Bifidobacterium breve NKU BB 1-13 and application thereof

Abstract

The invention provides bifidobacterium breve NKU BB 1-13 and application thereof, and belongs to the technical field of microorganisms. The invention provides a bifidobacterium breve NKU BB 1-13, and the preservation number of the bifidobacterium breve NKU BB 1-13 is GDMCC No: 65957. In the invention, the bifidobacterium breve NKU BB 1-13 cannot generate nitrate reductase, shows negative ornithine and lysine decarboxylase, has no hemolytic activity, and is a safe strain. The bifidobacterium breve NKU BB 1-13 has the characteristics of acid resistance and cholate resistance, and can tolerate the digestion environment of gastrointestinal tracts. The bifidobacterium breve NKU BB 1-13 can relieve oxidative stress and inflammation of intestinal tracts in the aging process, meanwhile, can promote goblet cells to secrete intestinal barrier protein and improve the intestinal barrier function, and then can improve intestinal health in the aging process.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a short Bifidobacterium NKU BB 1-13 and its applications. Background Technology

[0002] Probiotics are a collective term for live microorganisms that can improve the balance of the host's gut microbiota and have a positive impact on the host's health. Numerous studies on probiotics have shown that the appropriate intake of probiotics has beneficial properties such as promoting the digestion and absorption of nutrients in the host, improving the host's immunity, maintaining the balance of the host's gut microbiota structure, assisting the host in anti-oxidation, and protecting the intestinal mucosal barrier.

[0003] Aging is considered a weakening of the functions of various tissues in the body and a decline in the body's ability to adapt to the environment. It is a rather complex biological process, and its regulatory mechanisms are not yet fully understood. Research suggests that aging is caused by the decline of a single gene or the immune system, with multiple processes combining and interacting at many levels. These processes occur not only at the cellular and molecular levels but also at the tissue and organ systems.

[0004] Currently, a wide range of interventions are available to alleviate aging or age-related diseases, including dietary regulation, calorie restriction, and probiotic or prebiotic interventions. Probiotic interventions, in particular, can not only shift the gut microbiota towards a beneficial state but also delay aging by enhancing the body's antioxidant capacity, reducing inflammation, regulating immunity, and improving cognitive abilities. For example, *Lactobacillus plantarum* LLY-606 can effectively prolong the lifespan of mice by reducing the levels of inflammatory factors, increasing antioxidant capacity, alleviating intestinal barrier dysfunction, and improving cognitive impairment. Similarly, *Bacillus belyssus* can enhance muscle strength, cognitive learning ability, reproductive capacity, and antioxidant capacity in animals, and can be used for anti-aging in animals.

[0005] Currently, there are no known Bifidobacterium breve strains that can be used to alleviate age-related symptoms. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the purpose of this invention is to provide a short Bifidobacterium that can improve intestinal health in the context of aging.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a short-lived Bifidobacterium (Bifidobacterium breve) Bifidobacterium breve NKU BB 1-13, the preservation number of the said Bifidobacterium NKU BB 1-13 is GDMCC No: 65957.

[0008] This invention provides a microbial preparation comprising the Bifidobacterium breve NKU BB 1-13 described in the above technical solution.

[0009] Preferably, the viability of *Bifidobacterium breve* NKU BB 1-13 in the microbial preparation is ≥5×10⁻⁶. 9 CFU / mL or ≥5×10 9 CFU / g.

[0010] This invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: The aforementioned Bifidobacterium NKU BB 1-13 was cultured in a culture medium to obtain a microbial preparation.

[0011] Preferably, the culture temperature is 36~38℃; the culture time is 24~48h.

[0012] This invention provides a biological agent comprising the above-described Bifidobacterium breve NKU BB 1-13 and excipients.

[0013] This invention provides the application of the above-described Bifidobacterium breve NKU BB 1-13 in the preparation of antioxidant products.

[0014] This invention provides the application of the above-described Bifidobacterium breve NKU BB 1-13 in the preparation of anti-aging and / or gut health improvement products.

[0015] Preferably, the product includes one or more of the following: food, medicine, and health products.

[0016] Preferably, the application includes any one or more of the following (1) to (4): (1) Relieves inflammation and / or structural deterioration of the intestine; (2) Improves intestinal oxidative stress levels; (3) Improves intestinal inflammation levels; (4) Improve the intestinal barrier.

[0017] The beneficial effects of this invention are: This invention provides a short-lived Bifidobacterium (Bifidobacterium breve) Bifidobacterium breveThe *Bifidobacterium breve* NKU BB 1-13 strain, with accession number GDMCC No: 65957, is described in this invention. In this invention, the *Bifidobacterium breve* NKU BB 1-13 strain does not produce nitrate reductase, exhibits ornithine and lysine decarboxylase negativity, and has no hemolytic activity, making it a safe strain. The *Bifidobacterium breve* NKU BB 1-13 strain is acid- and bile-salt resistant, allowing it to tolerate the digestive environment of the gastrointestinal tract. It possesses outstanding antioxidant capacity, effectively scavenging various oxidative free radicals such as DPPH, superoxide anions, and hydroxyl radicals, thus assisting the host in antioxidant processes. The *Bifidobacterium breve* NKU BB 1-13 strain can alleviate oxidative stress and inflammation in the intestine during aging, while also promoting the secretion of intestinal barrier proteins by goblet cells, thereby improving intestinal barrier function. The results of the embodiments of this invention show that the use of the aforementioned Bifidobacterium breve NKU BB 1-13 can alleviate inflammation and structural deterioration of the colon in mice under aging conditions; the use of the aforementioned Bifidobacterium breve NKU BB 1-13 can increase the SOD content in the colon of aged mouse models, reduce MDA production, alleviate colonic oxidative damage, reduce colonic COX-2 content, and reduce the occurrence of colonic inflammation; the aforementioned Bifidobacterium breve NKU BB 1-13 can stimulate the expression of three intestinal protein genes, MUC-2, E-cadherin, and Claudin-1, in aged mouse models, thereby improving intestinal barrier function.

[0018] Biological Preservation Instructions Bifidobacterium breve NKU BB 1-13, classified and named as follows: Bifidobacterium breve It was deposited on March 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 65957. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The colony morphology of Bifidobacterium breve NKU BB 1-13 grown on TPY agar medium is shown. Figure 2 The image shows the nitrate reductase test results for Bifidobacterium breve NKU BB 1-13. Figure 3The image shows the results of the ornithine decarboxylase test for Bifidobacterium breve NKU BB 1-13. Figure 4 The image shows the results of the lysine decarboxylase test for Bifidobacterium breve NKU BB 1-13. Figure 5 The image shows the results of the lysin activity test for Bifidobacterium breve NKU BB 1-13. Figure 6 The image shows the acid resistance test results of Bifidobacterium breve NKU BB 1-13. Figure 7 The graph shows the results of the bile salt tolerance test for Bifidobacterium breve NKU BB 1-13. Figure 8 The graph shows the results of the determination of the NKU BB 1-13DPPH clearance rate of Bifidobacterium breve. Figure 9 The graph shows the hydroxyl radical scavenging rate of Bifidobacterium breve NKU BB 1-13. Figure 10 The graph shows the superoxide anion scavenging rate of Bifidobacterium breve NKU BB 1-13. Figure 11 Animal experimental design diagram for studying the probiotic effects of Bifidobacterium breve NKU BB 1-13 in maintaining gut health in the context of aging; Figure 12 Figure 6 shows colon sections and HE staining results of mice in each group; Figure 13 This is a graph showing the comparison of SOD content in the colon of mice in each group in Example 6; Figure 14 This is a graph showing the comparison of MDA content in the colon of mice in each group in Example 6; Figure 15 This is a graph showing the comparison of COX-2 content in the colon of mice in each group in Example 6; Figure 16 This is a graph showing the differences in gene expression of the intestinal barrier protein MUC2 in the colon of mice in Example 6; Figure 17 This is a graph showing the differences in gene expression of the intestinal barrier protein E-cadherin in the mice of different groups in Example 6. Figure 18 This is a graph showing the differences in gene expression of the intestinal barrier protein Claudin-1 in the colon of mice in Example 6. Figure 19 The image shows the immunohistochemical images of three intestinal proteins in the colon of mice in each group in Example 6. Detailed Implementation

[0021] This invention provides a short-lived Bifidobacterium (Bifidobacterium breve) Bifidobacterium breveNKU BB 1-13, the preservation number of the said Bifidobacterium NKU BB 1-13 is GDMCC No: 65957.

[0022] The *Bifidobacterium breve* NKU BB 1-13 provided by this invention was isolated from infant fecal samples. On TPY agar medium, the colonies of *Bifidobacterium breve* NKU BB 1-13 generally appear as smooth, milky-white dots, with some showing milky-like protrusions. The bacterial cells of *Bifidobacterium breve* NKU BB 1-13 are short rod-shaped, with some exhibiting Y-shaped branching. *Bifidobacterium breve* NKIBB 1-13 is an anaerobic bacterium, a Gram-positive bacterium, and its 16S rRNA nucleotide sequence is shown in SEQ ID NO. 1.

[0023] The *Bifidobacterium breve* NKU BB 1-13 provided by this invention is sensitive to gentamicin, erythromycin, vancomycin, streptomycin, tetracycline, and penicillin G, and resistant to chloramphenicol, streptomycin, and ciprofloxacin. In this invention, the *Bifidobacterium breve* NKU BB 1-13 does not produce active nitrate reductase, exhibiting ornithine and lysine decarboxylase negativity, and has no hemolytic activity, making it a safe strain. The *Bifidobacterium breve* NKU BB 1-13 possesses acid and bile salt resistance, allowing it to tolerate the digestive environment of the gastrointestinal tract.

[0024] In this invention, *Bifidobacterium breve* NKU BB 1-13, as a probiotic of the *Bifidobacterium* genus, can actively regulate the intestinal microbial structure, promote the growth of beneficial intestinal microorganisms, and thus positively influence intestinal health. Through in vitro antioxidant capacity experiments, this invention found that compared to ordinary *Bifidobacterium* strains, *Bifidobacterium breve* NKU BB 1-13 has outstanding antioxidant capacity, possessing the ability to efficiently scavenge various oxidative free radicals such as DPPH, superoxide anions, and hydroxyl radicals, and has the ability to assist the host in antioxidant processes. In this invention, *Bifidobacterium breve* NKU BB 1-13 can alleviate oxidative stress and inflammation in the intestine during the aging process, while also promoting the secretion of intestinal barrier proteins by goblet cells, improving intestinal barrier function, and thus improving intestinal health during the aging process.

[0025] In summary, the Bifidobacterium breve NKU BB 1-13 provided by this invention is a safe strain, an edible fungus, and has the characteristics of being acid- and bile-salt resistant. Bifidobacterium breve NKU BB 1-13 has good in vitro antioxidant capacity, has the effect of improving intestinal health, and can be used to prepare food, functional food and / or pharmaceuticals, etc.

[0026] This invention provides a microbial preparation comprising *Bifidobacterium breve* NKU BB 1-13 as described in the above-mentioned technical solution. As an optional embodiment of this invention, the microbial preparation can be a liquid preparation or a solid preparation. As an optional embodiment of this invention, the viability of *Bifidobacterium breve* NKU BB 1-13 in the microbial preparation can be ≥5 × 10⁻⁶. 9 CFU / mL or ≥5×10 9 CFU / g, or 5×10 9 ~1×10 10 CFU / mL, or 5×10 9 ~1×10 10 CFU / g.

[0027] This invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: culturing the *Bifidobacterium breve* NKU BB 1-13 in a culture medium to obtain the microbial preparation. As an optional embodiment of this invention, the culture medium can be TPY medium; the culture temperature can be 36-38℃ or 37℃; the culture time can be 24-48 h, or 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 h.

[0028] This invention provides a biological agent comprising the *Bifidobacterium breve* NKU BB 1-13 described in the above-described technical solution and excipients. As an optional embodiment of this invention, the excipients include food-acceptable excipients or pharmaceutical-acceptable excipients.

[0029] This invention provides the application of *Bifidobacterium breve* NKU BB 1-13, as described in the above-mentioned technical solution, in the preparation of antioxidant products. As an optional embodiment of this invention, the product includes any one or more of food, pharmaceutical, and health products. Through in vitro antioxidant capacity experiments, this invention has found that, compared to common Bifidobacterium strains, *Bifidobacterium breve* NKIBB 1-13 exhibits outstanding antioxidant capacity, possessing the ability to efficiently scavenge various oxidative free radicals such as DPPH, superoxide anion, and hydroxyl radicals, and has the ability to assist the host in antioxidant processes.

[0030] This invention provides the application of *Bifidobacterium breve* NKU BB 1-13 described in the above-mentioned technical solution in the preparation of anti-aging and / or gut health improvement products. As an optional embodiment of this invention, the product includes any one or more of food, pharmaceuticals, and health products. As an optional embodiment of this invention, the application includes any one or more of the following (1) to (4): (1) alleviating intestinal inflammation and / or structural deterioration; (2) improving intestinal oxidative stress levels; (3) improving intestinal inflammation levels; (4) improving the intestinal barrier. This invention demonstrates through animal experiments that *Bifidobacterium breve* NKU BB 1-13 can alleviate intestinal oxidative stress and inflammation during the aging process, and can also promote the secretion of intestinal barrier proteins by goblet cells, thereby improving intestinal barrier function.

[0031] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: Isolation and identification of Bifidobacterium breve NKU BB 1-13 (1) Bifidobacterium breve NKU BB 1-13 was isolated from the feces of healthy infants. The specific steps are as follows: Take 1g of fresh infant feces and place it in a 15mL centrifuge tube. Dissolve and resuspend the feces in 10mL of sterile physiological saline. Centrifuge at 4000rpm and 4℃ for 10 min. Take 100μL of the supernatant and dilute it 10 times. 4 After dilution, another 100 μL of the diluted solution was spread evenly on a Bifidobacterium breve selection medium and incubated at 37°C for 24 h in an anaerobic workstation. Small, smooth, milky-white colonies on the plate were selected for purification until stable single colonies of bacteria were obtained, which were named strain NKU BB 1-13.

[0033] Preparation method of Bifidobacterium breve selection medium: The culture medium formula for screening Bifidobacterium breve (unit: g / L): peptone: 10.0; beef meal: 8.0; yeast extract: 4.0; glucose: 20.0; dipotassium hydrogen phosphate: 2.0; diammonium hydrogen citrate: 2.0; sodium acetate: 5.0; magnesium sulfate: 0.2; manganese sulfate: 0.04; Tween 80: 1.0; mupirocin: 0.05; pH 5.7±0.2.

[0034] After mixing the raw materials according to the above formula at 25℃, sterilize at 121℃ for 15 min, cool to 50℃, add 100 mL of sterile horse serum, pour into plates at a rate of 15 mL / plate, and air dry in a clean bench for 1 h.

[0035] (2) The strain NKU BB 1-13 was cultured on TPY agar medium, and its colony characteristics were observed. Gram staining was also performed on the strain. Results showed that strain NKU BB 1-13 is a Gram-positive bacterium. The colony morphology of strain NKU BB 1-13 on TPY agar medium was as follows: Figure 1 As shown, its colony morphology is generally a smooth, milky-white dot, with some showing milky protrusions. The bacterial cells are short rods, some exhibiting Y-shaped branching. Strain NKU BB 1-13 is an anaerobic bacterium.

[0036] (3) After activation and culture, strain NKU BB 1-13 was inoculated into TPY liquid medium and anaerobic cultured at 37℃ for 24 h. After washing several times with PBS, a sufficient amount of bacterial cells was collected. Genomic DNA was extracted using a bacterial DNA extraction kit, and the quality of the extracted DNA was tested. Genome sequencing and bioinformatics analysis were then performed. The 16S rRNA sequence of strain NKU BB 1-13 was obtained as shown in SEQ ID NO.1, as follows:

[0037] The sequencing results were compared with those in NCBI to preliminarily determine the species. Finally, strain NKU BB 1-13 showed 100% similarity to the subspecies of Bifidobacterium breve, and was further confirmed as Bifidobacterium breve. Bifidobacterium breve ), which is Bifidobacterium breve NKU BB 1-13.

[0038] Example 2: Detection of harmful metabolites from Bifidobacterium breve NKU BB 1-13 1. Nitrate reductase test Bifidobacterium breve NKU BB 1-13 was cultured at a 2% inoculum to the third generation stationary phase. A blank nitrate medium was used as a negative control. The culture was inoculated at a 1% inoculum (V / V) onto pre-prepared nitrate medium. After purging oxygen in an anaerobic workstation, the culture was incubated at 37°C for 48 hours. Then, 10 drops of 5% potassium iodide solution and 10 drops of 5% starch solution were added to the culture system, vortexed, and the color changes in each culture tube were observed and recorded. Each sample was tested in triplicate. Results are as follows: Figure 2 As shown, Bifidobacterium breve NKUBB 1-13 did not exhibit a color reaction in the nitrate reductase activity test, indicating that Bifidobacterium breve NKUBB 1-13 does not produce active nitrate reductase.

[0039] 2. Amino acid decarboxylase test The presence of amino acid decarboxylases in microorganisms can promote the metabolism of amino acids into biogenic amines that are harmful to humans. Bromocresol purple is added as an indicator to the culture medium for amino acid decarboxylase testing. The presence of the corresponding amino acid decarboxylase in the cultured microorganism can be determined based on its color change. If present, the culture medium changes color to purple or purplish-red, indicating a positive reaction; if absent, the culture medium remains yellow, indicating a negative reaction.

[0040] Bifidobacterium breve NKU BB 1-13 was cultured to the third generation stationary phase. Inoculated at 1% (v / v) into pre-prepared liquid ornithine decarboxylase medium and lysine decarboxylase medium, respectively. After aeration was eliminated in an anaerobic digester, the cultures were incubated at 37°C for 48 hours, and the color changes of the culture medium were observed and recorded. The negative control consisted of liquid ornithine decarboxylase and lysine decarboxylase medium without any exogenous substances, directly placed in an anaerobic digester after aeration was eliminated, and incubated at 37°C for 48 hours. Each sample was tested in triplicate. Results are as follows: Figures 3-4 As shown, Bifidobacterium breve NKU BB 1-13 turned yellow after being cultured in ornithine decarboxylase medium and lysine decarboxylase medium for 48 hours, indicating that Bifidobacterium breve NKU BB 1-13 was negative for ornithine and lysine decarboxylase.

[0041] 3. Hemolytic test Bifidobacterium breve NKU BB 1-13 was cultured to the third generation stationary phase. Staphylococcus aureus ATCC25923 was used as a positive control. Strands were applied to Columbia blood agar plates and incubated at 37°C in the dark for 48 hours. The formation of hemolytic zones on each plate was observed and recorded. A clear hemolytic zone around the bacterial colony indicated β-hemolysis, while a green area (α-hemolysis) or no area (γ-hemolysis) around the bacterial colony was considered indicative of non-hemolytic bacteria. Three replicates were prepared for each sample. Results are as follows: Figure 5 As shown in the figure, Figure A shows the colony morphology of Staphylococcus aureus on blood agar plates; Figure B shows the colony morphology of Bifidobacterium breve NKU BB1-13 on blood agar plates.

[0042] Depend on Figure 5 It was found that Bifidobacterium breve NKU BB 1-13 did not produce a hemolytic zone when grown on Columbia blood agar plates, indicating that it has no hemolytic activity.

[0043] The above experiments show that Bifidobacterium breve NKU BB 1-13 does not contain active nitrate reductase, is negative for ornithine and lysine decarboxylase, and has no hemolytic activity, indicating that Bifidobacterium breve NKU BB 1-13 is a safe strain that can be used in the food and pharmaceutical fields.

[0044] Example 3: Determination of the probiotic properties of Bifidobacterium breve NKU BB 1-13 1. Acid resistance test Bifidobacterium breve NKU BB 1-13 was cultured to the third generation stationary phase and inoculated at a 1% (v / v) rate into TPY medium at pH 3 and pH 4, and incubated in an anaerobic incubator at 37°C. Bacterial suspensions from 0 hours (control group) and 3 hours of culture were serially diluted to appropriate concentrations. 200 μL of each diluted suspension was spread onto TPY agar plates and incubated at 37°C until colonies appeared. Finally, the colonies were counted. Results are as follows: Figure 6 As shown.

[0045] from Figure 6 It can be seen that *Bifidobacterium breve* NKU BB 1-13 has good acid resistance. After culturing for 3 hours at pH 4, the bacterial count of *Bifidobacterium breve* NKU BB 1-13 increased, and the survival rate of *Bifidobacterium breve* NKU BB 1-13 was 102.4%. After culturing for 3 hours at pH 3, the colony count of *Bifidobacterium breve* NKU BB 1-13 decreased slightly, but it still had good activity, and the survival rate of *Bifidobacterium breve* NKU BB 1-13 was 96.2%.

[0046] 2. Test for tolerance to bile salts Bifidobacterium breve NKU BB 1-13 was cultured to the third generation stationary phase and inoculated at a 1% (v / v) inoculum into TPY liquid medium with bile salt concentrations of 0.05% and 0.1%, and incubated in an anaerobic incubator at 37°C. Bacterial suspensions from 0 hours and 3 hours of culture were serially diluted to appropriate concentrations, and 200 μL of each diluted suspension was spread onto TPY solid plates. The plates were then incubated in an anaerobic incubator at 37°C until colonies appeared. Finally, the colonies on the plates were counted. Results are as follows: Figure 7 As shown.

[0047] from Figure 7 It can be seen that *Bifidobacterium breve* NKU BB 1-13 has good bile salt tolerance. Specifically, under culture conditions with a bile salt content of 0.05%, the colony count of *Bifidobacterium breve* NKU BB 1-13 decreased slightly, with a survival rate of 97.2%. When the bile salt content increased to 0.1%, the colony count of *Bifidobacterium breve* NKU BB 1-13 also showed only a slight decrease, with a survival rate of 95.03%.

[0048] Example 4: Antibiotic susceptibility test of Bifidobacterium breve NKU BB 1-13 The antibiotic susceptibility of *Bifidobacterium breve* NKU BB 1-13 strains was assessed using the disk diffusion method. The specific procedure was as follows: *Bifidobacterium breve* NKU BB 1-13 strains were washed with PBS and resuspended at 1×10⁻⁶ ppm. 8 CFU / mL, the bacterial suspension to be tested was evenly spread on TPY plates using a sterile spreader, and then antibiotic susceptibility was assessed using 8 different antimicrobial susceptibility test discs. The relevant information for the antimicrobial susceptibility test discs is shown in Table 1.

[0049] After incubation at 37°C for 24 hours, the growth of the test strains around the antibiotic-sensitive discs will be inhibited, forming plaques of different diameters. The sensitivity of the strain to the antibiotic can be determined based on the size of the plaques.

[0050] Table 1. Information related to drug sensitivity tablets

[0051] The antibiotic sensitivity of Bifidobacterium breve NKU BB 1-13 is shown in Table 2.

[0052] Table 2. Antibiotic susceptibility test results of Bifidobacterium breve NKU BB 1-13

[0053] As shown in Table 2, based on the inhibition zone results, Bifidobacterium breve NKU BB 1-13 is sensitive to gentamicin, erythromycin, vancomycin, streptomycin, tetracycline, and penicillin G, but resistant to chloramphenicol, streptomycin, and ciprofloxacin.

[0054] Example 5: Evaluation of the antioxidant capacity of Bifidobacterium breve NKU BB1-13 The scavenging capacity of Bifidobacterium breve NKU BB1-13 and other Bifidobacterium breve strains against DPPH, hydroxyl radicals, and superoxide anions was assessed using a kit and normalized scores were performed to evaluate the antioxidant capacity of Bifidobacterium breve NKU BB1-13.

[0055] The preparation procedure for the test sample is as follows: the frozen bifidobacterium NKU BB 1-13 culture is inoculated into TPY liquid medium at a 2% inoculation volume (v / v) and cultured anaerobically at 37℃ for 24 h; after 24 h of culture, 1 mL of turbid culture is taken, centrifuged at 8000 rpm and 4℃ for 10 min, the precipitate in the centrifuge tube is collected, washed three times with PBS, and a test sample is obtained.

[0056] The preparation method for the control strain of Bifidobacterium breve is the same as above.

[0057] 1. DPPH removal ability 0.2 mM DPPH anhydrous ethanol solution was mixed with the test sample at a 1:1 (volume ratio) and incubated in the dark at room temperature for 30 minutes. Then, after centrifugation at 8000×g for 10 minutes, the absorbance of the separated supernatant was measured at 517 nm. The DPPH free radical scavenging activity (%) was calculated as follows: ; Where: AS - absorbance of the sample; Absorbance of the blank consisting of the AB sample and ethanol; The absorbance of the control consisting of AC-DPPH solution and deionized water.

[0058] This method was used to determine the levels of *Bifidobacterium breve* NKU BB 1-13 and standard strains of *Bifidobacterium breve* (…). B.breve The DPPH scavenging ability of ATCC15701 was measured, and the results are as follows: Figure 8 And Table 3.

[0059] Table 3. DPPH clearance rate of the two Bifidobacterium breve strains

[0060] Figure 8As shown in Table 3, the DPPH scavenging ability of the control strain prepared under the same conditions was comparable to that of Bifidobacterium breve NKU BB 1-13. The DPPH scavenging ability of Bifidobacterium breve NKU BB 1-13 was slightly higher than that of the control strain, indicating that Bifidobacterium breve NKU BB 1-13 also had a good DPPH scavenging effect.

[0061] 2. Hydroxyl radical scavenging ability Hydroxyl radicals act on biomolecules such as proteins, nucleic acids, and lipids in the body, causing damage to cell structure and function, and consequently leading to metabolic disorders and diseases. Hydroxyl radical scavenging capacity is one of the important indicators of a sample's antioxidant capacity and is widely used in the research of antioxidant health products and drugs. H2O2 / Fe 2+ Hydroxyl radicals are generated via the Fenton reaction, and o-phenanthroline-Fe 2+ Fe in aqueous solution 2+ Oxidized to Fe 3+ This leads to a decrease in absorbance at 536 nm. The degree to which the sample inhibits the rate of decrease in absorbance at 536 nm reflects the sample's ability to scavenge hydroxyl radicals.

[0062] The Fenton colorimetric method described above was used to determine the levels of *Bifidobacterium breve* NKU BB 1-13 and standard strains of *Bifidobacterium breve* (…). B.breve The hydroxyl radical scavenging ability of ATCC 15701 was measured, and the results are as follows: Figure 9 And Table 4.

[0063] Table 4. Hydroxyl radical scavenging rate of the two Bifidobacterium breve strains

[0064] Figure 9 As shown in Table 4, the hydroxyl radical scavenging ability of the control strain prepared under the same conditions was about 10.7%, while the hydroxyl radical scavenging ability of Bifidobacterium breve NKU BB 1-13 was slightly higher than that of the control strain, indicating that Bifidobacterium breve NKU BB 1-13 has a strong hydroxyl radical scavenging ability.

[0065] 3. Superoxide anion scavenging ability Reactive oxygen species, such as superoxide anions, play a role in immunity and signal transduction in living organisms. However, excessive accumulation can damage cell membranes and biomolecules, leading to abnormal cellular and tissue metabolism and causing various diseases. Superoxide anions react with hydroxylamine hydrochloride to produce NO. 2－ NO 2－The reaction of p-aminobenzenesulfonamide and naphthylethylenediamine hydrochloride produces a red azo compound with a characteristic absorption peak at 530 nm. The superoxide anion content in the sample can be calculated based on the A530 value. This allows for the determination of the superoxide anion scavenging rate, reflecting the superoxide anion removal capacity.

[0066] The superoxide anion scavenging capacity of *Bifidobacterium breve* NKU BB 1-13 and the standard strain of *Bifidobacterium breve* (B. breveATCC 15701) was determined using the hydroxylamine method described above. The results are as follows: Figure 10 As shown in Table 5.

[0067] Table 5. Superoxide anion scavenging rate of two Bifidobacterium breve strains

[0068] Figure 10 As shown in Table 5, the superoxide anion scavenging capacity of the control strain prepared under the same conditions was about 60%, while the superoxide anion scavenging capacity of Bifidobacterium breve NKU BB 1-13 was about 70% to 80% higher than that of the control strain. This indicates that Bifidobacterium breve NKU BB 1-13 has a strong superoxide anion scavenging capacity. The superoxide anion scavenging rates of the two Bifidobacterium breve strains are shown in Table 2.

[0069] Considering the overall effectiveness of Bifidobacterium breve NKU BB 1-13 in scavenging various oxidative free radicals, it can be concluded that Bifidobacterium breve NKU BB 1-13 has a probiotic effect in assisting the scavenging of oxidative free radicals and reducing the negative effects of exogenous oxidative stress.

[0070] Example 6: Study on the probiotic effects of Bifidobacterium breve NKU BB1-13 in maintaining gut health in the context of aging. 1. Animal Experiment Design The effects of Bifidobacterium breve NKU BB 1-13 on gut health in the context of aging were evaluated using in vivo experiments in mice.

[0071] (1) The preparation method of Bifidobacterium breve NKU BB 1-13 bacterial suspension is as follows: After activation and culture, strain NKU BB 1-13 was inoculated into TPY liquid medium and anaerobically cultured at 37℃ for 24 h to obtain a culture broth of *Bifidobacterium breve* NKU BB 1-13. The bacterial pellet of *Bifidobacterium breve* NKU BB 1-13 was obtained by centrifugation, and the bacterial cells were resuspended in physiological saline to adjust the viability to 5 × 10⁻⁶. 9 CFU / mL, used for subsequent tests.

[0072] (2) Eight-week-old male ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed at the Experimental Animal Center of Nankai University, where they were given a one-week acclimatization period. All experimental procedures were approved by the Animal Ethics Committee of Nankai University (ethics number: 2025-SYDWLL-000665). The mice were randomly divided into three groups (n=8 per group). The control group received an intraperitoneal injection of 0.9% NaCl solution. The model group and B.breve The B. breve group received daily injections of 300 mg / kg of D-galactose. Additionally, the B. breve group received 0.2 mL of 5 × 10⁻⁶ D-galactose daily. 9 Mice were administered CFU / mL NKU BB 1-13 via gavage, while the control and model groups received the same volume of 0.9% NaCl solution via gavage. All experimental groups were euthanized at week 15 after injection or gavage, and their colons were harvested for testing. The experimental design is as follows: Figure 11 As shown.

[0073] 2. Pathological analysis of mouse colon by HE staining After dissection of mice in each group, colon tissue was collected and soaked in paraformaldehyde fixative, then rinsed overnight. The samples were then dehydrated sequentially with 0%, 80%, and 90% ethanol solutions for 30 minutes each, followed by two 20-minute dehydration cycles with 95% and 100% ethanol solutions. The samples were then immersed in a mixture of equal parts pure ethanol and xylene for 15 minutes, and repeatedly immersed in pure xylene solution for 15 minutes each until clear. Finally, the samples were immersed in a 1:1 mixture of xylene and paraffin for 15 minutes, followed by 50-60 minutes each of paraffin I and paraffin II to remove the clearing agent. The treated samples were then embedded, sectioned, spread, baked, stained with hematoxylin and eosin (H&E), and mounted. Results of H&E staining of mouse colon are shown below. Figure 12 , Figure 12 The scale bar for the first row of the graph is 500 μm, and the scale bars for the second to fourth rows are 100 μm.

[0074] Depend on Figure 12 It can be seen that, under the same magnification, the colonic slices were counted for inflammatory infiltration (white arrows indicate normal structures, red arrows indicate crypt disappearance, green arrows indicate lymphocyte accumulation, the areas marked by dashed boxes correspond to the magnified images in rows 2-4, and dashed circles indicate the areas pointed to by the arrows); compared with the blank group and the Bifidobacterium breve intervention group, the number of abnormalities in the model group was significantly increased, indicating that Bifidobacterium breve NKU BB 1-13 alleviated the inflammation and structural deterioration of the colon in mice under the background of aging.

[0075] 3. The kit was used to assess the levels of oxidative stress and inflammation in the mouse colon. Accurately weigh 0.05 g of colon and heart into centrifuge tubes containing 0.5 mL of pre-chilled PBS. Homogenize the tissues at low temperature (500 Hz, 300 s, 4 °C), then centrifuge at 8000 rpm for 10 min at 4 °C. Determine the SOD and MDA content in the colon using a total superoxide dismutase (T-SOD) assay kit and a malondialdehyde (MDA) assay kit (Nanjing Jiancheng Biotechnology Institute, China); determine the COX-2 content in the colon using an ELISA kit (Enzyme-Linked Biotechnology). The methods and procedures were strictly performed according to the kit instructions. The results are as follows: Figures 13-15 As shown in Tables 6-8, the figure This indicates a significant difference.

[0076] Table 6. SOD content in the colon of mice in each group

[0077] Table 7. MDA content in the colon of mice in each group

[0078] Table 8. COX-2 content in the colon of mice in each group

[0079] Superoxide dismutase (SOD) and microalcoholic acid (MDA) are commonly used indicators for detecting oxidative stress and the intensity of oxidative damage in tissues and organs. SOD, as an important antioxidant enzyme, reflects the strength of the endogenous antioxidant capacity of tissues and organs; MDA, as lipid peroxides produced after tissues and organs are attacked by oxidative groups, reflects the intensity of exogenous oxidative stress experienced by tissues and organs. Figures 13-15 A significant decrease (P < 0.05) and a significant increase (P < 0.05) in SOD and MDA in the colon of D-galactose-induced aging mice were observed, indicating that the colon of aging mice was in a state of oxidative damage. Intervention with *Bifidobacterium breve* NKU BB 1-13 significantly improved this trend, increasing SOD levels while alleviating external oxidative stress and reducing MDA production, demonstrating the ameliorative effect of *Bifidobacterium breve* on oxidative damage in the colon of aging mice. COX-2 is an inflammatory factor observable in multiple tissues and organs; its elevated levels usually indicate inflammation. Measurements of COX-2 in the intestines of mice in each group showed that, compared to the control group, the high COX-2 levels in the colon of the model group indicated the accumulation of inflammation. Supplementation with *Bifidobacterium breve* NKU BB 1-13 reduced colonic COX-2 levels and decreased the incidence of colonic inflammation.

[0080] 4. Real-time quantitative PCR to determine the gene expression levels of three intestinal proteins in mouse colon 50 mg colon and heart samples were weighed, and total RNA was extracted using the Trizol method. cDNA was then obtained by reverse transcription using the RevertAid RT Reverse Transcription Kit (Thermo Scientific™). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed on a CFX96 (Bio-Rad) using SYBR Green. The RT-qPCR process included pre-denaturation (95℃, 30 s, 1 cycle); cycling (95℃, 10 s; 60℃, 30 s, 40 cycles); and melting curve signal acquisition using the instrument's default acquisition program. Primer sequences used in real-time quantitative PCR are shown in Table 9.

[0081] Table 9 Primer sequence list for real-time quantitative PCR of three intestinal barrier proteins

[0082] Results of real-time quantitative PCR determination of three intestinal proteins in mouse colon as follows: Figures 16-18 See Tables 10-12.

[0083] Table 10. Expression levels of MUC2 in the colon of mice in each group (relative expression levels)

[0084] Table 11. E-cadherin expression levels in the colon of mice in each group (relative expression levels)

[0085] Table 12. Claudin-1 expression levels in the colon of mice in each group (relative expression levels)

[0086] Figures 16-18 The results showed that, compared with the control group, the gene expression levels of three intestinal proteins MUC-2, E-cadherin, and Claudin-1 were significantly reduced in the D-galactose-induced model group mice (P < 0.05). Under the intervention of Bifidobacterium breve NKU BB1-13, the gene expression levels of three intestinal proteins MUC-2, E-cadherin, and Claudin-1 in the intervention group mice were significantly restored, indicating that supplementation with Bifidobacterium breve NKU BB1-13 can stimulate the gene expression levels of three intestinal proteins MUC-2, E-cadherin, and Claudin-1.

[0087] 5. Immunohistochemical assay to analyze the expression levels of three intestinal proteins in mouse colon. Paraffin-embedded tissue sections were fixed by baking at 60°C for 1 hour. After dewaxing, the sections were incubated overnight in a humidified chamber at 4°C with a specific primary antibody (primary antibody: MUC2, E-). The sections were then incubated with Cy3-conjugated goat anti-rabbit IgG (H+L) secondary antibody at room temperature for 60 min. DAB staining was performed for 5 min, followed by hematoxylin counterstaining for 1 min, and PBS soaking for 30 min to achieve blue reversion. The sections were dehydrated, air-dried, mounted with neutral resin, and images were acquired using an optical microscope. The results are as follows: Figure 19 .

[0088] from Figure 19 As can be seen, the control group mice showed extensive brown deposits in their colons, indicating normal high expression of three intestinal barrier proteins: MUC-2, E-cadherin, and Claudin-1. Normal high expression of these intestinal barrier proteins is crucial for maintaining intestinal barrier function, which ensures the barrier against harmful bacteria and their metabolites, preventing them from entering tissues, organs, or the bloodstream. In contrast, under the same field of view conditions, the area of ​​brown deposits observed in the colons of D-galactose-induced aging mice was significantly reduced, indicating a decline in colonic barrier function in aging mice. Intervention with *Bifidobacterium breve* NKU BB1-13 significantly stimulated the secretion of these three intestinal barrier proteins—MUC-2, E-cadherin, and Claudin-1—by colonic goblet cells, as evidenced by the increased brown deposits observed in the immunohistochemical staining of the colons of mice in the *Bifidobacterium breve* NKU BB1-13 intervention group.

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A short bifidobacterium ( Bifidobacterium breve NKU BB 1-13, characterized in that, The preservation number of the Bifidobacterium breve NKU BB 1-13 is GDMCC No: 65957.

2. A microbial preparation, characterized in that, Includes the Bifidobacterium breve NKU BB 1-13 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viability of *Bifidobacterium breve* NKU BB1-13 in the microbial preparation is ≥5×10⁻⁶. 9 CFU / mL or ≥5×10 9 CFU / g.

4. The method for preparing the microbial preparation according to claim 2 or 3, characterized in that, include: The aforementioned Bifidobacterium NKU BB 1-13 was cultured in a culture medium to obtain a microbial preparation.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 36~38℃; the culture time is 24~48h.

6. A biological agent, characterized in that, Includes the Bifidobacterium breve NKU BB 1-13 as described in claim 1 and excipients.

7. The use of Bifidobacterium breve NKU BB 1-13 as described in claim 1 in the preparation of antioxidant products.

8. The use of the Bifidobacterium breve NKU BB 1-13 as described in claim 1 in the preparation of anti-aging and / or gut health improvement products.

9. The application according to claim 7 or 8, characterized in that, The products include any one or more of the following: food, medicine, and health products.

10. The application according to claim 8, characterized in that, The application includes any one or more of the following (1) to (4): (1) Relieves inflammation and / or structural deterioration of the intestine; (2) Improves intestinal oxidative stress levels; (3) Improves intestinal inflammation levels; (4) Improve the intestinal barrier.

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