Compositions containing Lactobacillus rhamnosus strains and their uses

Abstract

This invention relates to the field of microbial technology, specifically to a composition comprising a strain of *Lactobacillus rhamnosus* and excipients, and its uses. The *Lactobacillus rhamnosus* strain has the accession number CGMCC No. 25682. The composition comprising *Lactobacillus rhamnosus* of this invention can regulate immunity, increasing the levels of cytokines TNF-α and IL-6; it possesses good antioxidant and anti-aging capabilities, increasing the levels of superoxide dismutase, glutathione peroxidase, and glutathione, decreasing malondialdehyde levels, and improving the scavenging rate of hydroxyl radicals and DPPH radicals; it can produce DPP-4 and α-glucosidase inhibitors and stimulate NCI-H716 cells to produce GLP-1, demonstrating potential for blood glucose regulation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a composition comprising a strain of Lactobacillus rhamnosus and its use. Background Technology

[0002] A weakened or deficient immune system often leads to a variety of localized or systemic infections. Mild cases may result in the common cold, while more serious infections like hepatitis B and C viruses can damage the liver and lead to liver cancer. The most severe consequence is HIV infection, which deprives the body of its ability to fight off pathogens. A weakened immune system can also cause pharyngitis, gastritis, enteritis, pneumonia, bronchitis, rhinitis, otitis media, hepatitis, mastitis, cancer, and skin infections.

[0003] The aging population is a serious global issue, and slowing down aging has become a major concern. With the development of science and technology, there is some consensus on the mechanisms of aging. The most studied theory is the free radical-oxidative stress theory, which posits that under normal conditions, the body experiences a surge in free radicals (superoxide anion radicals (O2)). 2- The body maintains a balance between free radicals (such as hydroxyl radicals (OH)) and the antioxidant system. Once the body is stimulated, the content of free radicals increases or the ability to scavenge free radicals decreases, the level of antioxidant enzymes decreases, and the body's cellular components are damaged, leading to lipid peroxidation, protein damage, and oxidative stress, eventually resulting in apoptosis and aging.

[0004] Some probiotics are known to regulate immunity, provide the body with antioxidant enzymes and antioxidants, eliminate excess free radical buildup, actively cope with oxidative stress, and delay aging.

[0005] Currently, research on Lactobacillus rhamnosus strains in enhancing immunity, as well as their antioxidant and anti-aging effects, is insufficient. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention provides a composition containing Lactobacillus rhamnosus strain, which exhibits excellent properties in enhancing immunity, as well as anti-oxidation and anti-aging effects.

[0007] On one hand, the present invention provides a composition comprising a strain of Lactobacillus rhamnosus and excipients, wherein the Lactobacillus rhamnosus strain has the accession number CGMCC No. 25682.

[0008] In one example, the composition may be a pharmaceutical composition, and the excipients may be pharmaceutically acceptable excipients, stabilizers, or diluents, etc., but are not limited thereto.

[0009] On the other hand, the present invention provides the use of the composition described above in the preparation of a medicament for regulating immunity.

[0010] In one example, the composition is used to increase the levels of cytokines TNF-α and / or IL-6.

[0011] The present invention also provides the use of the composition described above in the preparation of medicaments for scavenging free radicals, responding to oxidative stress, antioxidation, and anti-aging.

[0012] In one example, the composition is used to increase the levels of superoxide dismutase, glutathione peroxidase, and glutathione, and / or decrease the level of malondialdehyde.

[0013] In one example, the composition is used to improve the scavenging rate of hydroxyl radicals and DPPH radicals.

[0014] The present invention also provides the use of the composition described above in the preparation of a medicament for producing DPP-4 and α-glucosidase inhibitors, and / or stimulating NCI-H716 cells to produce GLP-1.

[0015] In one example, the composition is used to lower blood sugar.

[0016] The present invention also provides the use of the composition described above for increasing the content of lactic acid or short-chain fatty acids.

[0017] Preferably, the short-chain fatty acid is acetic acid.

[0018] In one example, the composition is used to maintain the balance of the gut microbiota.

[0019] Beneficial effects

[0020] The composition of the present invention containing *Lactobacillus rhamnosus* strain can regulate immunity and increase the levels of cytokines TNF-α and IL-6; it has good 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 radicals; it can produce DPP-4 and α-glucosidase inhibitors and stimulate NCI-H716 cells to produce GLP-1, and has the potential to regulate blood sugar. Attached Figure Description

[0021] Figure 1 The diagram shows the RAPD clustering analysis of Lactobacillus rhamnosus strain HOM1213 constructed based on the UPGMA method and some commercial Lactobacillus rhamnosus strains.

[0022] Figure 2 The inhibition rate of the Lactobacillus rhamnosus strain HOM1213 of the present invention against DPP-4 and α-glucosidase is shown.

[0023] Figure 3 The in vitro antioxidant capacity of the Lactobacillus rhamnosus strain HOM1213 of the present invention is demonstrated.

[0024] Figure 4 The effects of the Lactobacillus rhamnosus strain HOM1213 of the present invention on in vivo antioxidant and anti-aging indicators in mice with oxidative damage model are shown.

[0025] Figure 5 A process flow diagram of the present invention is shown.

[0026] Microbial Preservation Instructions

[0027] The Lactobacillus rhamnosus HOM1213 strain of the present invention was deposited on September 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number CGMCC No. 25682.

[0028] The Lactobacillus rhamnosus HOM1213 strain of the present invention was submitted to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0029] The identification conclusions are as follows: Under the conditions of this laboratory, based on a comprehensive analysis of experimental data including cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and pheS gene sequence of the submitted bacterial strain, and referring to relevant research papers in *Bergey's Manual of Systematic Bacteriology* and the *International Journal of Systematic and Evolutionary Microbiology*, the identification result of the submitted bacterial strain (strain number: HOM1213) is: *Lacticaseibacillus rhamnosus*. Synonym: *Lactobacillus rhamnosus*.

[0030] The cell morphology of this strain is rod-shaped; its physiological and biochemical characteristics are Gram-positive, catalase-negative (-), and 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 Implementation

[0031] This invention discloses the strains, characteristics, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0032] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the technical solution of the present invention will be further described below in conjunction with specific embodiments, but this is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are implemented according to conventional methods and conditions in the art.

[0033] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods.

[0034] Unless otherwise specified, all reagents and materials used in this invention are prepared using conventional methods or obtained commercially.

[0035] As identified above, Lactobacillus rhamnosus and Lactaseibacillus rhamnosus are synonyms of the same species. Therefore, in this invention, Lactobacillus rhamnosus and Lactaseibacillus rhamnosus can be used interchangeably.

[0036] The Lactobacillus rhamnosus strain HOM1213 used in this invention was isolated from the feces of healthy infants. Its physicochemical properties include: an optimal growth temperature of 37°C; abundant metabolites; acid and bile salt resistance; antibacterial properties; immunomodulatory effects; blood sugar regulation; and antioxidant and anti-aging activities.

[0037] To further understand the present invention, the following detailed description of the *Lactobacillus rhamnosus* and its uses provided by the present invention is provided in conjunction with embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0038] Example 1: Isolation and identification of Lactobacillus rhamnosus strain HOM1213

[0039] (1) Preparation of culture medium

[0040] MRS liquid culture medium:

[0041] Mix 1 L of MRS medium (OXOID, CM1163) and double-distilled water thoroughly. Sterilize at 121℃ for 20 min and set aside.

[0042] Modified MRS solid culture medium:

[0043] MRS medium (OXOID, CM1163), 0.05 g bromocresol green (Shanghai Sangon Biotech), 1 L double-distilled water, mixed thoroughly. Adjust pH to 5.5, sterilize at 121℃ for 20 min, and set aside.

[0044] (2) Isolation of strains

[0045] Weigh 1g of healthy infant feces and mix thoroughly with 9mL of 0.9% physiological saline. Perform serial dilutions using the 10-fold dilution method, selecting two suitable dilutions. Spread 100μL of each sample onto a modified MRS solid plate and incubate anaerobically at 37℃ for 72h. Select single colonies with smooth surfaces, milky white or milky yellow coloration around the edges, streak, and purify. Observe colony morphology under a microscope. Select single colonies for pure culture in MRS liquid medium, preserve the culture with glycerol, and label it HOM1213.

[0046] (3) Identification of strains

[0047] The selected bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 24 h. Total bacterial DNA was extracted from the culture, and 16S rRNA gene amplification was performed using universal primers 27F and 1492R. PCR amplification and agarose gel electrophoresis were then performed, followed by gel extraction and sequencing (Shanghai Sangon Biotech). The strain HOM1213 was then identified as *Lactobacillus rhamnosus* by comparison with the NCBI database using BLAST. The 16S rRNA gene sequence of strain HOM1213 is shown in SEQ ID NO:1.

[0048] The sequence of the 16S rRNA gene of strain HOM1213 was determined as follows:

[0049] TGCAGTCGAACGAGTTCTGATTATTGAAAGGTGCTTGCATCTTGATT

[0050] TAATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCC

[0051] CTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCC

[0052] AAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGG

[0053] ATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG

[0054] CAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGA

[0055] GACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAA

[0056] TGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTC

[0057] GGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGT

[0058] TGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCA

[0059] GCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCG

[0060] TAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCT

[0061] TAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGG

[0062] ACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAA

[0063] CACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCG

[0064] AAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAA

[0065] ACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCT

[0066] AACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTC

[0067] AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT

[0068] CGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCT

[0069] GAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGT

[0070] TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG

[0071] CAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACT

[0072] GCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCC

[0073] CCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTG

[0074] CGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGAC

[0075] TGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGG

[0076] ATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG

[0077] TCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTT

[0078] AGGGAGCGAGCCGTCT

[0079] (4) Identification of strains by random amplified polymorphic DNA (RAPD)

[0080] 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.

[0081] DNA was extracted from the preserved bacterial strain. Using the strain DNA as a template, five primers were used to amplify polymorphic DNA fragments by PCR using OPA-02: TGC CGA GCTG; OPA-18: AGG TGA CCG T; OPL-07: AGG CGG GAA C; OPL-16: AGG TTG CAG G; and OPM-05: GGGAAC GTG T. The resulting fragments exhibited different DNA differences after gel electrophoresis and were analyzed using clustering analysis software. The results are shown below. Figure 1 .like Figure 1 As shown, the RAPD clustering analysis diagram of Lactobacillus rhamnosus strain HOM1213 is different from that of commercial Lactobacillus rhamnosus strains, and is unique.

[0082] Table 1

[0083]

[0084]

[0085] Example 2: Preparation process of active bacterial powder

[0086] (1) Culture of strains

[0087] The strain was inoculated into MRS liquid medium and incubated at 37°C for 24 hours, activating it for two generations. When inoculated into MRS medium and incubated at 37°C, the viable count reached 1*10⁻⁶. 10 CFU / mL or higher.

[0088] (2) Freeze drying

[0089] The fermentation broth of *Lactobacillus rhamnosus* strain HOM1213 was centrifuged to collect bacterial sludge. The sludge was washed with 0.9% sterile saline solution, mixed with the aforementioned preservative (100 g / L skim milk powder), and freeze-dried in a freeze dryer. The bacterial cake was then pulverized using a fine grinder to obtain the freeze-dried bacterial powder. The viable count of the freeze-dried bacterial powder was higher than 3.0 × 10⁻⁶. 11CFU / g.

[0090] Example 3 Gastrointestinal transit capacity test

[0091] (1) Isolation and activation of strains

[0092] The commercial strain *Lactobacillus rhamnosus* GG was selected as a control and purchased from ATCC (American Type Culture Collection). Each strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours, activating twice before use.

[0093] (2) Preparation of artificial gastric juice

[0094] 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 for later use.

[0095] (3) Preparation of artificial intestinal fluid

[0096] Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of water, and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution. Separately, dissolve 10g of pancreatic enzyme and 3g of ox bile (BD Difco) in an appropriate amount of water. Mix the two solutions, dilute with water to 1000mL, and filter under sterile conditions using a 0.22μm sterile filter membrane.

[0097] (4) Evaluation of the survival ability of the strain in simulated artificial gastrointestinal fluid

[0098] Take 1 mL of bacterial culture from each strain, centrifuge, collect the bacterial cells, add to 10 mL of artificial gastric fluid, mix well, and immediately count the viable cells, recorded as T0. Incubate at 37℃ for 3 hours, then count the viable cells again, recorded as T1. Centrifuge the sample, add 10 mL of artificial intestinal fluid, mix well, and incubate at 37℃ for 3 hours for viable cell counting, recorded as T2. The survival rate is calculated using the following formula:

[0099]

[0100]

[0101] Wherein, T0 is the number of viable bacteria (CFU / mL) of the test strain before treatment (0h); T1 is the number of viable bacteria of the test strain after treatment with artificial gastric fluid for 3h; and T2 is the number of viable bacteria of the test strain after treatment with artificial gastric fluid and artificial intestinal fluid for 3h.

[0102] As shown in Table 2, after 3 hours of treatment with simulated gastric fluid, the survival rate of Lactobacillus rhamnosus HOM1213 reached over 98%. After further treatment with simulated intestinal fluid for another 3 hours, the survival rate was still over 80%, indicating that Lactobacillus rhamnosus HOM1213 has a high survival rate in the intestine.

[0103] Table 2. Survival rate of strains in simulated gastrointestinal fluid

[0104]

[0105] Example 4 Antibiotic Sensitivity

[0106] The drug susceptibility test was performed according to the KB agar method recommended by the National Committee on Clinical Standards of Medicine (NCCLS), as follows:

[0107] (1) Commercial strain Lactobacillus rhamnosus GG was selected as the control strain. HOM1213 and GG were inoculated into MRS liquid medium and cultured at 37°C for 24 hours. After three generations of continuous activation, they were ready for use.

[0108] (2) Take 1 mL of bacterial culture (1.5 × 10⁻⁶) 8 15 mL of MRS solid medium (CFU / mL) was placed in a sterile petri dish, mixed thoroughly, and after the plate solidified, standard antibiotic susceptibility test strips were placed on it. The plate was then incubated at 37°C for 48 hours, and the diameter of the inhibition zone was measured and recorded.

[0109] According to the CLSI criteria, the results are shown in Table 3. Using *Lactobacillus rhamnosus* GG as a commercial strain control, the results showed that HOM1213 was resistant to vancomycin, gentamicin, kanamycin, and ampicillin, moderately resistant to streptomycin, and sensitive to the remaining four antibiotics. *Lactobacillus* exhibits natural resistance to vancomycin.

[0110] Table 3. Results of susceptibility testing of Lactobacillus rhamnosus HOM1213 to nine antibiotics

[0111]

[0112] S (susceptible) indicates sensitivity; I (intermediate) indicates moderate sensitivity; R (resistance) indicates drug resistance.

[0113] Example 5: Test on the ability to inhibit common pathogenic bacteria

[0114] (1) Activation of pathogenic bacteria

[0115] 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). The pathogenic strains were purchased from ATCC (American Type Culture Collection).

[0116] The pathogenic strains were inoculated into nutrient agar medium and cultured at 37°C with shaking at 200 rpm for 12 h. The indicator strains were then prepared with fresh medium to an OD of 0.1 for later use.

[0117] (2) Activation of strains

[0118] In this experiment, the commercial strain *Lactobacillus rhamnosus* GG was selected as the control strain. Both the commercial strain GG and the HOM1213 strain isolated in the previous examples were inoculated into MRS liquid medium at a 3% inoculum size and cultured statically at 37°C for 24 hours. After two activation cycles, the fermentation broth was obtained. The broth was centrifuged at 11000 rpm for 10 min, and the supernatant was used for an antibacterial test. The MRS liquid medium served as a negative control.

[0119] (3) Plate preparation

[0120] Pour the sterilized nutrient agar culture into a petri dish, add 100 μL of indicator bacterial solution to the culture medium, mix well, and let it stand to solidify.

[0121] (4) Antibacterial test

[0122] Using sterile forceps, gently place the Oxford cups onto the plate, maintaining a certain distance between the wells. Add 150 μL of fermentation supernatant to each well, and after diffusion at 4°C for 12 hours, incubate at 37°C for 18 hours. Observe and measure the diameter of the inhibition zone. The results are shown in Table 4.

[0123] Table 4. Inhibitory effects of each group on pathogenic bacteria

[0124]

[0125] Note: "–" indicates no antibacterial activity, <11mm; "+" indicates 11mm ≤ inhibition zone <16mm; "++" indicates 16mm ≤ inhibition zone <23mm; "+++" indicates ≥23mm.

[0126] As shown in Table 4, HOM1213 exhibits inhibitory effects against all five pathogenic bacteria. Compared to the commercial strain GG, HOM1213 demonstrates stronger antibacterial activity, particularly in its inhibition of Salmonella and Listeria monocytogenes.

[0127] Example 6: Lactic acid and short-chain fatty acid content

[0128] (1) Preparation of strain supernatant

[0129] The commercial strain *Lactobacillus rhamnosus* GG was selected as the control strain. The strain HOM1213 isolated in Example 1 and the commercial strain GG were inoculated at 3% each into MRS liquid medium and cultured at 37°C for 24 hours. After three generations of activation, the supernatant was collected after centrifugation at 11,000 rpm and 4°C for later use.

[0130] (2) Determination of lactic acid, acetic acid and formic acid content

[0131] Take 4 mL of supernatant, add 2 drops of phenolphthalein, adjust the color with KOH solution until a slight color change occurs, and make up to 5 mL with deionized water. Centrifuge at 11,000 rpm and 4 °C for 10 minutes. The sample is ready for use. Take samples of different concentrations of lactic acid, acetic acid, and formic acid into different 10 mL headspace vials, add 2 mL of phosphate buffer solution, 2 mL of derivatization reagent, and 1 mL of acetone, seal the vials, and react in a 100 °C water bath for 40 min. After cooling to room temperature, take the derivatized sample into a 2 mL centrifuge tube, add an equal volume of isooctane for vortex extraction, take the isooctane layer, and perform gas chromatography (GC) analysis. The results are shown in Table 5.

[0132] Table 5. Determination of lactic acid, acetic acid and formic acid content

[0133]

[0134] **: p<0.01 compared with the MRS control group

[0135] As shown in Table 5, strains HOM1213 and GG both 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.

[0136] Example 7 In vitro cytokine secretion assay

[0137] The commercial strain *Lactobacillus rhamnosus* GG was selected as the control strain. *Lactobacillus rhamnosus* strain HOM1213 isolated in Example 1 and the commercial strain GG were each inoculated at a 3% inoculum into MRS liquid medium and fermented at 37°C for 24 h, with three consecutive generations of activation. After centrifugation at 11000 rpm for 10 min, the bacterial cells were collected and adjusted to the working concentration (approximately 5.0 × 10⁻⁶) using antibiotic-free DMEM complete medium.5 CFU / mL). Mouse macrophages RAW264.7 (approximately 5 × 10⁻⁶ CFU / mL) were used. 5 Add 1 mL of cells / mL to each well of a 24-well culture plate. After 2 hours of adhesion, discard the culture medium and add 1 mL of bacterial culture medium to each well. A blank control group is included, with 1 mL of DMEM culture medium added. After co-culturing for 24 hours, collect the supernatant.

[0138] The levels of TNF-α and IL-6 in the cell supernatant were determined using enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.

[0139] Table 6. HOM1213 promotes the secretion of cytokines by mouse macrophages

[0140]

[0141] Note: *: p < 0.05 compared with the blank control group; **: p < 0.01 compared with the blank control group.

[0142] The results are shown in Table 6. Lactobacillus rhamnosus HOM1213 significantly increased the secretion of TNF-α and IL-6 by RAW264.7 cells. This indicates that Lactobacillus rhamnosus possesses potential immunomodulatory capabilities.

[0143] Example 8: Exploration of in vitro hypoglycemic efficacy and mechanism

[0144] (1) Improved MRS liquid culture medium

[0145] MRS medium (OXOID) is prepared by adding 0.05% L-cysteine ​​hydrochloride to the pre-made medium, stirring thoroughly, and sterilizing at 121°C for 20 minutes.

[0146] (2) Activation of bacterial strains and sample processing

[0147] The commercial strain *Rhamnosus casei* GG was selected as a positive control. The HOM1213 strain isolated in Example 1 and the commercial strain GG were inoculated into fresh modified MRS liquid medium at an inoculum of 1% of the total culture medium volume. The cultures were then anaerobically cultured at 37°C for 24 h, activating for three generations. The bacterial suspensions were centrifuged at 11,000 rpm for 10 min and washed three times with phosphate-buffered saline (PBS). The viable count was adjusted to 4 × 10⁻⁶. 10 CFU / mL, anaerobic culture for 8 h, centrifuge at 11000 rpm for 10 min, and collect the supernatant for later use.

[0148] (3) Assay for DPP-4 inhibition and α-glucosidase inhibition

[0149] The hypoglycemic function was screened in vitro using a DPP-4 inhibitor screening kit (Abnova#KA1311) and an α-glucosidase inhibitor screening kit (Biovision#K938). The inhibition rates of each sample against these two enzymes were calculated, and the results are shown in Table 7. Figure 2 .

[0150] Table 7. Inhibition rates of DPP-4 and α-glucosidase in each group

[0151]

[0152] **: There was a significant difference compared with the HOM1213 group (p<0.01)

[0153] From Table 7 and Figure 2 It is evident that strain HOM1213 exhibits high inhibition rates against DPP-4 and α-glucosidase. In both of these indicators, the HOM1213 group showed higher rates than the GG group. In conclusion, strain HOM1213 possesses the potential to regulate blood glucose, which may stem from the production of high levels of DPP-4 and α-glucosidase inhibitors.

[0154] Example 9: HOM1213 strain stimulates NCI-H716 cells to secrete GLP-1

[0155] (1) Culture of NCI-H716 cells

[0156] This experiment used NCI-H716 cells (purchased from the Cell Bank of the Chinese Academy of Sciences). NCI-H716 cells were grown in suspension in an incubator at 37°C and 5% CO2 in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (Hyclone) and 1% penicillin and streptomycin (Hyclone).

[0157] (2) Activation of bacterial strains and sample processing

[0158] Commercial strain *Lactobacillus rhamnosus* GG was selected as a positive control. *Lactobacillus rhamnosus* HOM1213 isolated in Example 1 and commercial strain GG were inoculated at 3% in MRS liquid medium and cultured statically at 37°C for 24 h, activating for 3 generations. Each bacterial culture was centrifuged at 11000 rpm for 10 min and washed three times with Krebs buffer (Sigma). The viable count was adjusted to 1 × 10⁻⁶. 10 CFU / mL, for later use.

[0159] (3) GLP-1 endocrine assay

[0160] NCI-H716 cells were fed at a concentration of 1.5 × 10⁻⁶. 6Seeds were planted at a density of 10 cells / well in 24-well Corning plates coated with matrix gel, and then added to endocrine differentiation medium. The plates were incubated at 37°C and 5% CO2 for 2 days for endocrine differentiation experiments. The endocrine differentiation medium was DMEM (Gibco) medium containing 10% fetal bovine serum and 1% antibiotics, and high in glucose.

[0161] Two days later, the DMEM medium was replaced with Krebs-Ringer buffer, which contained 1×10⁻⁶ kJ / L. 10 The probiotic strain with CFU / mL was cultured for 2 hours, then centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. 50 μg / mL of benzyl sulfonyl fluoride (Roche) and 10 μg / mL of sitagliptin (Sigma) were added to the supernatant, and the concentration of GLP-1 was detected using an ELISA kit (Raybiotech). The results are shown in Table 8.

[0162] Table 8. GLP-1 secretion yield of NCI-H716 cells stimulated by various bacterial strains

[0163]

[0164] **: There was a significant difference compared with the HOM1213 group (p<0.01)

[0165] As shown in Table 8, strain 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 strain GG of *Lactobacillus rhamnosus*, strain HOM1213 showed a highly significant difference (p < 0.01). These results indicate that *Lactobacillus rhamnosus* strain HOM1213 has the potential to lower blood glucose levels, and the mechanism may be through stimulating intestinal L cells to produce high levels of GLP-1. GLP-1 is an incretin that, in a glucose concentration-dependent manner, promotes insulin secretion from pancreatic β cells and inhibits glucagon secretion from α cells, thus effectively lowering postprandial blood glucose. When glucose concentration is below normal, it does not stimulate insulin secretion, thereby preventing hypoglycemia.

[0166] Example 10 In vitro antioxidant capacity test

[0167] (1) Activation of microbial strains

[0168] Lactobacillus rhamnosus strains HOM1213 and GG were inoculated into MRS liquid medium and fermented at 37°C for 24 h. After three generations of activation, the cells were centrifuged at 11000 rpm for 10 min, washed with PBS buffer, resuspended, and centrifuged again. This process was repeated three times. The bacterial count was adjusted to 2*10^6 cells / year. 10 CFU / ml available for use.

[0169] (2) Detection of antioxidant indicators

[0170] The indicators included total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), hydroxyl radical scavenging (·OH), and DPPH radical scavenging. The DPPH radical scavenging assay used a colorimetric method. The principle is that the radical scavenger donates an electron to pair with the lone pair electron of the DPPH radical, causing its own purple color to change to yellow, and its absorbance at 517 nm to decrease. The degree of change is linearly related to the degree of radical scavenging; that is, the stronger the radical scavenging ability of the scavenger, the lower the absorbance. Other indicators were determined using kits purchased from Nanjing Jiancheng Company, according to the instructions. 0.05% vitamin C was used as a positive control. The antioxidant indicators of HOM1213 and GG bacterial suspensions were measured, and the results are shown in Table 9. Figure 3 .

[0171] Table 9. Detection of in vitro antioxidant capacity

[0172]

[0173] Note: ND: Not detected; **: Significantly different from group HOM1213 (p<0.01).

[0174] From Table 9 and Figure 3 It is known that *Lactobacillus rhamnosus* HOM1213 exhibits high T-AOC, SOD, and GSH-Px activities, as well as high hydroxyl radical and DPPH radical scavenging rates, particularly demonstrating outstanding performance in SOD enzyme activity, hydroxyl radical scavenging, and DPPH radical scavenging. This indicates that HOM1213 possesses excellent antioxidant activity.

[0175] Example 11: Improving antioxidant levels and delaying aging in mice

[0176] (1) Mouse source

[0177] Thirty healthy, SPF-grade female mice (22-26g) bred by Beijing Huafukang Biotechnology Co., Ltd. were selected from Kunming. The experimental animals were housed in the SPF-grade animal room of the Health Food Functional Testing Center, School of Applied Arts and Sciences, Beijing Union University, and their maintenance feed was produced by Beijing Huafukang Biotechnology Co., Ltd.

[0178] (2) D-galactose oxidative damage model

[0179] Thirty mice were divided into three groups of ten each. Two groups of mice were used to induce a malondialdehyde (MDA) model by subcutaneous injection of D-galactose (prepared with physiological saline) at a dose of 300 mg / kg body weight (BW) into the neck and back, at a dose of 0.1 mL / 10 g, once daily for six weeks. Blood samples were collected to measure MDA levels. MDA levels were then randomly divided into two groups based on MDA levels: a model control group and a HOM1213 group. The third group served as a blank control group, receiving an equal volume of physiological saline subcutaneously injected into the neck and back daily during the modeling period.

[0180] (3) Dosage design

[0181] After successful modeling, mice were orally administered samples once daily at a gavage volume of 10 mL / kg body weight (BW). The HOM1213 group received 1×10⁻⁶ BW. 9 CFU bacterial solution was administered, while sterile water was used instead of the sample in the blank control group and the model control group. The daily gavage volume was the same as that of the sample group. All groups were given a maintenance diet. After 45 days of continuous gavage, various indicators in the mice were measured. While administering the sample, the HOM1213 group and the model control group continued to be injected with the same dose of D-galactose, while the blank control group was injected with an equal volume of physiological saline.

[0182] The specific experimental methods were based on the "Technical Specifications for Inspection and Evaluation of Health Foods" (2012 edition), and the experimental results were statistically analyzed using SPSS software.

[0183] (4) Index Measurement

[0184] All reagent kits required for the experiment were purchased from Nanjing Jiancheng Biotechnology Institute.

[0185] A. Determination of serum malondialdehyde (MDA) content in lipid peroxidation-related serum

[0186] 0.5 mL of blood was collected from the inner canthal venous plexus of mice, centrifuged at 3000 r / min for 10 min, and the malondialdehyde content in the serum was determined using a kit. The results are shown in Table 12.

[0187] B. Determination of protein carbonyl content in liver tissue protein oxidation products

[0188] A certain amount of liver tissue was taken and rinsed in ice-cold physiological saline to remove surface bloodstains, then dried with filter paper. The protein carbonyl content was determined using a protein carbonyl reagent kit (A087-1-2, Nanjing Jiancheng Bioengineering). Specific method: Reagent I was added at a weight-to-volume ratio of 1:9, and the mixture was mechanically homogenized under ice bath conditions. The homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant (10% liver tissue homogenate) was collected for analysis. 450 μL of the 10% liver tissue homogenate was taken, and 50 μL of Reagent II was added. After incubation at room temperature for 10 min, the mixture was centrifuged at 11000 rpm for 10 min, and the supernatant was collected for analysis. The results are shown in Table 13.

[0189] C. Antioxidant enzymes

[0190] Blood of 0.5 mL was collected from the inner canthal venous plexus of aged mice, centrifuged at 3000 r / min for 10 min, and the superoxide dismutase (SOD) activity in the serum was measured using a superoxide dismutase activity kit. The results are shown in Table 14.

[0191] Blood was collected in 10 μL from the inner canthal venous plexus of aged mice and diluted with distilled water to 1 mL to prepare a 1:99 hemolysate. Glutathione peroxidase (GSH-Px) activity was measured using a glutathione peroxidase kit, and the results are shown in Table 15.

[0192] D. Determination of content of antioxidant-containing reduced glutathione (GSH)

[0193] A certain amount of liver was cut off, rinsed with physiological saline, dried, weighed, and minced. The homogenate was then placed in a glass homogenizer and homogenized with cold physiological saline to prepare a 10% tissue homogenate. The homogenate was centrifuged at 2500 r / min for 10 min, and the supernatant was collected. The GSH content was determined using a GSH kit. The results are shown in Table 16.

[0194] Table 10. Serum malondialdehyde (MDA) content in mice after modeling

[0195]

[0196] **: p<0.01 between the model control group and the blank control group.

[0197] As shown in Table 10, after 6 weeks of subcutaneous injection of D-galactose to induce a D-galactose model in mice, the model group was randomly divided into a model control group and a sample group (HOM1213) based on the serum malondialdehyde (MDA) content. There was no significant difference between the HOM1213 group and the model control group (p>0.05), indicating that the serum MDA content in mice was relatively balanced among the model groups. Compared with the blank control group, the serum MDA content in the model control group was significantly higher (p<0.01), indicating that the D-galactose oxidative damage model was successfully established.

[0198] Table 11. Changes in mouse body weight before and after administration of the sample

[0199]

[0200] As shown in Table 11, there were no significant differences in mouse body weight between the model control group and the blank control group before and 45 days after oral administration of the sample (p>0.05), and no significant differences between the sample group and the model control group (p>0.05). Therefore, the sample had no adverse effect on mouse body weight.

[0201] Table 12. Effects of HOM1213 group on serum malondialdehyde in mice

[0202]

[0203] **: p < 0.01 between the sample group and the model control group.

[0204] As shown in Table 12, after oral administration of the sample to mice for 45 days, the serum malondialdehyde (MDA) content in the HOM1213 group was significantly lower than that in the model control group (p<0.01). This indicates that the HOM1213 sample can significantly reduce the MDA content in the serum of mice with D-galactose oxidative damage model. MDA is a major product of polyunsaturated fatty acid peroxidation. MDA levels are widely used to determine the level of oxidative damage in vivo.

[0205] Table 13. Effects of HOM1213 group on protein carbonyl content in mouse liver tissue

[0206]

[0207] Protein carbonylation is a type of protein oxidative damage, referring to the transformation of amino acid residue side chains into carbonyl products after attack by oxygen free radicals. Carbonyl modification can cause changes in protein structure, leading to cell and tissue lesions and participating in aging and apoptosis. As shown in Table 13, after oral administration of mouse samples for 45 days, compared with the model control group, the carbonyl content of liver tissue proteins in the HOM1213 group was reduced, but the differences were not statistically significant (p>0.05).

[0208] Table 14. Effects of HOM1213 group on serum superoxide dismutase (SOD) activity in mice

[0209]

[0210] *: p<0.05 between the sample group and the model control group.

[0211] As shown in Table 14, after oral administration of the sample to mice for 45 days, serum SOD activity was significantly increased in the HOM1213 group compared with the model control group (p<0.05). HOM1213 can increase the body's SOD level to resist oxidative stress.

[0212] Table 15. Effects of HOM1213 group on whole blood glutathione peroxidase activity in mice

[0213]

[0214] **: p < 0.01 between the sample group and the model control group.

[0215] As shown in Table 15, after oral administration of the mouse samples for 45 days, the whole blood glutathione peroxidase activity in the HOM1213 group increased significantly (p<0.01), indicating that the HOM1213 group can increase the whole blood glutathione peroxidase activity in D-galactose oxidative damage model mice to resist oxidative stress.

[0216] Table 16. Effects of HOM1213 group on reduced glutathione content in mouse liver tissue

[0217]

[0218] *: p < 0.05 between the sample group and the model control group.

[0219] As shown in Table 16, after oral administration of the sample to mice for 45 days, the content of reduced glutathione in the liver tissue of the HOM1213 group was significantly increased compared with the model control group (p<0.05). This indicates that HOM1213 can increase the content of reduced glutathione in the liver tissue of mice with D-galactose oxidative damage model.

[0220] According to the "Methods for Testing and Evaluation of Functions of Health Foods (2022 Edition)" and the "Methods for Determining Antioxidant Animal Experiments," if three out of the four indicators—lipid oxidation products, protein oxidation products, antioxidant enzymes, and antioxidant substances—are positive, the test sample can be determined to contribute to a positive result in antioxidant animal experiments. This indicates that *Lactobacillus rhamnosus* HOM1213 possesses antioxidant and anti-aging functions.

[0221] Figure 4This invention demonstrates the effects of the *Lactobacillus rhamnosus* strain HOM1213 on antioxidant and anti-aging indicators in mice with an oxidative damage model. After oral administration of samples to mice for 45 days, compared with the model control group, the HOM1213 group showed significantly increased serum superoxide dismutase (SOD) activity (p<0.05), highly significantly increased whole blood glutathione peroxidase (GSH-Px) activity (p<0.01), significantly increased liver tissue reduced glutathione (GSH) content (p<0.05), and highly significantly decreased serum malondialdehyde (MDA) content (p<0.01). While liver tissue protein carbonyl content decreased, the difference was not significant (p>0.05). Therefore, the HOM1213 group significantly increased serum SOD and GSH-Px activities and GSH levels, and significantly decreased MDA content in mice with D-galactose oxidative damage.

[0222] The entire process of this invention is as follows Figure 5 The process flowchart is shown below.

[0223] Example 12 Fermented Milk Experiment

[0224] Weigh 12g of skim milk powder, 2g of glucose, and 2g of yeast powder. Add double-distilled water to make up to 100mL, stir well, homogenize with a high-pressure homogenizer (60℃, 22MPa), sterilize at 95℃ for 10min, and cool to 37℃ for later use.

[0225] Lactobacillus rhamnosus HOM1213 was inoculated into MRS liquid medium and cultured at 37°C for 18 hours. This method was repeated twice to obtain a highly viable bacterial culture for later use.

[0226] The bacterial suspension was eluted and resuspended with 0.9% physiological saline, and then subjected to a 1×10⁻⁶ solution. 7 Inoculate cow's milk at a dose of CFU / mL, mix thoroughly, and incubate at a constant temperature of 37℃ for 16 hours to obtain fermented dairy products. The fermented milk contains more than 6.0 × 10⁻⁶ active Lactobacillus rhamnosus. 8 With a concentration of CFU / mL and a pH of up to 4.50, it has a rich, mellow, and sweet-and-sour taste.

[0227] Example 13 Fermented Oat Milk Experiment

[0228] Weigh 120g of enzymatically hydrolyzed oat flour and 20g of yeast powder, add double-distilled water to make up to 1000mL, homogenize at 200bar, sterilize at 90℃ for 10min, and cool to 37℃ for later use.

[0229] Lactobacillus rhamnosus seed culture HOM1213 was inoculated into MRS liquid medium and cultured at 37°C for 18 hours. After two subcultures, a high concentration of bacterial culture was obtained and stored at low temperature for later use.

[0230] The bacterial suspension was eluted and resuspended with 0.9% physiological saline, and then subjected to a 1×10⁻⁶ solution. 7 Inoculate the oat milk with an inoculum concentration of CFU / mL, mix thoroughly, and incubate at a constant temperature of 37℃ for 6 hours to obtain fermented oat milk. The viable count of Lactobacillus rhamnosus in the fermented milk is higher than 5.0 × 10⁻⁶. 8 With CFU / mL, the pH can be reduced to 4.35, resulting in a rich flavor.

[0231] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition comprising a strain of Lactobacillus rhamnosus and excipients, wherein the Lactobacillus rhamnosus strain has the accession number CGMCC No. 25682. in, The Lactobacillus rhamnosus strain possesses antioxidant and anti-aging capabilities, increases the levels of superoxide dismutase, glutathione peroxidase, and glutathione, decreases malondialdehyde levels, and enhances the scavenging rate of hydroxyl radicals and DPPH radicals; it also has the potential to regulate blood sugar, produce DPP-4 and α-glucosidase inhibitors, and stimulate NCI-H716 cells to produce GLP-1; and it has the ability to produce a high level of lactic acid.

2. Use of the composition according to claim 1 in the preparation of a medicament for regulating immunity.

3. The use according to claim 2, characterized in that, The composition is used to increase the levels of cytokines TNF-α and / or IL-6.

4. Use of the composition according to claim 1 in the preparation of a medicament for scavenging free radicals, responding to oxidative stress, antioxidation, and anti-aging.

5. The use according to claim 4, characterized in that, The composition is used to increase the levels of superoxide dismutase, glutathione peroxidase and glutathione, and / or decrease the level of malondialdehyde.

6. The use according to claim 5, characterized in that, The composition is used to improve the scavenging rate of hydroxyl radicals and DPPH radicals.

7. Use of the composition according to claim 1 in the preparation of a medicament for producing DPP-4 and α-glucosidase inhibitors, and / or stimulating NCI-H716 cells to produce GLP-1.

8. The use according to claim 7, characterized in that, The composition is used to help lower blood sugar.

9. Use of the composition according to claim 1 in the preparation of a medicament for increasing the content of lactic acid or short-chain fatty acids.

10. The use according to claim 9, characterized in that, The short-chain fatty acid is acetic acid.

11. The use according to claim 9, characterized in that, The composition is used to maintain the balance of intestinal flora.

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