Application of bifidobacterium breve probiotics in scavenging free radicals and relieving oxidative aging

Abstract

The application belongs to the technical field of fermentation and postbiotic preparation, and particularly relates to application of Bifidobacterium breve postbiotic in removing free radicals and relieving oxidative aging. The application finds through oxidative free radical removal experiment that the Bifidobacterium breve NKU BB 1-2 postbiotic has good oxidative free radical removal capacity, and has good removal capacity for hydroxyl radicals, superoxide anions and DPPH free radicals. Meanwhile, through component analysis, it is found that the Bifidobacterium breve NKU BB 1-2 postbiotic contains various biological active substances with antioxidant and anti-aging properties such as flavonoids, indoles, amino acids, bile acids and ceramides. And cell model experiment verifies that the Bifidobacterium breve NKU BB 1-2 postbiotic can significantly reduce oxidative damage suffered by cells, improve the antioxidant capacity of cells, and thus relieve oxidative aging.

Description

Technical Field

[0001] This invention belongs to the field of fermentation and postbiotic preparation technology, specifically relating to the application of Bifidobacterium breve postbiotic in scavenging free radicals and alleviating oxidative aging. Background Technology

[0002] Anti-aging research has always been a hot topic in the scientific community. With the increasing aging of the population, people's need to delay aging and maintain health is becoming more and more urgent. Oxidative aging is one of the core concepts of modern aging theory. Its core idea is that life activities or special environments will continuously produce oxidative free radicals, such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. These oxidative free radicals will constantly attack key biomolecules in cells and have a negative impact on key life activities of tissues and organs. Young individuals usually have a sophisticated antioxidant defense system (such as superoxide dismutase SOD, glutathione, vitamin C / E, etc.) that can effectively scavenge or neutralize these oxidative free radicals to achieve a dynamic balance between oxidation and antioxidation.

[0003] Although aging is a normal life process of organisms, the occurrence of aging often leads to a shift in redox homeostasis towards oxidation. The weakening of the body's antioxidant capacity and the increase in the generation of endogenous free radicals accelerate aging. The acceleration of aging further aggravates this oxidation process. This continuous and gradually aggravated oxidative stress leads to the accumulation of damage to biomolecules, such as increased DNA mutations, protein misfolding and loss of function, and lipid peroxidation that damages cell membrane structure.

[0004] Among all organ systems in the body, the gut, due to its unique physiological environment and function, has become a focal point of research in oxidative aging. The gut's high metabolic rate, high exposure, and vast microbial ecosystem make it highly susceptible to oxidative stress. Localized oxidative damage in the gut leads to barrier dysfunction and dysbiosis, which in turn triggers systemic inflammation and oxidative stress, creating a vicious cycle that drives systemic aging. Understanding the gut's central role in oxidative aging and researching how to maintain intestinal redox homeostasis, protect the intestinal barrier, and regulate healthy flora has become a key breakthrough and cutting-edge research area for delaying aging and preventing age-related diseases.

[0005] The International Society for the Study of Probiotics and Prebiotics (ISAPP) defines postbiotics as "preparations of non-living microorganisms and / or their components that are beneficial to the health of the host," requiring the presence of inactivated microbial cells or cell components (such as cell wall fragments, peptidoglycans, etc.) and clinically validated health benefits. Compared to live probiotic preparations, postbiotics offer higher stability, stronger safety profiles, and a wider range of applications, including but not limited to regulating gut microbiota, enhancing intestinal barrier function, and modulating immunity and metabolism. Current research on the application of postbiotics mainly focuses on two main viewpoints: one is that postbiotics... It should be considered as a whole mixture of various beneficial substances. Secondly, it is believed that the metabiotic components should be clearly defined, and certain beneficial components should be purified when necessary. However, the ISAPP consensus on metabiotics stipulates that an effective metabiotic must contain inactivated microbial cells or cellular components that are beneficial to the host. Microbial fermentation broth metabolites (fermentation supernatant) are not a necessary condition. That is, according to the ISAPP consensus document, although fermentation broth metabolites have been experimentally verified to be beneficial to the host, they cannot be called metabiotics, but should be called cell-free supernatant. This provides scientific support for researchers in this field to study metabiotics.

[0006] Bifidobacterium breve is a Gram-positive anaerobic bacillus belonging to the family Actinobacteriaceae and the genus Bifidobacterium. It is widely used in research on fermented dairy products, dietary supplements, and clinical nutrition. Live Bifidobacterium breve has a variety of effects and benefits, mainly including regulating the balance of intestinal flora, enhancing immunity, improving digestive function, and reducing the risk of intestinal diseases. However, there is very little research on the postbiotic effects of Bifidobacterium breve, especially on its role in alleviating oxidative aging and scavenging free radicals, which needs further development. Summary of the Invention

[0007] The purpose of this invention is to provide the application of Bifidobacterium breve postbiotic in scavenging free radicals and alleviating oxidative aging. The Bifidobacterium breve NKU BB 1-2 postbiotic preparation can effectively scavenge free radicals in the body, and has the functions of anti-oxidation and alleviating oxidative aging, with significant effects.

[0008] This invention provides a postbiotic preparation of Bifidobacterium breve NKU BB 1-2, comprising a suspension of inactivated Bifidobacterium breve NKU BB 1-2; the preservation number of Bifidobacterium breve NKU BB 1-2 is GDMCC No:65956.

[0009] Preferably, the bacterial concentration in the inactivated Bifidobacterium NKU BB 1-2 bacterial suspension is 1 × (10⁻⁶)⁻¹. 6 ~10 9 CFU / mL.

[0010] The present invention also provides a method for preparing the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 as described in the above technical solution, comprising the following steps: resuspending the cells of Bifidobacterium breve NKU BB 1-2 with sterile physiological saline or sterile PBS buffer to obtain a resuspension of Bifidobacterium breve NKU BB 1-2 cells;

[0011] The bacterial suspension of Bifidobacterium breve NKU BB 1-2 was inactivated at 70-80℃ for 10-30 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0012] Preferably, the method for obtaining the Bifidobacterium breve NKU BB 1-2 cells includes the following steps: fermenting Bifidobacterium breve NKU BB 1-2 to obtain a fermentation culture broth of Bifidobacterium breve NKU BB 1-2;

[0013] The fermentation broth of Bifidobacterium breve NKU BB 1-2 was subjected to solid-liquid separation, and the precipitate was collected to obtain the bacterial cells of Bifidobacterium breve NKU BB 1-2.

[0014] The present invention also provides the application of the Bifidobacterium breve NKU BB 1-2 postbiotic preparation described in the above technical solution or the preparation method described in the above technical solution in the preparation of antioxidant products.

[0015] Preferably, the antioxidant product includes products that have one or more functions such as scavenging free radicals, delaying oxidative aging, and reducing cell damage.

[0016] The present invention also provides an antioxidant product, the active ingredient of which includes a postbiotic preparation of Bifidobacterium breve NKU BB 1-2; the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 is the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 described in the above technical solution or the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 prepared by the preparation method described in the above technical solution.

[0017] Preferably, the content of the Bifidobacterium breve NKU BB 1-2 postbiotic preparation in the antioxidant product is 90.0 wt.%~99.9 wt.%.

[0018] Beneficial effects:

[0019] This invention provides a postbiotic preparation of *Bifidobacterium breve* NKU BB 1-2, comprising a resuspension of inactivated *Bifidobacterium breve* NKU BB 1-2 bacterial cells; the preservation number of *Bifidobacterium breve* NKU BB 1-2 is GDMCC No:65956. Furthermore, this invention also provides a method for preparing the postbiotic preparation of *Bifidobacterium breve* NKU BB 1-2 described above, comprising the following steps: resuspending *Bifidobacterium breve* NKU BB 1-2 bacterial cells in sterile physiological saline or sterile PBS buffer to obtain a resuspension of *Bifidobacterium breve* NKU BB 1-2 bacterial cells; inactivating the resuspension of *Bifidobacterium breve* NKU BB 1-2 bacterial cells at 70-80℃ for 10-30 min to obtain the postbiotic preparation of *Bifidobacterium breve* NKU BB 1-2. This invention, through oxidative free radical scavenging experiments, discovered that *Bifidobacterium breve* NKU BB 1-2 postbiotic possesses excellent oxidative free radical scavenging ability, showing good scavenging capacity against hydroxyl radicals, superoxide anions, and DPPH radicals. Furthermore, component analysis revealed that *Bifidobacterium breve* NKU BB 1-2 postbiotic contains various bioactive substances with antioxidant and anti-aging properties, including flavonoids, indoles, amino acids, bile acids, and ceramides. Cell model experiments verified that *Bifidobacterium breve* NKU BB 1-2 postbiotic can significantly reduce oxidative damage to cells, enhance cellular antioxidant capacity, and thus alleviate oxidative aging.

[0020] Biological Preservation Information

[0021] Bifidobacterium breve NKU BB 1-2, biologically classified as Bifidobacterium breve It was deposited on March 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:65956. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 A statistical graph showing the hydroxyl radical scavenging capacity of the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 and the control strain in Test Example 1;

[0024] Figure 2 A statistical graph showing the superoxide anion scavenging capacity of the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 and the control strain in Test Example 1;

[0025] Figure 3A statistical chart showing the DPPH free radical scavenging capacity of the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 and the control strain in Test Example 1.

[0026] Figure 4 A statistical chart of indole components in the prebiotic preparation of Bifidobacterium breve NKU BB 1-2 in Test Example 2;

[0027] Figure 5 A statistical chart of amino acid composition in the prebiotic preparation of Bifidobacterium breve NKU BB 1-2 in Test Example 2;

[0028] Figure 6 A statistical chart of bile acid components in the prebiotic preparation of Bifidobacterium breve NKU BB 1-2 in Test Example 2;

[0029] Figure 7 A statistical chart of ceramide components in the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 in Test Example 2;

[0030] Figure 8 A statistical chart of flavonoid components in the prebiotic preparation after testing Bifidobacterium breve NKU BB 1-2 in Example 2;

[0031] Figure 9 The results show the differences in indolelacic acid and L-phenylalanine in the post-bifidobacterium NKU BB 1-2 post-biotic preparations under different heat treatment conditions in Test Example 2;

[0032] Figure 10 The results of the differences in 3-oxo-5-beta-cholic acid and dehydrolubeol in the post-bifidobacterium NKU BB 1-2 biotic preparations under different heat treatment conditions in Example 2 were as follows:

[0033] Figure 11 The staining images of β-galactosidase in different groups in test example 3;

[0034] Figure 12 This is a statistical graph showing the area of ​​the blue region stained with β-galactosidase in different groups in test example 3.

[0035] Figure 13 A statistical graph showing the results of SOD assay in different groups of cells in Test Example 3;

[0036] Figure 14 A statistical graph showing the results of MDA assays in different groups of cells in Test Example 3;

[0037] Figure 15 A statistical graph showing the results of CAT assays in different groups of cells in Test Example 3;

[0038] Figure 16A statistical graph showing the results of GSH-PX assays in different groups of cells in Test Example 3;

[0039] exist Figures 12-16 middle," "Indicates significant difference" P <0.05, "Indicates significant difference" P <0.01, "Indicates significant difference" P <0.001. Detailed Implementation

[0040] This invention provides a postbiotic preparation of Bifidobacterium breve NKU BB 1-2, comprising a suspension of inactivated Bifidobacterium breve NKU BB 1-2; the preservation number of Bifidobacterium breve NKU BB 1-2 is GDMCC No:65956.

[0041] The Bifidobacterium breve NKU BB 1-2 described in this invention was isolated from infant fecal samples, and in vitro antioxidant capacity assessment experiments showed that Bifidobacterium breve NKU BB 1-2 has good oxidative free radical scavenging ability.

[0042] As one embodiment, the bacterial concentration in the inactivated Bifidobacterium NKU BB 1-2 bacterial suspension of the present invention is 1×(10⁻⁶)⁻¹. 6 ~10 9 (CFU / mL); As another embodiment, the bacterial concentration in the inactivated Bifidobacterium NKU BB 1-2 bacterial suspension of the present invention is 1×10⁻⁶ CFU / mL. 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL or 1×(10) 6 ~10 9 Any concentration within the range of CFU / mL.

[0043] The present invention also provides a method for preparing the post-biotic preparation of Bifidobacterium breve NKU BB 1-2 as described in the above technical solution, comprising the following steps: resuspending Bifidobacterium breve NKU BB 1-2 cells with sterile physiological saline or sterile PBS buffer to obtain a resuspension of Bifidobacterium breve NKU BB 1-2 cells; inactivating the resuspension of Bifidobacterium breve NKU BB 1-2 cells at 70~80℃ for 10~30 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0044] In one embodiment, the present invention ferments and culturees *Bifidobacterium breve* NKU BB 1-2 to obtain a fermentation culture broth of *Bifidobacterium breve* NKU BB 1-2. In one embodiment, the fermentation and culture temperature is 36-38°C; in another embodiment, the fermentation and culture temperature is 37°C. In one embodiment, the fermentation and culture time is 46-50 hours; in another embodiment, the fermentation and culture time is 47-48 hours; in another embodiment, the fermentation and culture reaches the mid-logarithmic growth phase of *Bifidobacterium breve* NKU BB 1-2; in another embodiment, the fermentation and culture reaches an OD value of 2.0 for *Bifidobacterium breve* NKU BB 1-2. In one embodiment, the culture medium for the fermentation and culture is TPY liquid medium. In one embodiment, the inoculum amount of *Bifidobacterium breve* NKU BB 1-2 is 1%-3% of the culture medium volume; in another embodiment, the inoculum amount of *Bifidobacterium breve* NKU BB 1-2 is 2% of the culture medium volume.

[0045] After obtaining the fermentation culture broth of *Bifidobacterium breve* NKU BB 1-2, as one embodiment, the present invention performs solid-liquid separation on the fermentation culture broth of *Bifidobacterium breve* NKU BB 1-2, collects the precipitate, and obtains the *Bifidobacterium breve* NKBBB 1-2 cells. As one embodiment, the solid-liquid separation can be performed by centrifugation; as another embodiment, the centrifugation speed can be 4000g; the centrifugation speed can be 10min.

[0046] After obtaining the *Bifidobacterium breve* NKU BB 1-2 cells, as one embodiment, the *Bifidobacterium breve* NKU BB 1-2 cells are washed 2-3 times. As one embodiment, the washing is performed using a resuspending agent. As one embodiment, the resuspending agent is sterile physiological saline or sterile PBS buffer.

[0047] After obtaining the *Bifidobacterium breve* NKU BB 1-2 bacterial cells, the present invention resuspends the *Bifidobacterium breve* NKU BB 1-2 bacterial cells in sterile physiological saline or sterile PBS buffer to obtain a *Bifidobacterium breve* NKU BB 1-2 bacterial resuspension. As one embodiment, the concentration of the *Bifidobacterium breve* NKU BB 1-2 bacterial resuspension is 1 × (10⁻⁶)⁻¹. 6 ~10 9 (CFU / mL); As another embodiment, the concentration of the Bifidobacterium breve NKU BB 1-2 bacterial suspension of the present invention is 1×10 CFU / mL. 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8CFU / mL, 1×10 9 CFU / mL or 1×(10) 6 ~10 9 Any concentration within the range of CFU / mL.

[0048] After obtaining the resuspension of *Bifidobacterium breve* NKU BB 1-2, this invention inactivates the resuspension at 70-80℃ for 10-30 minutes to obtain the post-biotic preparation of *Bifidobacterium breve* NKU BB 1-2. As one embodiment, the method of resuspensing the *Bifidobacterium breve* NKU BB 1-2 before inactivation ensures uniform heating of the resuspension, resulting in more thorough inactivation and disruption of the bacteria, avoiding the problem of incomplete inactivation that occurs with direct heat inactivation of the bacteria. As one embodiment, the inactivation temperature can be 70-75℃; as another embodiment, the inactivation temperature can be 70℃. As one embodiment, the inactivation time can be 10-20 minutes; as another embodiment, the inactivation time can be 10 minutes. The heat treatment conditions described above for inactivation at 70°C for 10 minutes in this invention can ensure that the high-abundance active ingredients are deactivated as little as possible during the heat treatment process.

[0049] This invention also provides the application of the *Bifidobacterium breve* NKU BB 1-2 postbiotic preparation described in the above-described technical solutions, or the *Bifidobacterium breve* NKU BB 1-2 postbiotic preparation prepared by the preparation method described in the above-described technical solutions, in the preparation of antioxidant products. As one embodiment, the antioxidant product of this invention possesses one or more functions including scavenging free radicals, delaying oxidative aging, and reducing cell damage; as another embodiment, the antioxidant product possesses the functions of scavenging free radicals and / or delaying oxidative aging; as yet another embodiment, the oxidative aging can be the alleviation of D-galactose-induced oxidative aging of Caco-2 cells, and the D-galactose-induced Caco-2 cell oxidative aging model can simulate the alleviating effect of postbiotics on the oxidative aging of intestinal epithelial cells after ingestion in real-world application scenarios. The postbiotic preparation of Bifidobacterium breve NKU BB 1-2 described in this invention has been evaluated in vitro and shows good oxidative group scavenging ability and a large number of antioxidant bioactive components. Compared with other Bifidobacterium breve strains, Bifidobacterium breve NKU BB 1-2 has a better oxidative group scavenging ability. Furthermore, the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 has been verified by cell experiments to have the effect of assisting in antioxidation and alleviating oxidative aging.

[0050] This invention also provides an antioxidant product, the active ingredient of which includes a Bifidobacterium breve NKU BB 1-2 postbiotic preparation; the Bifidobacterium breve NKU BB 1-2 postbiotic preparation is the Bifidobacterium breve NKU BB 1-2 postbiotic preparation described in the above-mentioned technical solution or the Bifidobacterium breve NKU BB 1-2 postbiotic preparation prepared by the preparation method described in the above-mentioned technical solution. As one embodiment, the content of the Bifidobacterium breve NKU BB 1-2 postbiotic preparation in the antioxidant product is 90.0 wt.%~99.9 wt.%. This invention does not specifically limit the type of excipients in the antioxidant product; conventional excipients in the art can be added as needed.

[0051] 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.

[0052] In the following examples, the TPY liquid culture medium (components g / L) contained: hydrolyzed casein 10.0 g, plant peptone 5.0 g, yeast extract 2.0 g, glucose 5.0 g, L-cysteine ​​0.5 g, dipotassium hydrogen phosphate 2.0 g, magnesium chloride 0.5 g, zinc sulfate 0.25 g, calcium chloride 0.15 g, ferric chloride 0.0001 g, and Tween-80 1.0 g.

[0053] Unless otherwise specified, the following embodiments are all conventional methods;

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] Example 1

[0056] A method for preparing a post-biotic preparation of Bifidobacterium breve NKU BB1-2, comprising the following steps:

[0057] Bifidobacterium breve NKU BB 1-2 was inoculated into TPY liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) at an inoculation rate of 2% of the culture medium volume. Fermentation was carried out at 37℃ for 48 hours until the OD value of Bifidobacterium breve NKU BB 1-2 reached 2.0, yielding the Bifidobacterium breve NKU BB 1-2 fermentation broth. The fermentation broth was centrifuged at 4000g for 10 minutes, and the precipitate was collected. The precipitate was washed twice with sterile physiological saline and then resuspended in sterile physiological saline to obtain a bacterial resuspension. The concentration of Bifidobacterium breve NKU BB 1-2 in the bacterial resuspension was 1×10⁻⁶. 9 CFU / mL.

[0058] The bacterial resuspension was heat-treated at 70℃ for 10 min to obtain a post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0059] Comparative Example 1

[0060] A method for preparing a live bacterial suspension of Bifidobacterium breve NKU BB 1-2, comprising the following steps:

[0061] A live bacterial suspension of *Bifidobacterium breve* NKU BB 1-2 was prepared using the method described in Example 1, and the concentration of *Bifidobacterium breve* NKU BB 1-2 in the bacterial suspension was 1 × 10⁻⁶. 9 CFU / mL.

[0062] Comparative Example 2

[0063] A method for preparing a post-biotic preparation of Bifidobacterium breve NKU BB1-2, comprising the following steps:

[0064] A bacterial resuspension was prepared using the method described in Example 1, and the concentration of Bifidobacterium breve NKU BB 1-2 in the bacterial resuspension was 1×10⁻⁶. 9 CFU / mL;

[0065] The bacterial resuspension was subjected to ultrasonic treatment at 300W for 10 seconds at 10 seconds for 20 minutes to obtain the Bifidobacterium breve NKU BB 1-2 post-biotic preparation.

[0066] Comparative Example 3

[0067] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, except that the bacterial resuspension was heat-treated at 60°C for 10 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0068] Comparative Example 4

[0069] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, the difference being that the bacterial resuspension was heat-treated at 60°C for 30 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0070] Example 2

[0071] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, except that the bacterial resuspension was heat-treated at 70°C for 30 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0072] Example 3

[0073] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, except that the bacterial resuspension was heat-treated at 80°C for 10 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0074] Example 4

[0075] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, the difference being that the bacterial resuspension was heat-treated at 80°C for 30 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

[0076] Comparative Example 5

[0077] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, except that Bifidobacterium breve NKU BB 1-2 in Example 1 was replaced with Bifidobacterium breve (B. breve) ATCC 15700 (purchased from China Industrial Microbial Culture Collection Center (CICC)).

[0078] Comparative Example 6

[0079] The post-biotic preparation of Bifidobacterium breve NKU BB 1-2 was prepared using the preparation method in Example 1, except that Bifidobacterium breve NKU BB 1-2 in Example 1 was replaced with Bifidobacterium breve ATCC 15701 (purchased from China Industrial Microbial Culture Collection Center (CICC)).

[0080] Test Example 1

[0081] The evaluation of the oxidative group scavenging ability of the post-biotic preparations of *Bifidobacterium breve* NKU BB 1-2 prepared in Examples 1, 1-2, and 5-6 was conducted as follows:

[0082] 1. Hydroxyl radical scavenging ability

[0083] Hydroxyl radicals act on biomolecules such as proteins, nucleic acids, and lipids in the body, causing damage to cell structure and function, which in turn leads to metabolic disorders and diseases. The ability to scavenge hydroxyl radicals is one of the important indicators of the antioxidant capacity of a substance being tested.

[0084] 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.

[0085] Based on the above principle, the hydroxyl radical scavenging capacity of the live Bacillus brevis NKU BB 1-2 suspension in Comparative Example 1 and the post-biotic preparations of Bacillus brevis NKU BB 1-2 in Comparative Examples 2, 1, 5, and 6 were determined using the Fenton colorimetric method. The results are as follows: Figure 1 As shown in Table 1, in Figure 1 In the above, NKU BB 1-2, NKU BB 1-2 post-gene (thermal inactivation), NKU BB 1-2 post-gene (ultrasound), B.breve ATCC 15700 and B.breve ATCC 15701 refer to the results in Comparative Example 1, Example 1, Comparative Example 2, Comparative Example 5 and Comparative Example 6, respectively. Figure 2 and Figure 3 Similarly.

[0086] Table 1 shows the hydroxyl radical scavenging rates of the live bacterial suspension of *Bifidobacterium breve* NKU BB 1-2 in Comparative Example 1, and the postbiotic preparations of *Bifidobacterium breve* from Examples 1, 2, and 5-6.

[0087]

[0088] Depend on Figure 1 As shown in Table 1, the hydroxyl radical scavenging abilities of the post-biotics of the standard strains of *Bifidobacterium breve* prepared under the same conditions were 11.58±1.35% and 6.62±1.08%, respectively. However, the hydroxyl radical scavenging ability of the heat-treated post-biotics of *Bifidobacterium breve* NKU BB 1-2 was significantly higher than that of the two standard strains of *Bifidobacterium breve* post-biotics. The hydroxyl radical scavenging ability of the heat-treated post-biotics of *Bifidobacterium breve* NKU BB 1-2 was significantly higher than that of the live strain of *Bifidobacterium breve* NKU BB 1-2. The hydroxyl radical scavenging ability of the heat-treated post-biotics of *Bifidobacterium breve* NKU BB 1-2 was not significantly different from that of the ultrasonically treated post-biotics of *Bifidobacterium breve* NKIBB 1-2.

[0089] 2. Superoxide anion scavenging ability

[0090] 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-Under the action of p-aminobenzenesulfonamide and naphthylethylenediamine hydrochloride, a red azo compound is generated, which has a characteristic absorption peak at 530 nm. The content of superoxide anion in the sample can be calculated based on the A530 value, and then the scavenging rate of superoxide anion can be obtained, reflecting the superoxide anion scavenging ability.

[0091] Based on the above, the superoxide anion scavenging capacity of the live bacterial suspension in Comparative Example 1 and the postbiotic preparations of Bifidobacterium breve prepared in Comparative Example 2, Example 1, and Comparative Examples 5-6 was determined using the hydroxylamine method. The results are as follows: Figure 2 As shown in Table 2.

[0092] Table 2 shows the superoxide anion scavenging capacity of the *Bifidobacterium breve* NKU BB 1-2 live bacterial suspension in Comparative Example 1, and the postbiotic preparations of *Bifidobacterium breve* from Examples 1, 2, and 5-6.

[0093]

[0094] Figure 2 As shown in Table 2, the superoxide anion scavenging abilities of the two Bifidobacterium breve strains prepared under the same conditions were 72.48±1.70 and 56.67±1.02, respectively. The superoxide anion scavenging ability of the heat-treated Bifidobacterium breve NKU BB 1-2 postbiotic preparation was higher than that of the two wild-type Bifidobacterium breve strains. The superoxide anion scavenging ability of the heat-treated Bifidobacterium breve NKU BB 1-2 postbiotic preparation was significantly higher than that of the live Bifidobacterium breve NKU BB 1-2 strain. The superoxide anion scavenging ability of the heat-treated Bifidobacterium breve NKU BB 1-2 postbiotic was not significantly different from that of the ultrasonically treated Bifidobacterium breve NKU BB 1-2 postbiotic.

[0095] 3. DPPH removal ability

[0096] Mix the 0.2 mM DPPH anhydrous ethanol solution with the sample at a 1:1 volume ratio, incubate in the dark at room temperature for 30 min, then centrifuge at 8000×g for 10 min, and measure the absorbance of the separated supernatant at 517 nm. The DPPH free radical scavenging activity (%) is calculated as follows:

[0097]

[0098] Where: AS - absorbance of the sample;

[0099] Absorbance of the blank consisting of the AB sample and ethanol;

[0100] The absorbance of the control composed of AC-DPPH solution and deionized water

[0101] The method was used to determine the viable bacterial suspension in Comparative Example 1 and the postbiotic preparations of Bifidobacterium breve prepared in Comparative Example 2, Example 1, and Comparative Examples 5-6. The results are as follows: Figure 3 And Table 3.

[0102] Table 3 shows the DPPH scavenging rates of the *Bifidobacterium breve* NKU BB 1-2 live bacterial suspension in Comparative Example 1, and the postbiotic preparations of *Bifidobacterium breve* from Examples 1, 2, and 5-6.

[0103]

[0104] Depend on Figure 3 As shown in Table 3, the DPPH scavenging capacity of the two standard strains of Bifidobacterium breve prepared under the same conditions was about 70%. The DPPH scavenging capacity of the NKU BB 1-2 postbiotic was significantly higher than that of the standard strain postbiotic. The DPPH scavenging capacity of the heat-treated NKU BB 1-2 postbiotic was significantly higher than that of the live NKU BB 1-2 strain. The DPPH scavenging capacity of the heat-treated NKU BB 1-2 postbiotic was not significantly different from that of the ultrasonically treated NKU BB 1-2 postbiotic.

[0105] In summary, the evaluation of the oxidative group scavenging ability of heat-inactivated Bifidobacterium breve NKU BB1-2 postbiotics mainly involved three comparisons: 1. Comparison of the oxidative group scavenging ability between the NKU BB1-2 postbiotics and live Bifidobacterium breve NKU BB1-2 bacteria; 2. The oxidative group scavenging ability of the NKU BB1-2 postbiotics prepared by the two postbiotic preparation methods (heat inactivation and ultrasonication); 3. The oxidative group scavenging ability of the NKU BB1-2 postbiotics compared with that of two other standard Bifidobacterium breve postbiotic strains. The following conclusions can be drawn: 1. The postbiotic of *Bifidobacterium breve* NKU BB 1-2 has a superior oxidative group scavenging ability compared to live *Bifidobacterium breve* NKU BB 1-2; 2. The two different preparation methods, heat inactivation and ultrasonication, have little effect on the oxidative group scavenging ability of the postbiotic of *Bifidobacterium breve* NKU BB 1-2. Considering industrial production conditions and costs, this invention selects heat inactivation to prepare the postbiotic of *Bifidobacterium breve* NKU BB 1-2; 3. Compared with postbiotics of other *Bifidobacterium breve* strains, the postbiotic of *Bifidobacterium breve* NKU BB 1-2 exhibits a superior oxidative group scavenging ability. Therefore, it is reasonable to infer that the postbiotic of *Bifidobacterium breve* NKU BB 1-2 obtained through heat inactivation treatment has excellent antioxidant capacity and potential probiotic properties to alleviate oxidative aging.

[0106] Test Example 2

[0107] Component analysis of the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 in Example 1 and component analysis of postbiotic preparations of Bifidobacterium breve NKU BB 1-2 prepared under different heat treatment conditions

[0108] 1. Non-targeted metabolomics determination of the components of the post-biotic preparation of Bifidobacterium breve NKU BB 1-2 in Example 1, the steps are as follows:

[0109] (1) Sample pretreatment

[0110] Cell (bacterial) samples were placed in EP tubes, and 300 μL of 80% methanol aqueous solution was added. The samples were then rapidly frozen in liquid nitrogen for 5 min, thawed on ice, vortexed for 30 s, and sonicated for 6 min. The samples were then centrifuged at 5000 rpm and 4℃ for 1 min. The supernatant was transferred to a new centrifuge tube and freeze-dried into a dry powder. The powder was dissolved in 10% methanol aqueous solution according to the volume of the sample and then analyzed by LC-MS.

[0111] (2) Mass spectrometry conditions

[0112] The scan range was selected as m / z 100-1500; the ESI source settings were as follows: Spray Voltage: 3.5kV; Sheath gas flow rate: 35psi; Aux gas flow rate: 10L / min; Capillary Temp: 320℃; S-lens RF level: 60℃; Aux gas heater temp: 350℃; Polarity: positive, negative; the MS / MS secondary scan was a data-dependent scan.

[0113] (3) Data processing and metabolite identification

[0114] The data files were converted to mzXML format using ProteoWizard. XCMS was first used for peak extraction and quantification. Peak alignment was performed for different samples using parameters such as retention time and mass-to-charge ratio. Then, the data was compared with a high-quality secondary spectrum database based on a set mass deviation of 10 ppm and information on adducted ions to identify metabolites. Background ions were then removed using a blank sample database, finally yielding the identification and relative quantification results of the metabolites. Data processing was performed using a Linux operating system (CentOS version 6.6) and software R and Python. The identified metabolites were annotated using the KEGG database (https: / / www.genome.jp / kegg / pathway.html), the HMDB database (https: / / hmdb.ca / metabolites), and the LIPIDMaps database (http: / / www.lipidmaps.org / ).

[0115] 2. Postbiotic component analysis of Bifidobacterium breve NKU BB 1-2

[0116] The non-targeted metabolic assay of *Bifidobacterium breve* NKU BB1-2 revealed a complex biogenic composition. Therefore, considering component abundance, antioxidant properties, and anti-aging effects, a classification analysis was performed on bioactive components with extensive applications or research. These components were mainly categorized into indoles and their derivatives, amino acids, bile acids, ceramides, and flavonoids. The results are as follows: Figures 4-8 As shown in Tables 4-8.

[0117] Table 4. Content data of indole and its derivative metabolites

[0118]

[0119] Table 5. Data on the content of amino acid metabolites

[0120]

[0121] Table 6. Data on the content of bile acid metabolites.

[0122]

[0123] Table 7. Data on the content of ceramide metabolites

[0124]

[0125] Table 8. Data on the content of flavonoid metabolites

[0126]

[0127] Depend on Figures 4-8As shown in Tables 4-8, among indole and its derivatives, indole-lactic acid (ILA) and D-tryptophan account for a relatively high proportion. Indole-lactic acid has significant effects in antioxidation, anti-inflammation, immunomodulation, and anti-tumor. It can not only scavenge free radicals, but also induce the expression of antioxidant enzymes and activate endogenous antioxidant pathways by binding to aryl hydrocarbon receptors (AhR). At the same time, it can significantly alleviate the process of oxidative aging by alleviating inflammation and improving immune aging. Similarly, D-tryptophan can also directly neutralize free radicals through its indole structure, reduce lipid peroxidation and DNA oxidative damage, and protect cell integrity. Among amino acid metabolites, short peptides composed of multiple amino acids and phenylalanine are present in relatively high proportions. Both possess unique biological activities in antioxidation and anti-aging. Short peptides composed of multiple amino acids can directly scavenge free radicals and also positively influence cells and tissues through various pathways. Phenylalanine (Phe-Pro) is a common component of collagen repeat sequences (such as Gly-XY, where X / Y is often Pro / Hyp), directly participating in maintaining the stability of the collagen triple helix structure. Collagen is the core structural protein of skin, bone, and connective tissue, and its integrity directly affects the tissue's anti-aging ability. Among bile acid metabolites, 3-oxo-5-beta-cholic acid is the most abundant.

[0128] As products of bile acid metabolism, gut microbiota-derived bile acid metabolites have been shown to participate in the body's bile acid metabolism, which is closely related to antioxidation, immune enhancement, and aging relief. Ceramide metabolites and flavonoid metabolites are two classes of bioactive substances with antioxidant and anti-aging effects that have been widely proven and applied. Ceramides can significantly improve endothelial aging, while flavonoids usually have a strong ability to scavenge free radicals, which can greatly reduce the negative impact of exogenous free radicals on tissues and organs.

[0129] 3. Analysis of differences in the biogenic active components of *Bifidobacterium breve* NKU BB1-2 under different heat treatment conditions in Examples 1, 2-4, and Comparative Examples 3-4.

[0130] To determine the optimal heat treatment conditions for *Bifidobacterium breve* NKU BB 1-2, i.e., to ensure complete heat inactivation of live cells while minimizing the loss of active ingredients due to heat treatment, non-targeted metabolomics analysis was used to analyze the differences in post-biotic active ingredients of *Bifidobacterium breve* NKIBB 1-2 under six heat treatment conditions: 60℃ for 10 min (Comparative Example 3); 60℃ for 30 min (Comparative Example 4); 70℃ for 10 min (Example 1); 70℃ for 30 min (Example 2); 80℃ for 10 min (Example 3); and 80℃ for 30 min (Example 4). The analysis primarily focused on the abundance changes of the most abundant metabolites among the common active ingredients, including indole-lactate (indole and its derivatives), L-phenylalanine (amino acids and their derivatives), 3-oxo-5-beta-cholic acid (bile acids), and dehydrolucopyroyl leaf alcohol (flavonoids). The content changes of these four active ingredients under different heat treatment conditions are shown below. Figures 9-10 As shown in Table 9, colony counts were performed on the post-biotics of Bifidobacterium NKU BB 1-2 prepared under each heat treatment condition.

[0131] Table 9. Metaphytic bacterial colony counts under different heat treatment conditions.

[0132]

[0133] Combination Figures 9-10 The results in Table 9 show that 70℃ for 10 min is the optimal heat treatment condition for Bifidobacterium breve NKU BB1-2. Based on this, extending the heat treatment time and increasing the heat treatment temperature will lead to varying degrees of loss of active ingredients; while decreasing the heat treatment temperature will result in incomplete inactivation of live bacterial cells, and live bacterial cells will remain in the metabiotic.

[0134] Test Example 3

[0135] Evaluation of the probiotic potential of Bifidobacterium breve NKU BB1-2 postbiotics in Example 1 to alleviate oxidative aging using a D-galactose-induced Caco-2 cell oxidative senescence model.

[0136] 1. D-galactose-induced oxidative senescence model in Caco-2 cells

[0137] Caco-2 cells were seeded in 96-well plates and cultured in a CO2 incubator for 24 hours to allow them to adhere. When the cells reached approximately 80% confluence under a microscope, the old culture medium was removed, and the cells were washed twice with sterile PBS. Serum-free DMEM medium was then added, and the cells were starved in a CO2 incubator for 12 hours. After starvation, the old culture medium was discarded, and the cells were washed twice with PBS. Serum-free medium containing D-galactose at final concentrations of 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM was added, respectively. After 24 hours of stimulation, the medium was removed, and fresh serum-free medium was added, followed by 10% CCK8 reagent. The cells were cultured in a CO2 incubator for 1 hour, and the absorbance was measured at 450 nm. The calculation method followed the CCK8 reagent kit instructions from Beyotime Biotechnology Co., Ltd. Based on the calculation results, the D-galactose concentration at which cell viability was approximately 50% was selected as the induction dose, thus establishing a D-galactose-induced senescence model in Caco-2 cells. Based on the results of the CCK-8 assay, Caco-2 cells can be induced to undergo oxidative senescence by stimulating them with serum-free medium containing 500 mM D-galactose for 24 h, thus establishing a Caco-2 cell oxidative senescence model.

[0138] 2. Determination of the dosage and method of intervention for Bifidobacterium breve NKU BB 1-2 postbiotic intervention

[0139] A gradient concentration of Bifidobacterium breve NKU BB 1-2 postbiotic formulations was prepared. The specific preparation method is as follows: the postbiotic concentration was quantified by the bacterial content per milliliter of liquid before heat treatment. Ten [units of concentration] were prepared before heat treatment. 4 10 5 10 6 10 7 10 8 10 9 Different concentrations of Bifidobacterium breve NKU BB 1-2 postbiotic preparations were prepared by heat treatment using a sterile saline resuspension of live bacteria at CFU / mL.

[0140] Caco-2 cells were seeded in 96-well plates and cultured in a CO2 incubator for 24 hours to allow them to adhere. When the cells reached approximately 80% confluence under a microscope, the old culture medium was removed, and the cells were washed twice with sterile PBS. Serum-free DMEM medium was then added, and the cells were starved in a CO2 incubator for 12 hours. After starvation, the old culture medium was discarded, and the cells were washed twice with PBS. Serum-free medium containing different concentrations of Bifidobacterium breve NKBBB 1-2 was added, and the cells were cultured for 24 hours. Fresh serum-free medium was then added, followed by 10% CCK8 reagent. After 1 hour of incubation in a CO2 incubator, the absorbance was measured at 450 nm and calculated according to the CCK8 reagent kit instructions from Beyotime Biotechnology Co., Ltd. The results were used to assess whether different concentrations of the metabiotic had a toxic effect on Caco-2 cells. According to the CCK-8 test results, all concentrations of the metabiotic showed no toxic effect on the cells; therefore, 10% CCK8 was selected as the optimal concentration. 9 The post-biotics prepared from CFU / mL live bacteria were used as high-dose intervention cells, and 10 6 The post-biotic prepared from CFU / mL live bacteria was used as the low-dose intervention group for cells. The intervention was carried out by co-culturing the cells with the post-biotic and D-galactose for 24 h to simulate the intervention scenario of post-biotic administration during the process of oxidative aging.

[0141] 3. Determination of β-galactosidase content in cells undergoing senescence

[0142] Caco-2 cells were seeded in 24-well plates. Modeling and intervention were performed as described above. The control group was starved and then cultured in fresh serum-free DMEM. After intervention, the cell culture medium was aspirated, the cells were washed once with sterile PBS, and stained with 0.5 mL of β-galactosidase fixative for 15 min at room temperature. After washing three times with sterile PBS, 500 μL of staining working solution was added, and the plates were sealed with Parafilm sealing film and incubated overnight at 37°C. Microscopic observation was performed, and images were taken of the center of each well to assess the senescence of Caco-2 cells in each group. The results are as follows: Figure 11 As shown, the upper and lower figures of the same group represent two repetitions of the same group, indicating that the results are not accidental.

[0143] Figure 11 The staining images of Caco-2 cells in each group clearly show that, under the same field of view, the model group had more senescent cells stained blue-green compared to the control group. In the high-dose intervention group and the low-dose intervention group treated with Bifidobacterium breve NKU BB 1-2, the blue-green color was significantly reduced, indicating a decrease in the number of senescent cells. This senescence-relieving effect was observed in both high-dose and low-dose groups, with a slightly better dose-response relationship at the high-dose level.

[0144] Furthermore, Figure 12 The blue-green area was quantified using ImageJ graphics processing software. The blue-green area in the field of view of each group of cells was measured, revealing a significantly higher blue-green area in the model group, accurately reflecting increased cellular senescence. The post-biotic intervention group not only reversed this oxidative senescence, but the high-dose intervention group also showed a better effect in alleviating cellular senescence than the control group. Measurement of β-galactosidase content in cellular senescence indicated the beneficial effect of *Bifidobacterium breve* NKU BB1-2 post-biotics in alleviating Caco-2 cell senescence.

[0145] 4. Measurement of cell biochemical indicators

[0146] Caco-2 cells were seeded in 12-well plates. Modeling and intervention were performed as described above. After intervention, the cell culture medium was aspirated, and the cells were washed twice with sterile PBS. 100 μL of cell lysis buffer was added for lysis, and the cells were collected in 1.5 mL centrifuge tubes. The cells were centrifuged at 4°C and 12000 × g for 10 min. The supernatant was aliquoted into new centrifuge tubes for determination of the levels of antioxidant enzymes SOD, MDA, CAT, and GSH-PX in the cells. The levels were measured using commercially available assay kits from Nanjing Jiancheng Biotechnology Research Institute, following the kit instructions. The test results are as follows: Figures 13-16 As shown.

[0147] Tests on SOD and MDA in cells showed that, compared with the control group, the model group cells had significantly reduced SOD activity and increased MDA content; tests on CAT and GSH-PX in cells showed that the activities of CAT and GSH-PX in the model group cells were also significantly reduced. SOD, CAT, and GSH-PX are all important components of the cell's own antioxidant defense system, playing an important role in scavenging oxidative free radicals and reducing the negative effects of oxidative stress; MDA can reflect the degree of negative effects of oxidative stress on cells, and a high MDA value often indicates stronger oxidative stress damage.

[0148] This indicates that under the intervention of D-galactose, cells experienced extremely strong oxidative stress, resulting in a state where oxidation exceeded antioxidation, thus triggering accelerated cellular senescence. The intervention of Bifidobacterium breve NKU BB 1-2 postbiotic significantly alleviated this negative trend, manifested by increased SOD, CAT, and GSH-PX activities and decreased MDA content. Measurements of four indicators related to oxidative damage showed that the Bifidobacterium breve NKU BB 1-2 postbiotic assisted Caco-2 cells in clearing exogenous oxidative stimuli, enhancing endogenous antioxidant capacity, alleviating cellular oxidative stress, and thus mitigating the probiotic effect of Caco-2 cell senescence.

[0149] From the above examples, it can be concluded that the Bifidobacterium breve NKU BB 1-2 postbiotic preparation of the present invention can effectively scavenge free radicals in the body, and has the functions of anti-oxidation and alleviating oxidative aging, with significant effects.

[0150] 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 1-2 post-biotic formulation, characterized in that, The contents include a suspension of inactivated Bifidobacterium breve NKU BB 1-2; the preservation number of Bifidobacterium breve NKU BB 1-2 is GDMCC No:65956; The bacterial cell concentration in the inactivated bifidobacterium NKU BB 1-2 bacterial suspension was 1×(10⁻⁶). 6 ~10 9 CFU / mL; The Bifidobacterium breve postbiotic preparation contains flavonoids, indoles, amino acids, bile acids, and ceramides. The Bifidobacterium breve postbiotic preparation is prepared by inactivating the Bifidobacterium breve NKU BB 1-2 bacterial suspension at 70~80℃ for 10~30min.

2. The method for preparing the post-biotic preparation of Bifidobacterium breve NKU BB 1-2 as described in claim 1, characterized in that, The procedure includes the following steps: resuspending Bifidobacterium breve NKU BB 1-2 cells in sterile physiological saline or sterile PBS buffer to obtain a resuspension of Bifidobacterium breve NKU BB 1-2 cells; The bacterial suspension of Bifidobacterium breve NKU BB 1-2 was inactivated at 70-80℃ for 10-30 min to obtain the post-biotic preparation of Bifidobacterium breve NKU BB 1-2.

3. The preparation method according to claim 2, characterized in that, The method for obtaining the Bifidobacterium breve NKU BB 1-2 cells includes the following steps: fermenting Bifidobacterium breve NKU BB 1-2 to obtain the fermentation culture broth of Bifidobacterium breve NKU BB 1-2; The fermentation broth of Bifidobacterium breve NKU BB 1-2 was subjected to solid-liquid separation, and the precipitate was collected to obtain the bacterial cells of Bifidobacterium breve NKU BB 1-2.

4. The application of the Bifidobacterium breve NKU BB 1-2 postbiotic preparation according to claim 1 or the preparation method according to claim 2 or 3 in the preparation of antioxidant products.

5. The application according to claim 4, characterized in that, The antioxidant products include those that possess one or more functions such as scavenging free radicals, delaying oxidative aging, and reducing cell damage.

6. An antioxidant product, characterized in that, The active ingredient includes a postbiotic preparation of Bifidobacterium breve NKU BB 1-2; the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 is the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 as described in claim 1 or the postbiotic preparation of Bifidobacterium breve NKU BB 1-2 prepared by the preparation method described in claim 2 or 3.

7. The antioxidant product according to claim 6, characterized in that, The content of the Bifidobacterium breve NKU BB 1-2 postbiotic preparation in the antioxidant product is 90.0 wt.%~99.9 wt.%.

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