Phytobacterium plantarum and application thereof in preparation of product for improving retina maculopathy

Abstract

The invention belongs to the technical field of marine microorganisms, and discloses a plant lactobacillus and an application of the plant lactobacillus in preparation of products for improving retina maculopathy, the plant lactobacillus NHNK-627 is preserved in China Center for Type Culture Collection on February 28, 2025, and the preservation number of the plant lactobacillus NHNK-627 is CCTCC NO: M 2025323. The invention further discloses a preparation method of the plant lactobacillus NHNK-627 for improving the retina maculopathy of the retina maculopathy of the retina maculopathy of the retina maculopathy of the retina maculopathy of the retina maculopathy of the retina maculopathy. NHNK-627 has the functions of improving retina oxidation resistance, enhancing retina barrier, inhibiting retina cell apoptosis, reducing retina neovascularization and relieving retina inflammatory response, in addition, an NHNK-627 thallus biological membrane can improve the colonization ability of nasal epithelial cells and intestinal epithelial cells, and can be used for preparing products for improving retina maculopathy.

Description

Technical Field

[0001] The invention belongs to the technical field of marine microorganisms, and particularly relates to a strain of Lactobacillus plantarum and its application in preparing a product for improving retinal macular degeneration. Background Art

[0002] Mucosal membranes of the human body, such as the mouth, nasopharynx, and vagina, each harbor their own commensal bacterial communities. The tissues lining the surface of the eye are also mucosal. The ocular surface sits at the interface between the environment and the host immune system and is constantly exposed to microorganisms such as bacteria, fungi, and viruses. When the ocular surface microbiome is out of balance, eye diseases can develop. The eye harbors a core microbiome that varies depending on age, geographic region, ethnicity, contact lens wear, and disease status. This core microbiome is limited to four bacterial species: Staphylococci, diphtheroids, Propionibacterium acnes, and Streptococci.

[0003] Eye diseases long thought to be purely hereditary may be caused in part by bacteria escaping from the intestine to the retina. Damage to the retinal barrier allows bacteria to migrate from the intestine to the retina, leading to secondary retinal degeneration, which can be alleviated with antibiotic treatment. Tight junctions exist between retinal pigment epithelial cells, forming the outer blood-retinal barrier between the retina and the choroid, preventing abnormal choroidal components from entering the retina. This outer barrier is composed of retinal pigment epithelial (RPE) cells and their junctional complexes. These cells are bounded by gap junctions at the top, zonula occludens in the middle, and zonula adhesions at the bottom. The zonula occludens are the most important.

[0004] Choroidal neovascularization (CNV), whether occurring in the macula, around the optic disc, or in the periphery, can lead to visual impairment, particularly in age-related macular degeneration (AMD). Currently, the main treatment and prevention options for macular degeneration (MD) are drugs that inhibit vascular endothelial growth factor (VEGF), requiring long-term intravitreal injections, or photodynamic therapy, which uses lasers of specific wavelengths to seal abnormal blood vessels. However, these treatments can damage the peripheral retina, leading to visual field loss and are expensive. Metabolites produced by beneficial bacteria colonizing the gut can be absorbed, exerting systemic effects on the body with high safety and low cost. Furthermore, nasal microbial colonization may have a more direct regulatory effect on head organs. Products containing beneficial bacteria may offer a more ideal treatment and prevention option for MD.

[0005] Marine microorganisms are among the earliest known sources of marine bioactive substances, including algae, bacteria, fungi, and protozoa. These bioactive substances have important applications in pharmaceuticals, food, cosmetics, and other fields. Therefore, providing a product developed using microbial technology that can improve macular degeneration is of great practical significance. Summary of the Invention

[0006] In response to the high risks and high costs of existing treatment options for retinal macular degeneration, the present invention provides a strain of Lactobacillus plantarum and its application in preparing products for improving retinal macular degeneration. The Lactobacillus plantarum can improve the survival rate of oxidatively damaged human retinal pigment epithelial cells, improve the retinal antioxidant capacity, alleviate retinal inflammatory reactions caused by oxidative damage, strengthen the retinal barrier, reduce retinal neovascularization, inhibit retinal cell apoptosis, improve the survival rate of oxidatively damaged intestinal epithelial cells, improve the antioxidant capacity of intestinal epithelial cells, maintain the intestinal barrier, improve the colonization ability of intestinal epithelial cells, improve the colonization ability of nasal epithelial cells, and form a biofilm, thereby achieving the effect of preventing and treating retinal macular degeneration, and can be used to prepare products for improving retinal macular degeneration.

[0007] The present invention first provides a plant lactobacillus ( Lactiplantibacillus plantarum ), the plant lactobacillus is plant lactobacillus NHNK-627, which was deposited in the China Center for Type Culture Collection on February 28, 2025, and its deposit number is CCTCC NO: M 2025323.

[0008] The present invention further provides a product of the Lactobacillus plantarum, which is one of the following: live bacteria, fermentation / secretion products, inactivated bacteria, and biofilm of Lactobacillus plantarum NHNK-627.

[0009] The preparation method of the live bacteria comprises: inoculating the plant lactobacillus into a culture medium, culturing at 37° C. for 48 hours to obtain a fermentation liquid, centrifuging to obtain a precipitate, and washing the precipitate to obtain the live bacteria; The preparation method of the fermentation / secretion product comprises: inoculating the plant lactobacillus into a culture medium, culturing at 37° C. for 48 hours to obtain a fermentation liquid, centrifuging to obtain a supernatant, and filtering at a precision of 0.22 μm to obtain a filtrate which is the fermentation / secretion product; The preparation method of the inactivated bacteria comprises the following steps: inoculating the plant lactobacillus into a culture medium, culturing at 37° C. for 48 h to obtain a fermentation liquid, centrifuging to obtain a precipitate, washing the precipitate, and inactivating at 121° C. for 15-30 min to obtain the inactivated bacteria.

[0010] The present invention also provides the use of the Lactobacillus plantarum or a product thereof in preparing a product for improving retinal macular degeneration.

[0011] Preferably, the mechanism by which the product improves retinal macular degeneration includes any one or more of the following: 1) Improve the survival rate of oxidatively damaged human retinal pigment epithelial cells; 2) Improve retinal antioxidant capacity; 3) Alleviate retinal inflammatory response; 4) Strengthen the retinal barrier; 5) Reduce retinal neovascularization; 6) Inhibit retinal cell apoptosis.

[0012] Improving the retinal antioxidant capacity includes any one or two of the following methods: 1) Increase the secretion of glutathione (GSH) in human retinal pigment epithelial cells; 2) Upregulating the expression of antioxidant-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the antioxidant-related gene is the nuclear factor E2-related factor 2 gene NRF2 and / or heme oxygenase 1 gene HO-1 .

[0013] The method for alleviating retinal inflammatory response comprises down-regulating the expression of pro-inflammatory factor-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the pro-inflammatory factor-related genes in the human retinal pigment epithelial cells include interleukin-8 gene IL- 8 , interleukin-6 gene IL-6、 interleukin-1β gene IL-1β, Tumor necrosis factor alpha gene TNF - α Any one or more of .

[0014] The method for enhancing the retinal barrier comprises upregulating the expression of barrier-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the barrier-related genes in the human retinal pigment epithelial cells are zonula occludens-1 genes. ZO-1 and / or claudin 4 gene CLD4 .

[0015] The method for reducing retinal neovascularization comprises down-regulating the expression of neovascularization-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the neovascularization-related genes in human retinal pigment epithelial cells are vascular endothelial growth factor genes. VEGF .

[0016] The inhibition of retinal cell apoptosis includes down-regulating the expression of apoptosis-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the apoptosis-related gene is the tumor protein 53 gene. P53 and / or cyclin-dependent kinase inhibitor genesP21 .

[0017] Preferably, the mechanism by which the product improves retinal macular degeneration includes any one or more of the following: 1) Improve the survival rate of oxidatively damaged intestinal epithelial cells; 2) Improve the antioxidant capacity of intestinal epithelial cells; 3) Maintain the intestinal barrier.

[0018] The improving the antioxidant capacity of intestinal epithelial cells includes improving the secretion of glutathione (GSH) in the intestinal epithelial cells.

[0019] The maintenance of the intestinal barrier comprises upregulating the expression of barrier genes of intestinal epithelial cells, wherein the barrier genes of intestinal epithelial cells are OCLD and / or CLD4 .

[0020] Preferably, the mechanism by which the product improves retinal macular degeneration includes any one or more of the following: 1) Improve the colonization ability of intestinal epithelial cells; 2) Improve the colonization ability of nasal epithelial cells; 3) Formation of biofilm.

[0021] Biological Deposit Description The biological deposit information in this application is as follows: Storage time: February 28, 2025 Name of depository: China Center for Type Culture Collection Deposit number: CCTCC NO: M 2025323 Address of the depository: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, China Classification name: Lactobacillus plantarum NHNK-627 ( Lactiplantibacillus plantarum NHNK-627).

[0022] The present invention adopts Lactobacillus plantarum NHNK-627, which has the effects of improving the survival rate of oxidatively damaged human retinal pigment epithelial cells, improving the retinal antioxidant capacity, alleviating the retinal inflammatory response caused by oxidative damage, strengthening the retinal barrier, reducing retinal neovascularization, inhibiting retinal cell apoptosis, improving the survival rate of oxidatively damaged intestinal epithelial cells, improving the intestinal epithelial cell antioxidant capacity, maintaining the intestinal barrier, improving the intestinal epithelial cell colonization ability, improving the nasal epithelial cell colonization ability, and forming a biofilm, thereby achieving the effect of preventing and treating retinal macular degeneration. The present invention can be used to prepare products for improving retinal macular degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a diagram showing the detection results of Lactobacillus plantarum NHNK-627 in Gram staining microscopy in Example 1 of the present invention; Figure 2 Figure 3 shows the experimental results of Lactobacillus plantarum NHNK-627 improving the survival of ARPE-19 cells damaged by H2O2 in Example 3 of the present invention, wherein A represents normal ARPE-19 cells, B represents oxidatively damaged ARPE-19 cells in the control group, and C, D, and E represent oxidatively damaged ARPE-19 cells treated with live NHNK-627 suspension, fermentation product, and inactivated bacteria in the experimental group, respectively. Figure 3 Graphs showing the results of an experiment to determine the adhesion ability of Lactobacillus plantarum NHNK-627 to RPMI-2650 nasal epithelial cells in Example 10 of the present invention, wherein A shows the experimental results of treatment with a live bacterial suspension of NHNK-627, and B shows the experimental results of treatment with a biofilm bacterial suspension of NHNK-627; Figure 4 These are graphs showing the experimental results of determining the adhesion ability of Lactobacillus plantarum NHNK-627 to human intestinal epithelial cells Caco-2 in Example 11 of the present invention, wherein A shows the experimental results of NHNK-627 live bacterial suspension treatment, and B shows the experimental results of NHNK-627 biofilm bacterial suspension treatment. DETAILED DESCRIPTION

[0024] The present invention provides a strain of plant lactobacillus and an application for preparing a product for improving retinal maculopathy. Those skilled in the art can learn from the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0025] The Lactobacillus plantarum NHNK-627 described in the present invention is derived from the intestine of Abalone diversicolor and is identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum This strain is Gram-positive and rod-shaped under a microscope. It grows on MRS plates and forms round, smooth, opaque, white colonies with neat edges. It grows uniformly in MRS liquid medium and forms a white precipitate after prolonged storage. The optimal growth temperature is 37°C.

[0026] The biological deposit information of Lactobacillus plantarum NHNK-627 involved in this application is as follows: Storage time: February 28, 2025 Name of depository: China Center for Type Culture Collection Deposit number: CCTCC NO: M 2025323 Address of the depository: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, China Classification name: Lactobacillus plantarum NHNK-627 ( Lactiplantibacillus plantarum NHNK-627).

[0027] Furthermore, in the application described in the present invention, the Lactobacillus plantarum NHNK-627 provided by the present invention exists in the form of non-sterilized live bacteria or sterilized inactivated bacteria, or in the form of fermentation / secretory products (i.e., supernatant), or in the form of biofilms, or in the form of derivatives. The derivative forms are preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation / secretory products, live bacteria, inactivated bacteria, and biofilms.

[0028] The preparation method of the live bacteria is as follows: Lactiplantibacillus plants ) inoculated into a culture medium, cultured at 37° C. for 48 h to obtain a fermentation broth, centrifuged to obtain a precipitate, and washed the precipitate to obtain the viable bacteria; The preparation method of the fermentation / secretion product is as follows: the plant lactobacillus ( Lactiplantibacillus plants ) was inoculated into the culture medium, cultured at 37°C for 48 h, and the fermentation broth was obtained. The supernatant was collected by centrifugation and filtered with a precision of 0.22 μm. The obtained filtrate was the fermentation / secretion product; The preparation method of the inactivated bacteria is as follows: Lactiplantibacillus plants ) was inoculated into the culture medium, cultured at 37°C for 48 h to obtain a fermentation broth, centrifuged to obtain a precipitate, washed the precipitate, and inactivated at 121°C for 15-30 min to obtain the inactivated bacteria.

[0029] In the following examples, the MRS solid culture medium and MRS liquid culture medium used were both from Qingdao Haibo Biotechnology Co., Ltd., the DMEM culture medium was from Beijing Solebaugh Technology Co., Ltd., and the sterile PBS used was from Hefei White Shark Biotechnology Co., Ltd. (0.01 M, pH = 7.2).

[0030] It should be noted that, unless otherwise specified, the reagents and consumables used in the present invention are all common commercially available products. The present invention will now be further described with reference to the embodiments.

[0031] Example 1 Isolation of NHNK-627 The sample was obtained from an adult, 5 cm long, 3-day-fasted abalone from a farm in Qingdao. The digestive tract was dissected using sterile tools. After rinsing three times with sterile saline, the intestinal tissue was broken up by homogenization and grinding. The homogenate was collected in 1 ml of sterile saline and then graded and diluted. The supernatant was streaked onto MRS solid medium and incubated at 37°C for 24-48 hours. White colonies were selected and repeatedly inoculated and screened until a uniform single colony was obtained, designated NHNK-627. Gram staining microscopy: NHNK-627 is Gram-positive and appears rod-shaped under a microscope. It grows on MRS plates and forms small white, smooth, round, and opaque colonies. It grows evenly in MRS liquid medium and forms a white precipitate after prolonged storage. Figure 1 shown.

[0032] Example 2 Nucleic Acid Identification of NHNK-627 1. 16S rDNA gene sequence analysis: Single colonies were picked and transferred to MRS liquid medium. After incubation at 37°C overnight, the cells were collected by centrifugation at 12,000 rpm for 1 minute and the DNA extraction kit (Beijing Solebold Technology Co., Ltd.) was used. The primers used were bacterial universal primers 27F and 1492R. The PCR amplification system consisted of a 50 μL system with a 95°C pre-denaturation for 5 minutes, 35 cycles of 94°C for 15 seconds, 57°C for 15 seconds, and 72°C for 40 seconds, followed by an extension at 72°C for 10 minutes. 2. Sequence analysis results The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). The results showed that the NHNK-627 strain was Lactobacillus plantarum ( Lactiplantibacillus plantarum ).

[0033] Example 3 NHNK-627 improves the survival rate of oxidatively damaged ARPE-19 cells 1. Preparation of NHNK-627 A single colony of NHNK-627 was picked and placed in fresh MRS solid medium (Qingdao Haibo Biotechnology Co., Ltd.) and cultured at 37°C for 48 h. The OD value was adjusted to 0. 600 = 0.5, centrifuged at 5000 rpm for 10 min, and the supernatant was taken, and then filtered with a 0.22 μm filter membrane. The obtained filtrate was the fermentation product; Collect the above centrifuged precipitated bacteria (i.e., live bacteria), wash them twice with sterile PBS, resuspend them in DMEM medium and adjust the OD 600 = 0.5, to obtain a live bacterial suspension; Some of the viable bacterial pellets were washed twice with sterile PBS and then sterilized by autoclave at 121°C for 15 min. The pellets were then resuspended in DMEM medium and adjusted to OD 600 =0.5, and inactivated bacteria were obtained.

[0034] 2. Culture of human retinal pigment epithelial cells ARPE-19 ARPE-19 cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, cultured at 37°C and 5% CO2, and passaged or plated after the cells reached 80-90% confluence.

[0035] 3. NHNK-627 improves the relative survival rate of ARPE-19 cells damaged by H2O2 ARPE-19 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate and cultured at 37°C for 12 h until the cells adhered. After the culture was completed, the culture medium in the 96-well cell culture plate was removed, and 100 μL of DMEM medium containing 1.5 mmol / L H2O2 was added to each well of the control group and NHNK-627 group (including the fermentation product group, the live bacterial suspension group, and the inactivated bacterial group). The oxidative damage model was established at 37°C for 3 h. Subsequently, the DMEM medium containing H2O2 was removed, and 100 μL of DMEM medium containing 1% (v / v) NHNK-627 live bacterial suspension / fermentation product / inactivated bacterial body was added to each well of the NHNK-627 group, and an equal volume of DMEM medium (100 μL) was added to each well of the control group. Three parallels were set up for each group. The cells were cultured at 37°C and 5% CO2 for 24 h. Figure 2 10 μL of CCK-8 solution was then added to each well and incubated at 37°C for 3 h. After incubation, the absorbance at 450 nm was measured using a microplate reader to quantify the number of viable cells.

[0036] Calculation formula: Relative survival rate (%) = (absorbance value of NHNK-627 group / absorbance value of control group) × 100% The results are shown in Table 1: Table 1 NHNK-627 improves the relative survival rate of ARPE-19 cells damaged by H2O2 The results showed that NHNK-627 could increase the survival rate of human retinal pigment epithelial cells ARPE-19 under H2O2 oxidative damage and improve the antioxidant capacity of human retinal pigment epithelial cells.

[0037] Example 4 NHNK-627 increases the glutathione GSH content in retinal cells ARPE-19 1. Preparation of NHNK-627 The preparation methods of fermentation product, live bacterial suspension and inactivated bacterial bodies are as described in Example 3.

[0038] 2. Culture of human retinal pigment epithelial cells ARPE-19 The cell culture method is as described in Example 3.

[0039] 3. NHNK-627 increases glutathione (GSH) content in ARPE-19 cells with oxidative damage ARPE-19 cells were seeded at 1×10^6 cells / well in a 6-well cell culture plate and cultured at 37°C for 12 h until the cells adhered. After incubation, the culture medium was removed from the cell culture plate. 2 mL of DMEM medium containing 1.5 mmol / L H₂O₂ was added to each well of the control and NHNK-627 groups (including the fermentation product group, the live bacterial suspension group, and the inactivated bacterial group). The cells were incubated at 37°C for 3 h to establish an oxidative damage model. Subsequently, the H₂O₂-containing DMEM medium was removed, and 1.9 mL of DMEM medium and 100 μL of NHNK-627 live bacterial suspension / fermentation product / inactivated bacterial suspension were added to each well of the NHNK-627 group. An equal volume of DMEM medium (2 mL) was added to each well of the control group. The cells were cultured at 37°C and 5% CO₂ for 24 h. After incubation, the cell culture supernatant was retained and glutathione (GSH) content was measured according to the instructions of a reduced glutathione assay kit (Nanjing Jiancheng Bioengineering Institute).

[0040] Calculation formula: GSH relative growth rate (%) = (GSH content in NHNK-627 group - GSH content in control group) / GSH content in control group × 100% The results are shown in Table 2: Table 2 NHNK-627 increases GSH content in ARPE-19 cells The results showed that NHNK-627 could increase the glutathione GSH content secreted by human retinal pigment epithelium ARPE-19, with a relative growth rate of 13.70%~21.51%, thereby improving the antioxidant capacity of cells.

[0041] Example 5 NHNK-627 regulates the expression of inflammation, barrier function, anti-oxidation, and apoptosis-related genes in retinal ARPE-19 cells induced by oxidative damage 1. Preparation of NHNK-627 The preparation methods of fermentation product, live bacterial suspension and inactivated bacterial bodies are as described in Example 3.

[0042] 2. Culture of human retinal pigment epithelial cells ARPE-19 The cell culture method is as described in Example 3.

[0043] 3. NHNK-627 regulates the expression of inflammation, barrier function, antioxidant, and apoptosis-related genes in ARPE-19 cells ARPE-19 cells were seeded at 1×10^6 cells / well in a 6-well cell culture plate and cultured at 37°C for 12 h until the cells adhered. After incubation, the culture medium was removed from the cell culture plate. 2 mL of DMEM medium containing 1.5 mmol / L H₂O₂ was added to each well of the control and NHNK-627 groups (including the fermentation product group, the live bacterial suspension group, and the inactivated bacterial group). The cells were incubated at 37°C for 3 h to establish an oxidative damage model. Subsequently, the H₂O₂-containing DMEM medium was removed, and 1.9 mL of DMEM medium and 100 μL of the live NHNK-627 bacterial suspension / fermentation product / inactivated bacterial suspension were added to each well of the NHNK-627 group. An equal volume of DMEM medium (2 mL) was added to each well of the control group. The cells were cultured at 37°C under 5% CO₂ for 24 h. After the incubation, 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. Total RNA was extracted according to the instructions of Trizol total RNA extraction reagent (Beijing Solaibao Technology Co., Ltd.) and the concentration and purity were determined. After the extraction was completed, the RNA was reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR was used to determine the pro-inflammatory factor-related genes TNF-α, IL-8, IL-6, IL-1β , barrier-related genes ZO-1 and CLD4 , apoptosis-related genes P53 and P21 , antioxidant-related genes NRF2 and HO-1 and angiogenesis-related genes VEGF The relative expression of the control gene is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0044] Formula: F=2 -ΔΔCT ,in: △CT 实验 =CT 实验 -CT 内参（实验）; △CT 对照 =CT 对照 -CT 内参（对照）; △△CT=△CT 实验 -△CT 对照 The results are shown in Table 3, Table 4 and Table 5: Table 3 NHNK-627 live bacterial suspension regulates gene expression in ARPE-19 cells induced by oxidative damage Table 4 NHNK-627 fermentation products regulate gene expression in ARPE-19 cells induced by oxidative damage Table 5 NHNK-627-inactivated bacteria regulate gene expression in ARPE-19 cells induced by oxidative damage The results showed that NHNK-627 could regulate gene expression in ARPE-19 cells induced by oxidative damage and downregulate genes related to pro-inflammatory factors. TNF-α, IL-8, IL-6 and IL-1β downregulates the expression of apoptosis-related genes P53 and P21 downregulates the expression of angiogenesis-related genes VEGF Upregulation of barrier-related genes ZO-1 and CLD4 and antioxidant-related genes NRF2 and HO-1 It can alleviate the inflammatory response caused by oxidative damage, strengthen the retinal barrier and reduce retinal neovascularization, while inhibiting retinal cell apoptosis and improving the antioxidant capacity of retinal cells, thereby preventing macular degeneration.

[0045] Example 6 NHNK-627 improves the survival rate of oxidatively damaged Caco-2 cells 1. Preparation of NHNK-627 The preparation methods of fermentation product, live bacterial suspension and inactivated bacterial bodies are as described in Example 3.

[0046] 2. Cell Culture Caco-2 cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, cultured at 37°C and 5% CO2, and passaged or plated after the cells reached 80-90% confluence.

[0047] 3. NHNK-627 improves the relative survival rate of Caco-2 cells damaged by H2O2 Caco-2 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate and cultured at 37°C for 12 h until the cells adhered. After incubation, the culture medium was removed from the cell culture plates. 100 μL of DMEM medium containing 1.5 mmol / L H₂O₂ was added to each well of the control and NHNK-627 groups (including the fermentation product group, the live bacterial suspension group, and the inactivated bacterial group) and incubated at 37°C for 3 h to establish an oxidative damage model. Subsequently, the H₂O₂-containing DMEM medium was removed, and 100 μL of DMEM medium containing 1% (v / v) NHNK-627 live bacterial suspension / fermentation product / inactivated bacterial suspension was added to each well of the NHNK-627 group. An equal volume of DMEM medium (100 μL) was added to each well of the control group. Each group was set up in triplicate. After incubation at 37°C and 5% CO₂ for 24 h, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 3 h. After incubation, the absorbance at 450 nm was measured using a microplate reader to quantify the number of viable cells.

[0048] Calculation formula: Relative survival rate (%) = (absorbance value of NHNK-627 group / absorbance value of control group) × 100% The results are shown in Table 6: Table 6 NHNK-627 improves the relative survival rate of Caco-2 cells damaged by H2O2 The results showed that NHNK-627 intervention could increase the survival rate of Caco-2 cells after H2O2 oxidative damage. Compared with the control group, the relative survival rate of the NHNK-627 group was between 101.24% and 129.87%.

[0049] Example 7 NHNK-627 increases the glutathione (GSH) content in Caco-2 cells 1. Preparation of NHNK-627 The preparation method of NHNK-627 fermentation product and live bacterial suspension is as described in Example 3.

[0050] 2. Culture of Human Intestinal Epithelial Cells Caco-2 The Caco-2 cell culture method was as described in Example 6.

[0051] 3. NHNK-627 increases glutathione (GSH) content in Caco-2 cells Caco-2 cells were seeded at 1×10⁶ cells / well in a 6-well cell culture plate and cultured at 37°C for 12 h until the cells adhered. After incubation, the culture medium was removed, and 2 mL of DMEM medium containing 1.5 mmol / L H₂O₂ was added to each well of the control and NHNK-627 groups. The cells were incubated at 37°C for 3 h to establish an oxidative damage model. Subsequently, the H₂O₂-containing DMEM medium was removed, and 1.9 mL of DMEM medium and 100 μL of NHNK-627 fermentation product or live bacterial suspension were added to each well of the NHNK-627 group. An equal volume of DMEM medium (2 mL) was added to each well of the control group. The cells were cultured at 37°C and 5% CO₂ for 24 h. After incubation, the cell culture supernatant was retained and assayed according to the instructions of a reduced glutathione assay kit (Nanjing Jiancheng Bioengineering Institute).

[0052] Calculation formula: GSH relative growth rate (%) = (GSH content in NHNK-627 group - GSH content in control group) / GSH content in control group × 100% The results are shown in Table 7: Table 7 NHNK-627 increases the glutathione GSH content in Caco-2 cells The results showed that NHNK-627 live bacteria and fermentation products could increase the content of glutathione (GSH) in Caco-2 cells, with a relative growth rate ranging from 11.69% to 37.00%, thereby improving the antioxidant capacity of intestinal epithelial cells.

[0053] Example 8 NHNK-627 regulates oxidative damage-induced Caco-2 cell barrier gene expression 1. Preparation of NHNK-627 The preparation method of fermentation product and live bacterial suspension is as described in Example 3.

[0054] 2. Culture of Human Intestinal Epithelial Cells Caco-2 The Caco-2 cell culture method was as described in Example 6.

[0055] 3. NHNK-627 regulates the expression of barrier-related genes in Caco-2 cells induced by oxidative damage Caco-2 cells were seeded at 1×10^6 cells / well in a 6-well cell culture plate and cultured at 37°C for 12 h until the cells adhered. After the culture, the culture medium in the cell culture plate was removed, and 2 mL of DMEM medium containing 1.5 mmol / L H2O2 was added to each well of the control group and NHNK-627 group, and incubated at 37°C for 3 h to establish the oxidative damage model. Subsequently, the DMEM medium containing H2O2 was removed, and 1.9 mL of DMEM medium and 100 μL of NHNK-627 fermentation product or live bacterial suspension were added to each well of the NHNK-627 group, and an equal volume of DMEM medium (2 mL) was added to each well of the control group. The cells were cultured at 37°C and 5% CO2 for 24 h. After the 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. Total RNA was extracted using Trizol total RNA extraction reagent (Beijing Solebold Technology Co., Ltd.) and the concentration and purity were determined. After extraction, the cells were reverse transcribed into cDNA and purified. GAPDH As the internal reference gene, the barrier gene was determined by qPCR OCLD 、 CLD4 The relative expression of the gene is F=1, using 2 -ΔΔCT The F value of each sample was calculated by this method.

[0056] The results are shown in Tables 8 and 9: Table 8 NHNK-627 fermentation products regulate the expression of barrier genes in Caco-2 cells induced by oxidative damage Table 9 NHNK-627 live bacterial suspension regulates the expression of barrier genes in Caco-2 cells induced by oxidative damage The results showed that NHNK-627 could upregulate Caco-2 cell barrier genes OCLD and CLD4 expression, thereby maintaining the integrity of the intestinal barrier.

[0057] Example 9 Biofilm formation by NHNK-627 1. NHNK-627 biofilm formation A single colony of NHNK-627 was picked and placed in MRS liquid medium, cultured at 37°C for 24 h, and then adjusted to OD 600 = 0.2, 100 μL of bacterial solution was added to each well of a 96-well plate, with 3 replicates per group, and then cultured at 37°C for 24 h; 2. Crystal violet staining After incubation, discard the supernatant and wash twice with 100 μL of sterile PBS per well. Then, add 100 μL of 4% paraformaldehyde fixative to each well and fix at room temperature for 30 min. Discard the fixative and add 100 μL of crystal violet to each well. Stain at room temperature for 30 min. After staining, wash twice with sterile PBS and air dry. Add 100 μL of anhydrous ethanol to each well, let stand for 1 min, and measure the absorbance at 600 nm.

[0058] The results are shown in Table 10: Table 10 Biofilm formation amount of NHNK-627 The results showed that NHNK-627 600 =0.2 was able to form a biofilm by culturing in MRS medium at 37℃ for 24 h.

[0059] Example 10 NHNK-627 biofilm improves colonization ability of nasal epithelial cells RPMI-2650 1. Preparation of NHNK-627 live bacterial suspension Pick a single colony of NHNK-627 and place it in fresh MRS liquid medium. Culture at 37°C for 24 h. Centrifuge at 5000 rpm for 10 min to collect the cells, wash twice with sterile PBS, and resuspend the cells in DMEM medium and adjust the OD value. 600 =0.5 to obtain a live bacterial suspension.

[0060] 2. Preparation of NHNK-627 biofilm suspension A single colony of NHNK-627 was picked and placed in MRS liquid medium, cultured at 37°C for 24 h, and then adjusted to OD 600 =0.2 was added to the bacterial culture dish and then cultured at 37°C for 24 hours. After the culture was completed, the upper culture medium was discarded, and the bottom biofilm was resuspended after washing twice with sterile PBS. The bacteria were collected by centrifugation at 5000 rpm for 10 minutes, and the bacteria were resuspended in DMEM medium and the OD was adjusted. 600 =0.5 to obtain biofilm bacterial suspension.

[0061] 3. NHNK-627 biofilm improves bacterial colonization ability on RPMI-2650 cells RPMI-2650 cells were seeded in DMEM supplemented with 10% (v / v) FBS serum and cultured at 37°C in 5% CO2. When cell confluency reached 80%, 1 mL of 0.25% trypsin (i.e., 0.25 g of trypsin per 100 mL, purchased from Shanghai Biotech Co., Ltd.) was added to each flask and digested at room temperature for 5 min. When cells began to float in sheets, digestion was terminated by adding 5 mL of DMEM. Cells were harvested by centrifugation at 1200 rpm for 5 min and seeded at 1 × 10^6 cells / well into a 6-well plate containing a cell slide for overnight culture. After incubation, the supernatant was discarded, and the cells were washed twice with sterile PBS. 1 mL of DMEM supplemented with FBS serum and 1 mL of live bacterial suspension or biofilm suspension were added to each well and incubated for an additional 2 h. After incubation, the cells were washed twice with sterile PBS to remove unadhered bacteria. The cell slides were fixed in 4% paraformaldehyde for 15 min, then Gram-stained and photographed. Figure 3 shown.

[0062] The results showed that NHNK-627 biofilm could increase the adsorption of bacteria to nasal epithelial cells.

[0063] Example 11 NHNK-627 biofilm improves bacterial colonization ability in intestinal epithelial cells Caco-2 1. Preparation of NHNK-627 live bacteria and biofilm suspension The preparation method is as described in Example 10.

[0064] 2. NHNK-627 biofilm improves the ability of bacteria to colonize Caco-2 cells Caco-2 cells were inoculated in DMEM medium containing 10% (V / V) FBS serum and cultured at 37°C and 5% CO2. When the cell confluence reached 80%, 1 mL of 0.25% trypsin (i.e., 0.25 g of trypsin per 100 mL, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was added to each bottle of cells and digested at room temperature for 5 minutes. When the cells were observed to float in sheets, 5 mL of DMEM medium was immediately added to terminate the digestion, and the cells were collected by centrifugation at 1200 rpm for 5 minutes. The cells were inoculated into a 6-well plate containing cell slides at a rate of 1×10^6 cells / well for overnight culture. After the culture was completed, the supernatant was discarded, and the cells were washed twice with sterile PBS. 1 mL of DMEM medium without FBS serum and 1 mL of live bacterial suspension or biofilm bacterial suspension were added to each well and incubated for another 2 hours. After the incubation was completed, the cells were washed twice with sterile PBS to remove non-adherent bacteria. The slides were fixed in 4% mass concentration paraformaldehyde for 15 minutes, and then Gram-stained and photographed. Figure 4 shown.

[0065] The results showed that NHNK-627 biofilm could increase the adsorption of bacteria to intestinal epithelial cells.

[0066] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the present invention. These related improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum, characterized in that The plant lactobacillus is plant lactobacillus ( Lactiplantibacillus plantarum ) NHNK-627 was deposited in the China Center for Type Culture Collection on February 28, 2025, and its deposit number is CCTCC NO: M 2025323.

2. The product of Lactobacillus plantarum according to claim 1, which is one of live bacteria, fermentation / secretory products, inactivated bacteria, and biofilm of Lactobacillus plantarum NHNK-627.

3. Use of the product of Lactobacillus plantarum according to claim 1 or Lactobacillus plantarum according to claim 2 in preparing a product for improving retinal macular degeneration.

4. The use of the product for improving retinal macular degeneration according to claim 3, characterized in that: The mechanism includes any one or more of the following: 1) Improve the survival rate of oxidatively damaged human retinal pigment epithelial cells; 2) Improve retinal antioxidant capacity; 3) Alleviate retinal inflammatory response; 4) Strengthen the retinal barrier; 5) Reduce retinal neovascularization; 6) Inhibit retinal cell apoptosis.

5. The use of the product for improving retinal macular degeneration according to claim 4, characterized in that: Improving the retinal antioxidant capacity includes any one or two of the following methods: 1) Increase the secretion of glutathione (GSH) in human retinal pigment epithelial cells; 2) Upregulating the expression of antioxidant-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the antioxidant-related gene is the nuclear factor E2-related factor 2 gene NRF2 and / or heme oxygenase 1 gene HO-1 ; The method for alleviating retinal inflammatory response comprises down-regulating the expression of pro-inflammatory factor-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the pro-inflammatory factor-related genes in the human retinal pigment epithelial cells include interleukin-8 gene IL-8 , interleukin-6 gene IL-6, interleukin-1β gene IL-1β, Tumor necrosis factor alpha gene TNF - α Any one or more of the following; The method for enhancing the retinal barrier comprises upregulating the expression of barrier-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the barrier-related genes in the human retinal pigment epithelial cells are zonula occludens-1 genes. ZO-1 and / or claudin 4 gene CLD4 ; The method for reducing retinal neovascularization comprises down-regulating the expression of neovascularization-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the neovascularization-related genes in human retinal pigment epithelial cells are vascular endothelial growth factor genes. VEGF ; The inhibition of retinal cell apoptosis includes down-regulating the expression of apoptosis-related genes in oxidatively damaged human retinal pigment epithelial cells, wherein the apoptosis-related gene is the tumor protein 53 gene. P53 and / or cyclin-dependent kinase inhibitor genes P21 .

6. The use of the product for preparing and improving retinal macular degeneration according to claim 3, characterized in that: The mechanism includes any one or more of the following: 1) Improve the survival rate of oxidatively damaged intestinal epithelial cells; 2) Improve the antioxidant capacity of intestinal epithelial cells; 3) Maintain the intestinal barrier.

7. The use of the product for improving retinal macular degeneration according to claim 6, characterized in that: The improving the antioxidant capacity of intestinal epithelial cells includes improving the secretion of glutathione GSH in intestinal epithelial cells; The maintenance of the intestinal barrier comprises upregulating the expression of barrier genes of intestinal epithelial cells, wherein the barrier genes of intestinal epithelial cells are OCLD and / or CLD4 .

8. The use of the product for preparing and improving retinal macular degeneration according to claim 3, characterized in that: The mechanism includes any one or more of the following: 1) Improve the colonization ability of intestinal epithelial cells; 2) Improve the colonization ability of nasal epithelial cells; 3) Formation of biofilm.

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