Bifidobacterium breve NHNK-630 and use thereof in the preparation of a product for preventing and treating retinal macular degeneration

Abstract

The present application relates to the field of microbial technology, in particular to Bifidobacterium breve NHNK-630 and its application in preparing products for preventing and treating retinal macular degeneration. Bifidobacterium breve The present application isolates Bifidobacterium breve (Bifidobacterium breve) NHNK-630 from the feces of healthy female infants, and the live bacteria, inactivated bacteria or fermentation products of Bifidobacterium breve NHNK-630 have the functions of improving the antioxidant capacity of the retina, increasing the retinal barrier, reducing retinal apoptosis, reducing retinal neovascularization and reducing retinal inflammation; in addition, the biofilm of Bifidobacterium breve NHNK-630 itself can increase the colonization ability to intestinal epithelial cells, so that Bifidobacterium breve NHNK-630 can be used for preparing products for preventing and treating retinal macular degeneration.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Bifidobacterium breve NHNK-630 and its application in the preparation of products for the prevention and treatment of macular degeneration. Background Technology

[0002] Macular degeneration primarily affects the central area of ​​the retina (macula), leading to progressive vision loss. Age-related macular degeneration is closely related to aging, lifestyle, and dietary habits, and is more common in the elderly. Its typical pathological features include degenerative changes in retinal pigment epithelial cells and apoptosis of cells in the macular region.

[0003] Age-related macular degeneration (AMD) is classified into two types: wet and dry. The main pathological mechanisms of dry AMD include accumulated oxidative stress, chronic inflammation, and retinal pigment epithelial cell apoptosis. Excessive production of reactive oxygen species leads to protein misfolding, lipid peroxidation, and persistent activation of the immune system, thereby inducing retinal pigment epithelial cell damage and photoreceptor degeneration. The main pathological mechanisms of wet AMD include the growth of abnormal blood vessels originating from the choroidal layer of the eye into the retina, and fluid leakage from the blood into the retina.

[0004] Currently, the primary treatment for retinal inflammation in degenerative retinal diseases such as age-related macular degeneration (AMD) is intravitreal injection of triamcinolone acetonide. While it effectively inhibits retinal edema and inflammatory cell infiltration caused by inflammation, complications such as steroid-induced glaucoma and acute endophthalmitis are common. Anti-vascular endothelial growth factor (VEGF) therapy is widely recognized as the first-line clinical treatment for wet age-related macular degeneration. However, anti-VEGF agents are typically administered via intravitreal injection, which is unpopular with patients and can lead to side effects (e.g., red eye, eye pain, infection). The frequency or number of injections can also impose a financial burden on patients and result in decreased disease control. Summary of the Invention

[0005] To overcome the above problems, the present invention provides Bifidobacterium breve NHNK-630 and its application in the preparation of products for the prevention and treatment of macular degeneration.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium breve NHNK-630 was deposited on March 10, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025432.

[0007] The Bifidobacterium breve described in this invention ( Bifidobacterium breve NHNK-630, derived from the feces of a healthy female infant, was identified as Bifidobacterium breve by 16S rDNA analysis. Bifidobacterium breve This strain is Gram-positive and appears as rod-shaped or rod-shaped cells under a microscope. When grown on a Deman-Rogosa-Sharp (MRS) solid plate containing 0.1% (m / v) cysteine, it forms smooth, opaque colonies with neat edges and milky-white, round, raised surfaces. In MRS liquid medium containing 0.1% (m / v) cysteine, it grows in a uniformly turbid manner, and the cells form a white precipitate upon prolonged standing. The optimal growth temperature is 37°C.

[0008] A second aspect of the present invention provides a microecological preparation comprising the *Bifidobacterium breve* described in the first aspect (…). Bifidobacterium breve NHNK-630 strain, pure culture, inactivated bacterial cells, extract, fermentation product lysate, or a combination thereof.

[0009] A third aspect of the present invention provides the use of the microecological preparation described in the second aspect in the preparation of products for the prevention and treatment of macular degeneration.

[0010] In one or more embodiments, the prevention and treatment of macular degeneration includes: (a1) Enhances the antioxidant capacity of the retina; (a2) Increases the retinal barrier; (a3) Reduces retinal apoptosis; (a4) Reduces retinal neovascularization; (a5) Reduces retinal inflammation.

[0011] Preferably, the enhancement of retinal antioxidant capacity includes: increasing the secretion of glutathione (GSH) in human retinal pigment epithelial cells; increasing the survival rate of oxidatively damaged human retinal pigment epithelial cells; and upregulating the expression of antioxidant-related genes in oxidatively damaged human retinal pigment epithelial cells, namely nuclear factor e2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1).

[0012] Preferably, the enhancement of the retinal barrier includes upregulating the expression of barrier-related genes, namely, atresia zona 1 (ZO-1) and tight junction protein 4 (CLD4), in oxidatively damaged human retinal pigment epithelial cells.

[0013] Preferably, the reduction of retinal apoptosis includes: downregulating the expression of apoptosis-related genes in oxidatively damaged human retinal pigment epithelial cells, namely cell tumor antigen P53 (P53) and cell cycle-dependent protein kinase inhibitor 1A (P21).

[0014] Preferably, the reduction of retinal neovascularization includes: downregulating the expression of neovascularization-related genes, namely anti-vascular endothelial growth factor (VEGF), in human retinal pigment epithelial cells damaged by oxidative stress.

[0015] Preferably, the reduction of retinal inflammation includes: downregulating the expression of pro-inflammatory factor-related genes, namely interleukin-8 (IL-8) and interleukin-6 (IL-6), in human retinal pigment epithelial cells damaged by oxidative stress.

[0016] In one or more embodiments, the product includes a drug and a drug composition.

[0017] In a fourth aspect, the present invention provides a product for preventing and treating macular degeneration, comprising the microecological preparation described in the second aspect.

[0018] In one or more embodiments, the prevention and treatment of macular degeneration includes: (a1) Enhances the antioxidant capacity of the retina; (a2) Increases the retinal barrier; (a3) Reduces retinal apoptosis; (a4) Reduces retinal neovascularization; (a5) Reduces retinal inflammation.

[0019] Preferably, the enhancement of retinal antioxidant capacity includes: increasing the secretion of glutathione (GSH) in human retinal pigment epithelial cells; increasing the survival rate of oxidatively damaged human retinal pigment epithelial cells; and upregulating the expression of antioxidant-related genes in oxidatively damaged human retinal pigment epithelial cells, namely nuclear factor e2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1).

[0020] Preferably, the enhancement of the retinal barrier includes upregulating the expression of barrier-related genes, namely, atresia zona 1 (ZO-1) and tight junction protein 4 (CLD4), in oxidatively damaged human retinal pigment epithelial cells.

[0021] Preferably, the reduction of retinal apoptosis includes: downregulating the expression of apoptosis-related genes in oxidatively damaged human retinal pigment epithelial cells, namely cell tumor antigen P53 (P53) and cell cycle-dependent protein kinase inhibitor 1A (P21).

[0022] Preferably, the reduction of retinal neovascularization includes: downregulating the expression of neovascularization-related genes, namely anti-vascular endothelial growth factor (VEGF), in human retinal pigment epithelial cells damaged by oxidative stress.

[0023] Preferably, the reduction of retinal inflammation includes: downregulating the expression of pro-inflammatory factor-related genes, namely interleukin-8 (IL-8) and interleukin-6 (IL-6), in human retinal pigment epithelial cells damaged by oxidative stress.

[0024] In one or more embodiments, the product includes a drug and a drug composition.

[0025] The beneficial effects of this invention are as follows: This invention isolates and obtains Bifidobacterium breve from the feces of healthy female infants. Bifidobacterium breve NHNK-630, its live bacteria, inactivated bacteria, or fermentation products have the functions of improving the antioxidant capacity of the retina, increasing the retinal barrier, reducing retinal apoptosis, reducing retinal neovascularization, and reducing retinal inflammation; in addition, Bifidobacterium breve ( Bifidobacterium breve NHNK-630's own biofilm can increase its ability to colonize intestinal epithelial cells, therefore, Bifidobacterium breve ( Bifidobacterium breve NHNK-630 can be used to prepare products for the prevention and treatment of macular degeneration. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 Figure 1 shows the results of improving the relative survival rate of ARPE-19 cells damaged by H2O2 oxidation with Bifidobacterium breve NHNK-630. In the figure, A represents the normal state of ARPE-19 cells before H2O2 oxidation damage, B represents the result of the control group after H2O2 oxidation damage, C represents the result after treatment with live Bifidobacterium breve NHNK-630, and D represents the result after treatment with inactivated Bifidobacterium breve NHNK-630. The scale bars in A, B, C, and D are all 100 μm. Figure 2 To enhance the adhesion of Bifidobacterium breve NHNK-630 biofilm to intestinal epithelial cells, A represents the normal state of Caco-2 cells, B represents the adsorption of live Bifidobacterium breve NHNK-630 to Caco-2 cells, and C represents the adsorption of Caco-2 cells by Bifidobacterium breve NHNK-630 biofilm. The scale bars in A, B, and C are all 10 μm.

[0028] Preservation instructions: Bifidobacterium breve ( Bifidobacterium breve NHNK-630 was deposited on March 10, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025432, and classified as *Bifidobacterium breve* (NHNK-630). Bifidobacterium breve NHNK-630. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0031] Studies have shown that atresia zona 1 (ZO-1), tight junction protein 4 (CLD4), and atresia protein (OCLD) together constitute the core molecular network of tight junctions in the retinal pigment epithelium, assembling together to maintain the integrity of the extracorporeal blood-retinal barrier (oBRB). In retinal degenerative diseases, abnormal downregulation of retinal barrier-related genes often occurs, leading to increased oBRB permeability, allowing toxic plasma components to enter the retina and accelerating the degenerative process. The gut and retinal barrier are highly homologous in molecular mechanisms, communicating bidirectionally through the gut-eye axis. When both are damaged simultaneously, a systemic inflammatory cascade response is triggered. Abnormal immune cells from the gut may be activated and migrate to the retina, inducing local inflammatory responses in areas such as the macula, participating in the development of dry or wet age-related macular degeneration.

[0032] This invention isolates and obtains Bifidobacterium breve from the feces of healthy female infants. Bifidobacterium breve NHNK-630, with its live or inactivated bacteria, has the functions of improving the antioxidant capacity of the retina, increasing the retinal barrier, reducing retinal apoptosis, reducing retinal neovascularization, and reducing retinal inflammation; in addition, Bifidobacterium breve ( Bifidobacterium breve NHNK-630's own biofilm can increase its ability to colonize intestinal epithelial cells, therefore, Bifidobacterium breve ( Bifidobacterium breve NHNK-630 can be used to prepare products for the prevention and treatment of macular degeneration.

[0033] Enhancing the antioxidant capacity of the retina includes: increasing the secretion of glutathione (GSH) in human retinal pigment epithelial cells; increasing the survival rate of oxidatively damaged human retinal pigment epithelial cells; and upregulating the expression of antioxidant-related genes in oxidatively damaged human retinal pigment epithelial cells, namely nuclear factor e2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1).

[0034] Enhancing the retinal barrier includes upregulating the expression of barrier-related genes, namely, atresia zona 1 (ZO-1) and tight junction protein 4 (CLD4), in oxidatively damaged human retinal pigment epithelial cells.

[0035] Reducing retinal apoptosis includes downregulating the expression of apoptosis-related genes in oxidatively damaged human retinal pigment epithelial cells, namely cell tumor antigen P53 (P53) and cell cycle-dependent protein kinase inhibitor 1A (P21).

[0036] Reducing retinal inflammation includes downregulating the expression of pro-inflammatory genes related to oxidative damage to human retinal pigment epithelial cells, namely interleukin-8 (IL-8) and interleukin-6 (IL-6).

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] In the following examples, all reagents used were commercially available products.

[0039] Example 1 Bifidobacterium breve ( Bifidobacterium breve Separation of NHNK-630: Samples were collected from the feces of healthy female infants. The samples were placed in physiological saline and shaken to mix. The supernatant was streaked onto MRS solid plates containing 0.1% (m / v) cysteine. After anaerobic incubation at 37°C for 48 h, white colonies were picked and repeatedly inoculated and screened until uniform single colonies were obtained, which were named NHNK-630.

[0040] Gram staining and microscopic examination: Single colonies of NHNK-630 were streaked onto MRS solid plates containing 0.1% (m / v) cysteine ​​and anaerobically cultured at 37°C for 48 h. White single colonies were then picked for Gram staining and microscopic examination. Gram staining and microscopic examination showed that NHNK-630 was Gram positive and appeared as rods or bars under the microscope. When grown on MRS solid plates containing 0.1% (m / v) cysteine, it formed smooth, opaque colonies with neat edges and milky white, round, raised surfaces. In MRS liquid medium containing 0.1% (m / v) cysteine, it grew in a uniformly turbid manner, and after prolonged standing, the cells formed a white precipitate. The optimal growth temperature was 37°C.

[0041] The 16S rDNA gene sequence is shown in SEQ ID NO. 1. After homology comparison (BLASTN), NHNK-630 was determined to be a Bifidobacterium breve. Bifidobacterium breve ).

[0042] Example 2 Bifidobacterium breve NHNK-630 improves the survival rate of oxidatively damaged human retinal pigment epithelial cells ARPE-19: Oxidative stress damage to retinal pigment epithelial cells is one of the core factors driving the occurrence and progression of age-related macular degeneration (AMD). When retinal pigment epithelial cells are severely damaged and die, the photoreceptor cells in the macular region above them undergo apoptosis due to loss of nutritional support, leading to blurred central vision or scotomas, and ultimately permanent loss of central vision. Therefore, this embodiment uses an oxidative damage model of retinal pigment epithelial cells to evaluate the protective effect of Bifidobacterium breve NHNK-630 against age-related macular degeneration.

[0043] Single colonies of *Bifidobacterium breve* NHNK-630 were cultured in MRS liquid medium containing 0.1% (m / v) cysteine ​​and anaerobic at 37°C for 48 h. The culture medium was then adjusted to OD using MRS liquid medium. 600 =0.5, obtain the fermentation broth, centrifuge the fermentation broth, and obtain the supernatant and precipitate separately. Filter the supernatant through a 0.22 μm filter membrane to obtain the fermentation product of *Bifidobacterium breve* NHNK-630. Wash the precipitate twice with sterile phosphate-buffered saline (PBS) solution, then resuspend the cells in DMEM medium modified by Durbeco and adjust the OD. 600 =0.5, viable Bifidobacterium breve NHNK-630 was obtained. A portion of the viable Bifidobacterium breve NHNK-630 was taken and sterilized at 121℃ for 15 min to obtain inactivated Bifidobacterium breve NHNK-630.

[0044] Example 3 Bifidobacterium breve NHNK-630 enhances the antioxidant capacity of the retina: Culture of human retinal pigment epithelial cells ARPE-19: ARPE-19 cells were activated in DMEM medium containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin and streptomycin, and then cultured at 37°C and 5% CO2. After the cells reached 80-90% confluence, they were passaged or plated.

[0045] 3.1 Bifidobacterium breve NHNK-630 improves the relative survival rate of ARPE-19 cells damaged by H2O2 oxidation: ARPE-19 cells were divided at a rate of 1×10 4Cells were seeded per well in 96-well cell culture plates and cultured for 12 h until adherence. After culture, the culture medium was removed. The control group and the *Bifidobacterium breve* NHNK-630 group were each inoculated with 100 μL of DMEM medium containing 1.5 mmol / L H2O2 and cultured at 37°C for 3 h. The original culture medium was then removed. The *Bifidobacterium breve* NHNK-630 group was inoculated with 100 μL of DMEM medium containing 1% (v / v) live or inactivated *Bifidobacterium breve* NHNK-630, while the control group was inoculated with an equal volume of DMEM medium. Each group was divided into three replicates. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 3 h. The absorbance at 450 nm was measured using a microplate reader.

[0046] Calculation formula: Relative survival rate (%) = Bifidobacterium breve NHNK-630 group / control group × 100%.

[0047] The relative survival rate of ARPE-19 cells in the Bifidobacterium breve NHNK-630 group is as follows: Figure 1 As shown in Table 1.

[0048] Table 1. Relative survival rate of ARPE-19 cells in the *Bifidobacterium breve* NHNK-630 group.

[0049] In Table 1, the SD value represents the standard deviation.

[0050] From Table 1 and Figure 1 The results show that Bifidobacterium breve NHNK-630 can increase the survival rate of human retinal pigment epithelial cells ARPE-19 damaged by H2O2 oxidation and enhance the antioxidant capacity of human retinal pigment epithelial cells.

[0051] 3.2 Bifidobacterium breve NHNK-630 increases glutathione (GSH) content in human retinal pigment epithelial cells ARPE-19: ARPE-19 cells were divided at a rate of 1×10 6 Cells were seeded per well in 96-well cell culture plates and cultured for 12 h until adherence. After culture, the culture medium was removed. The control group and the *Bifidobacterium breve* NHNK-630 group were each inoculated with 100 μL of DMEM medium containing 1.5 mmol / L H2O2 and cultured at 37°C for 3 h. After culture, the supernatant was removed. The *Bifidobacterium breve* NHNK-630 group was inoculated with 1.9 mL of DMEM medium and 100 μL of *Bifidobacterium breve* NHNK-630 fermentation product or inactivated cells, while the control group was inoculated with an equal volume of DMEM medium and cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was collected, and the glutathione (GSH) content was measured.

[0052] Calculation formula: GSH relative growth rate (%) = Bifidobacterium breve NHNK-630 group / control group × 100%.

[0053] The relative growth rate of GSH in ARPE-19 cells of the Bifidobacterium breve NHNK-630 group is shown in Table 2.

[0054] Table 2. Relative growth rate of GSH in ARPE-19 cells of the *Bifidobacterium breve* NHNK-630 group.

[0055] In Table 2, the SD value represents the standard deviation.

[0056] The results in Table 2 show that the fermentation products and inactivated cells of Bifidobacterium breve NHNK-630 can increase the content of glutathione (GSH) secreted by human retinal pigment epithelium ARPE-19, with a relative growth rate of 114.93%~134.29%, thereby improving the antioxidant capacity of cells.

[0057] 3.3 Bifidobacterium breve NHNK-630 regulates gene expression in human retinal pigment epithelial cells ARPE-19 induced by oxidative damage: ARPE-19 cells were divided at a rate of 1×10 6 Cells were seeded per well in 96-well cell culture plates and cultured for 12 h until adherence. After culture, the culture medium was removed. The control group and the *Bifidobacterium breve* NHNK-630 group were each inoculated with 100 μL of DMEM medium containing 1.5 mmol / L H2O2 and cultured at 37°C for 3 h. After culture, the supernatant was removed. The *Bifidobacterium breve* NHNK-630 group was inoculated with 1.9 mL of DMEM medium and 100 μL of live *Bifidobacterium breve* NHNK-630, fermentation products, or inactivated cells. The control group was inoculated with an equal volume of DMEM medium and cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent was added to each well, and total RNA was extracted according to the reagent instructions. The concentration and purity were then determined. After extraction, the DNA was reverse transcribed into cDNA. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene, the expression levels of inflammatory genes IL-8 and IL-6, barrier-related genes ZO-1 and CLD4, apoptosis-related genes P5 and P21, antioxidant-related genes NRF2 and HO-1, and angiogenesis-related gene VEGF were determined using quantitative polymerase chain reaction (qPCR). The relative fold change of gene expression in the control group was F=1, and the F-value for each sample was calculated using the 2-ΔΔCT method.

[0058] Calculation formula: F = 2 - ΔΔCT, where: △CT experiment = CT experiment - CT internal reference (experiment); △CT control = CT control - CT internal reference (control); △△CT = △CT experiment - △CT control.

[0059] The expression levels of inflammatory genes IL-8 and IL-6, barrier-related genes ZO-1 and CLD4, apoptosis-related genes P5 and P21, antioxidant-related genes NRF2 and HO-1, and angiogenesis-related gene VEGF are shown in Tables 3 to 5.

[0060] Table 3. Regulation of gene expression in ARPE-19 cells under oxidative damage by live Bifidobacterium breve NHNK-630.

[0061] In Table 3, the SD values ​​represent the standard deviation.

[0062] Table 4. Regulation of gene expression in ARPE-19 cells under oxidative damage by fermentation products of Bifidobacterium breve NHNK-630.

[0063] In Table 4, the SD value represents the standard deviation.

[0064] Table 5. Regulation of gene expression in ARPE-19 cells under oxidative damage by inactivated Bifidobacterium breve NHNK-630 cells

[0065] In Table 5, the SD value represents the standard deviation.

[0066] The results in Tables 3-5 show that Bifidobacterium breve NHNK-630 can regulate gene expression in ARPE-19 cells induced by oxidative damage, downregulate the expression of pro-inflammatory cytokine-related genes IL-8 and IL-6, apoptosis-related genes P53 and P21, and angiogenesis-related gene VEGF, and upregulate the expression of barrier-related genes ZO-1 and CLD4, and antioxidant-related genes NRF2 and HO-1, thereby playing an anti-inflammatory role, enhancing the ocular barrier, and reducing ocular neovascularization, thus preventing and treating macular degeneration.

[0067] Example 4 Bifidobacterium breve NHNK-630 improves the survival rate of Caco-2 cells under oxidative damage: A close bidirectional link exists between retinal damage and intestinal oxidative stress, primarily mediated through the gut-retinal axis. Some eye diseases, long considered purely hereditary, may have their pathogenesis partly stemming from the escape and translocation of gut bacteria to the retina. Studies have shown that damage to the retinal-intestinal barrier can lead to bacterial migration from the gut to the retina, subsequently triggering secondary macular degeneration. Aging and gut microbiota dysbiosis exacerbate intestinal oxidative stress, which increases intestinal permeability by disrupting tight junction proteins, allowing harmful products to more easily penetrate into the bloodstream. When these harmful products accumulate in the eye via the bloodstream, they further aggravate retinal inflammation and damage. Therefore, this study uses a Caco-2 cell oxidative damage model to evaluate the protective effect of *Bifidobacterium breve* NHNK-630 on the intestinal barrier.

[0068] Caco-2 cells were activated in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin and streptomycin, and then cultured at 37°C and 5% CO2. After the cells reached 80-90% confluence, they were passaged or plated.

[0069] 4.1 Bifidobacterium breve NHNK-630 improves the relative survival rate of Caco-2 cells damaged by H2O2 oxidation: Caco-2 cells were divided into 1×10 4 Cells were seeded per well in 96-well cell culture plates and cultured for 12 h until adherence. After culture, the culture medium was removed. The control group and the *Bifidobacterium breve* NHNK-630 group were each inoculated with 100 μL of DMEM medium containing 1.5 mmol / L H2O2 and cultured at 37°C for 3 h. The original culture medium was then removed. The *Bifidobacterium breve* NHNK-630 group was inoculated with 100 μL of DMEM medium containing 1% (v / v) live *Bifidobacterium breve* NHNK-630, fermentation products, or inactivated cells. The control group was inoculated with an equal volume of DMEM medium. Each group was divided into three replicates. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 3 h. The absorbance at 450 nm was measured using a microplate reader.

[0070] Calculation formula: Relative survival rate (%) = Bifidobacterium breve NHNK-630 group / control group × 100%.

[0071] The relative survival rate of Caco-2 cells in the Bifidobacterium breve NHNK-630 group is shown in Table 6.

[0072] Table 6. Relative survival rate of Caco-2 cells in the Bifidobacterium breve NHNK-630 group.

[0073] In Table 6, the SD values ​​represent the standard deviation.

[0074] As shown in Table 6, Bifidobacterium breve NHNK-630 can increase the survival rate of Caco-2 cells damaged by H2O2 oxidation. Compared with the model group, the relative survival rate of the Bifidobacterium breve NHNK-630 group is 124.94%~148.45%.

[0075] 4.2 Bifidobacterium breve NHNK-630 increases glutathione (GSH) content in Caco-2 cells: Caco-2 cells were divided into 1×10 6 Cells were seeded per well in 96-well cell culture plates and cultured for 12 h until adherence. After culture, the culture medium was removed. The control group and the *Bifidobacterium breve* NHNK-630 group were each inoculated with 100 μL of DMEM medium containing 1.5 mmol / L H2O2 and cultured at 37°C for 3 h. After culture, the supernatant was removed. The *Bifidobacterium breve* NHNK-630 group was inoculated with 1.9 mL of DMEM medium and 100 μL of inactivated *Bifidobacterium breve* NHNK-630 cells, while the control group was inoculated with an equal volume of DMEM medium and cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was collected, and the glutathione (GSH) content was measured.

[0076] Calculation formula: GSH relative growth rate (%) = Bifidobacterium breve NHNK-630 group / control group × 100%.

[0077] The relative growth rate of GSH in Caco-2 cells of the Bifidobacterium breve NHNK-630 group is shown in Table 7.

[0078] Table 7. Relative growth rate of GSH in Caco-2 cells of the *Bifidobacterium breve* NHNK-630 group.

[0079] In Table 7, the SD value represents the standard deviation.

[0080] The results in Table 7 show that inactivated Bifidobacterium breve NHNK-630 cells can increase the GSH content of Caco-2 cells, with a growth rate between 121.33% and 129.09%, thereby enhancing the antioxidant capacity of the cells.

[0081] 4.3 Bifidobacterium breve NHNK-630 regulates oxidative damage-induced expression of Caco-2 cell barrier genes: Caco-2 cells were divided into 1×10 6Cells were seeded per well in 96-well cell culture plates and cultured for 12 h until adherence. After culture, the culture medium was removed. The control group and the *Bifidobacterium breve* NHNK-630 group were each inoculated with 100 μL of DMEM medium containing 1.5 mmol / L H2O2 and cultured at 37°C for 3 h. After culture, the supernatant was removed. The *Bifidobacterium breve* NHNK-630 group was inoculated with 1.9 mL of DMEM medium and 100 μL of live *Bifidobacterium breve* NHNK-630 or its fermentation product, while the control group was inoculated with an equal volume of DMEM medium and cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent was added to each well, and total RNA was extracted according to the reagent instructions. The concentration and purity were then determined. After extraction, the DNA was reverse transcribed into cDNA. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene, the expression levels of inflammatory genes IL-8 and IL-6, and barrier-related genes ZO-1, OCLD, or CLD4 were determined using quantitative polymerase chain reaction (qPCR). The relative fold change of gene expression in the control group was F=1, and the F value for each sample was calculated using the 2-ΔΔCT method.

[0082] Calculation formula: F = 2 - ΔΔCT, where: △CT experiment = CT experiment - CT internal reference (experiment); △CT control = CT control - CT internal reference (control); △△CT = △CT experiment - △CT control.

[0083] The expression levels of barrier-related genes ZO-1, OCLD, or CLD4 are shown in Tables 8 and 9.

[0084] Table 8. Regulation of Caco-2 cell barrier gene expression by fermentation products of Bifidobacterium breve NHNK-630 under oxidative damage.

[0085] In Table 8, the SD values ​​represent the standard deviation.

[0086] Table 9. Regulation of Caco-2 cell barrier gene expression induced by oxidative damage in live Bifidobacterium breve NHNK-630.

[0087] In Table 9, the SD value represents the standard deviation.

[0088] The results in Tables 8 and 9 show that Bifidobacterium breve NHNK-630 can upregulate the expression of Caco-2 cell barrier genes ZO-1, OCLD and CLD4, thereby maintaining the integrity of the intestinal barrier.

[0089] Example 5 Bifidobacterium breve NHNK-630 forms a biofilm: Single colonies of *Bifidobacterium breve* NHNK-630 were picked and cultured in MRS liquid medium containing 0.1% (m / v) cysteine. The culture was then incubated anaerobicly at 37°C for 24 h. The OD was adjusted using MRS medium. 600 =0.2. Add 100 μL of bacterial culture to each well of a 96-well plate, with 3 replicates per group, and then continue anaerobic culture at 37°C for 24 h.

[0090] After incubation, the supernatant was discarded, and each well was washed twice with 100 μL of sterile PBS solution. Then, 100 μL of 4% (w / v) paraformaldehyde fixative was added to each well, and the solution was fixed at room temperature for 30 min. The fixative was discarded, and 100 μL of crystal violet was added to each well, and the solution was stained at room temperature for 30 min. After staining, the wells were washed twice with sterile PBS solution and air-dried. 100 μL of anhydrous ethanol was added to each well, and the absorbance at 600 nm was measured after standing for 1 min. The results are shown in Table 10.

[0091] Table 10 Biofilm formation by Bifidobacterium breve NHNK-630

[0092] In Table 10, the SD values ​​represent the standard deviation.

[0093] The results in Table 10 show that Bifidobacterium breve NHNK-630 can form its own biofilm, with absorbance values ​​at 0D=600nm ranging from 2.26 to 3.12.

[0094] Example 6 Bifidobacterium breve NHNK-630 biofilm enhances adhesion to intestinal epithelial cells: Single colonies of *Bifidobacterium breve* NHNK-630 were picked and cultured in MRS liquid medium containing 0.1% (m / v) cysteine. The culture was then incubated anaerobicly at 37°C for 48 h. The culture was adjusted to OD using fresh MRS medium containing 0.1% (m / v) cysteine. 600 =0.2 mg / L was added to the bacterial culture dish, and then cultured at 37°C for 24 h. After the culture was completed, the supernatant was discarded, the cells were washed twice with sterile PBS solution, and the cells adhering to the culture dish were resuspended. The cells were collected by centrifugation (5000 rpm, 10 min), resuspended in DMEM medium, and the OD was adjusted. 600 =0.5, and a biofilm is obtained.

[0095] Caco-2 cells were seeded in DMEM medium containing 10% (v / v) FBS and cultured at 37°C and 5% CO2. When confluence reached 80%, cells were digested with trypsin and collected, then seeded into 6-well plates containing cell spreaders and cultured overnight. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. 1 mL of DMEM medium and 1 mL of live Bifidobacterium breve NHNK-630 bacteria or biofilm were added to each well, and the cells were cultured for another 2 h. After culture, the cells were washed twice with sterile PBS to remove unattached cells. The spreaders were fixed in 4% (w / v) paraformaldehyde for 15 min, followed by Gram staining and photographing. Figure 2 As shown.

[0096] The results showed that the Bifidobacterium breve NHNK-630 biofilm could increase the adhesion of the bacteria to intestinal epithelial cells.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A short bifidobacterium ( Bifidobacterium breve NHNK-630 was deposited on March 10, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025432.

2. A microecological preparation, characterized in that, Including the Bifidobacterium breve described in claim 1 ( Bifidobacterium breve NHNK-630 strain, pure culture, inactivated bacterial cells, extract, fermentation product lysate, or a combination thereof.

3. The use of the microecological preparation according to claim 2 in the preparation of products for the prevention and treatment of macular degeneration.

4. The application as described in claim 3, characterized in that, The prevention and treatment of macular degeneration includes: (a1) Enhances the antioxidant capacity of the retina; (a2) Increases the retinal barrier; (a3) Reduces retinal apoptosis; (a4) Reduces retinal neovascularization; (a5) Reduces retinal inflammation.

5. The application as described in claim 4, characterized in that, The enhancement of retinal antioxidant capacity includes: increasing glutathione secretion in human retinal pigment epithelial cells; increasing the survival rate of oxidatively damaged human retinal pigment epithelial cells; and upregulating the expression of antioxidant-related genes in oxidatively damaged human retinal pigment epithelial cells, namely nuclear factor E2-related factor 2 and heme oxygenase-1. The enhancement of the retinal barrier includes upregulating the expression of barrier-related genes, namely atresia zona 1 and tight junction protein 4, in oxidatively damaged human retinal pigment epithelial cells.

6. The application as described in claim 4, characterized in that, The reduction of retinal apoptosis includes: downregulating the expression of apoptosis-related genes in oxidatively damaged human retinal pigment epithelial cells, namely cell tumor antigen P53 and cell cycle-dependent protein kinase inhibitor 1A; The reduction of retinal neovascularization includes downregulating the expression of anti-vascular endothelial growth factor, a gene related to neovascularization in oxidatively damaged human retinal pigment epithelial cells.

7. The application as described in claim 4, characterized in that, The reduction of retinal inflammation includes downregulating the expression of pro-inflammatory genes, namely interleukin-8 and interleukin-6, in human retinal pigment epithelial cells damaged by oxidative stress.

8. The application as described in claim 3, characterized in that, The products include pharmaceuticals and pharmaceutical compositions.

9. A product for preventing and treating macular degeneration, characterized in that, Includes the microecological preparation described in claim 2.

10. The product as described in claim 9, characterized in that, The products include pharmaceuticals and pharmaceutical compositions.

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