213 strain group can increase the whole blood glutathione peroxidase activity of D-galactose oxidative damage model mice to resist the level of oxidative stress.

[0174] Table 16. Effects of Lactobacillus rhamnosus HOM1213 strain group on reduced glutathione content in mouse liver tissue *: Comparison between sample group and model control group p<0.05

[0175] As shown in Table 16, 45 days after oral administration of the samples to mice, the reduced glutathione content in the liver tissue of the Lactobacillus rhamnosus HOM1213 group was significantly increased compared to the model control group (p < 0.05). This indicates that the Lactobacillus rhamnosus HOM1213 strain can increase the reduced glutathione content in the liver tissue of mice with D-galactose oxidative damage model.

[0176] According to the antioxidant animal testing method in the Health Food Functional Testing and Evaluation Methods (2022 Edition), if three of the four indicators—lipid oxidation products, protein oxidation products, antioxidant enzymes, and antioxidant substances—are positive, the test sample can be considered positive for the antioxidant animal testing results. This indicates that the Lactobacillus rhamnosus HOM1213 strain has antioxidant and anti-aging properties.

[0177] Fig. 4 shows the influence of the rhamnosus lactobacillus HOM1213 strain on the antioxidant and anti-aging indexes in the oxidative damage model mice. After 45 days of oral administration of the mouse sample, compared with the model control group, the serum superoxide dismutase (SOD) activity of the rhamnosus lactobacillus strain HOM1213 group was improved, with significant difference (p < 0.05), the whole blood glutathione peroxidase (GSH-Px) activity was increased, with very significant difference (p < 0.01), the liver tissue reduced glutathione (GSH) content was increased, with significant difference (p < 0.05), the serum malondialdehyde (MDA) content was reduced, with very significant difference (p < 0.01), the liver tissue protein carbonyl content was reduced, but the difference was not significant (p> 0.05), thus, the HOM1213 group sample can significantly increase the serum SOD and GSH-Px activity, GSH level, and significantly reduce the MDA content in the D-galactose oxidative damage model mice.

[0178] The entire process of the present invention is shown in the process flow chart of FIG5 .

[0179] Example 12 Fermented milk test

[0180] Weigh 12 g skim milk powder, 2 g glucose, and 2 g yeast powder, add double-distilled water to 100 mL, stir evenly, homogenize with a high-pressure homogenizer (60°C, 22 MPa), sterilize at 95°C for 10 min, and cool to 37°C for use.

[0181] The seed liquid of the Lactobacillus rhamnosus HOM1213 strain isolated in Example 1 was inoculated into MRS liquid culture medium and cultured at 37° C. for 18 h. The culture was passaged twice according to this method to obtain a high-activity bacterial liquid for later use.

[0182] The bacterial solution was eluted with 0.9% saline and resuspended at 1×10 7 CFU / mL inoculation amount was inoculated into milk, mixed evenly, and kept at 37°C for 16 hours to obtain a fermented milk product. The content of active Lactobacillus rhamnosus in the fermented milk was higher than 6.0×10 8 CFU / mL, pH can reach 4.50, with a mellow taste, sweet and sour.

[0183] Example 13 Fermented oat milk test

[0184] Weigh 120 g of enzymatically hydrolyzed oatmeal powder and 20 g of yeast powder, add double-distilled water to 1000 mL, homogenize at 200 bar, sterilize at 90°C for 10 min, and cool to 37°C for later use.

[0185] The seed liquid of the Lactobacillus rhamnosus HOM1213 strain isolated in Example 1 was inoculated into MRS liquid culture medium, cultured at 37° C. for 18 h, subcultured twice to obtain a high-concentration bacterial liquid, and stored at low temperature for later use.

[0186] The bacterial solution was eluted with 0.9% saline and resuspended at 1×10 7 CFU / mL inoculum was inoculated into oat slurry, mixed evenly, and kept at 37°C for 6 hours to obtain fermented oat milk. The viable number of Lactobacillus rhamnosus in the fermented milk was higher than 5.0×10 8 CFU / mL, the pH can be reduced to 4.35, and the taste is rich.

[0187] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus strain, the deposit number of which is CGMCC No.25682.

2. The Lactobacillus rhamnosus strain according to claim 1, wherein The Lactobacillus rhamnosus strain comprises a 16S rRNA gene sequence represented by SEQ ID NO:

1.

3. A fermentation product obtained by fermenting the Lactobacillus rhamnosus strain according to claim 1 or 2.

4. The fermentation product according to claim 3, wherein The fermented product is a fermented dairy product; optionally, the content of active Lactobacillus rhamnosus in the fermented dairy product is 6.0×10 8 CFU / mL and above.

5. The fermentation product according to claim 3, wherein The fermented product is a fermented oatmeal dairy product; optionally, the viable count of Lactobacillus rhamnosus in the fermented oatmeal dairy product is 5.0×10 8 CFU / mL and above. A live bacterial preparation comprising the Lactobacillus rhamnosus strain according to claim 1 .

7. The live bacteria preparation according to claim 6, wherein The live bacteria preparation contains 3.0×10 11 CFU / g and above live bacteria.

8. A food or health product comprising the live bacteria preparation according to claim 6.

9. The food or health product according to claim 8, wherein: The food is selected from one or more of milk powder, solid beverage, fermented dairy product, milk-containing beverage and cheese.

10. The food or health product according to claim 9, wherein: The fermented dairy product is selected from the group consisting of fermented cow's dairy products and fermented oat dairy products.

11. Use of the Lactobacillus rhamnosus strain according to claim 1 in the preparation of drugs for scavenging free radicals, coping with oxidative stress, anti-oxidation and anti-aging.

12. The use according to claim 11, wherein The Lactobacillus rhamnosus strain is used to increase the levels of superoxide dismutase, glutathione peroxidase and glutathione, and reduce the level of malondialdehyde.

13. The use according to claim 12, wherein The Lactobacillus rhamnosus strain is used to remove excessive free radicals and improve the removal rate of hydroxyl free radicals and DPPH free radicals.

14. Use of the Lactobacillus rhamnosus strain according to claim 1 in the preparation of a drug for producing DPP-4 and α-glucosidase inhibitors and stimulating NCI-H716 cells to produce GLP-1.

15. The use according to claim 14, wherein The Lactobacillus rhamnosus strain is used to assist in lowering blood sugar.

16. Use of the Lactobacillus rhamnosus strain according to claim 1 in the preparation of a medicament for regulating immunity.

17. The use according to claim 16, wherein The Lactobacillus rhamnosus strain is used to increase the level of cytokines TNF-α and / or IL-6.

18. Use of the Lactobacillus rhamnosus strain according to claim 1 for increasing the content of lactic acid or short-chain fatty acids.

19. The use according to claim 18, wherein The short-chain fatty acid is acetic acid.

20. The use according to claim 18, wherein The Lactobacillus rhamnosus strain is used to maintain the balance of intestinal flora.

21. A method for preparing the live bacteria preparation according to claim 6 or 7, comprising the following steps: Expanding and culturing the Lactobacillus rhamnosus strain according to claim 6 or 7 in an optimized liquid culture medium; Collecting bacteria; The protective agent is added and resuspended, vacuum freeze-dried, and crushed to obtain an active bacterial agent.

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