Lactobacillus acidophilus for improving senile retinal degeneration as well as product and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus acidophilus for improving senile retinal degeneration as well as a product and application thereof. The lactobacillus acidophilus NHNK-620 disclosed by the invention is deposited in the China Center for Type Culture Collection, and the preservation number of the lactobacillus acidophilus NHNK-620 is CCTCC NO: M 20241762. Experiments show that the NHNK-620 has the functions of improving the oxidation resistance of the retina, increasing the retinal barrier, reducing the retinal apoptosis, reducing the retinal neovascularization and reducing the retinal inflammation, in addition, the NHNK-620 thallus biological membrane can improve the colonization capacity of nasal epithelial cells and intestinal epithelial cells, and the NHNK-620 can be used for preparing products for improving the senile retinal degeneration.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to Lactobacillus acidophilus for improving senile retinal degeneration, and products and applications thereof. Background Art

[0002] Mucosal sites of the body, such as the oral cavity, nasopharynx, and vagina, all harbor their own commensal flora. The tissues on the surface of the eye are also mucosal. The ocular surface is at the interface of the environment and the host immune system and is constantly exposed to an environment with microorganisms such as bacteria, fungi, and viruses. When the ocular surface microbiome is out of balance, eye diseases may occur. The eye contains a core microbiome that depends 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 that have long been considered purely hereditary may be caused in part by bacteria escaping from the intestine to the retina. Damage to the retinal and intestinal barriers allows bacteria to transfer from the intestine to the retina, causing secondary retinal degeneration, which can be alleviated by antibiotic treatment. There are tight junctions between retinal pigment epithelial cells, which constitute the outer barrier between the retina and the choroid, or the outer blood retinal barrier, which can prevent abnormal components from the choroid from entering the retina. The outer barrier is composed of retinal pigment epithelial (RPE) cells and their junctional complexes. The cells include gap junctions at the top, occluded zonules in the middle, and adhesion zonules at the bottom, among which the occluded zonules are the most important.

[0004] Choroidal neovascularization (CNV) will lead to visual impairment regardless of whether it occurs in the macula, around the optic disc, or in the periphery, especially in age-related macular degeneration.

[0005] Marine microorganisms are one of the earliest sources of marine bioactive substances known to people, including algae, bacteria, fungi, protozoa and other microorganisms. These bioactive substances have important application values ​​in various fields such as medicine, food, and cosmetics. Therefore, providing a product developed using microbial technology is of great practical significance. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides Lactobacillus acidophilus and products and applications for improving senile retinal degeneration, which are used to solve the technical problems in the prior art.

[0007] The present invention provides Lactobacillus acidophilus ( Lactobacillus acidophilus ), which is Lactobacillus acidophilus (Lactobacillus acidophilus )NHNK-620, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on August 9, 2024, and its preservation number is CCTCC NO: M 20241762, Lactobacillus acidophilus.

[0008] Further, according to the above-mentioned Lactobacillus acidophilus ( Lactobacillus acidophilus ) prepared by Lactobacillus acidophilus ( Lactobacillus acidophilus ) of live bacteria, inactivated bacteria or fermentation products.

[0009] Furthermore, the product also includes auxiliary materials and / or additives.

[0010] The present invention also provides the above-mentioned Lactobacillus acidophilus ( Lactobacillus acidophilus ) or the above-mentioned Lactobacillus acidophilus ( Lactobacillus acidophilus ) in the preparation of products for improving senile retinal degeneration.

[0011] Furthermore, the improvement of senile retinal degeneration includes at least one of improving retinal antioxidant capacity, increasing retinal barrier, reducing retinal apoptosis, reducing retinal neovascularization and reducing retinal inflammation.

[0012] Further, the improvement of age-related retinal degeneration includes at least one of the following a)-e): a) Improve the antioxidant capacity of the retina, increase the secretion of glutathione (GSH) in human retinal pigment epithelial cells; increase the survival rate of oxidatively damaged human retinal pigment epithelial cells; upregulate the antioxidant-related nuclear factor E2-related factor 2 gene in oxidatively damaged human retinal pigment epithelial cells NRF2 and heme oxygenase 1 gene HO-1 Expression; b) Increase retinal barrier and upregulate barrier-related zonula closure protein-1 gene in oxidatively damaged human retinal pigment epithelial cells ZO-1 and claudin 4 gene CLD4 Expression; c) Reduce retinal apoptosis and downregulate the apoptosis-related tumor protein 53 gene in oxidatively damaged human retinal pigment epithelial cells P53 cyclin-dependent kinase inhibitor gene P21 Expression; d) Reduce retinal neovascularization and downregulate the vascular endothelial growth factor gene associated with neovascularization of human retinal pigment epithelial cells damaged by oxidation VEGF Expression; e) Reduce retinal inflammation and downregulate the pro-inflammatory factor-related tumor necrosis factor α gene in oxidatively damaged human retinal pigment epithelial cells TNF-α、 Interleukin-8 gene IL-8、 Interleukin-6 gene IL-6 and interleukin-1β gene IL-1β expression.

[0013] The present invention also proposes the above-mentioned Lactobacillus acidophilus ( Lactobacillus acidophilus ) or Lactobacillus acidophilus as described above ( Lactobacillus acidophilus ) is used in increasing the colonization ability of bacterial biofilm on nasal epithelial cells and intestinal epithelial cells.

[0014] The Lactobacillus acidophilus disclosed in the present invention ( Lactobacillus acidophilus ), and its deposit number is CCTCCNO: M 20241762. Experiments show that NHNK-620 has the functions of improving retinal antioxidant capacity, increasing retinal barrier, reducing retinal apoptosis, reducing retinal neovascularization and reducing retinal inflammation. In addition, NHNK-620 bacterial biofilm can increase the colonization ability of nasal epithelial cells and intestinal epithelial cells. It can be used to prepare products to improve senile retinal degeneration.

[0015] Biological Deposit Description Lactobacillus acidophilus ( Lactobacillus acidophilus ) NHNK-620, was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on August 9, 2024, and its deposit number is CCTCC NO: M 20241762. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The figure is a graph showing the growth pattern of Lactobacillus acidophilus NHNK-620 on an MRS plate and the detection result of Gram staining microscopy in Example 1 of the present invention; Figure 2 This is a graph showing the experimental results of Lactobacillus acidophilus NHNK-620 improving the survival of ARPE-19 cells damaged by H2O2 oxidation in Example 3 of the present invention; Figure 3This is a graph showing the experimental results of measuring the adhesion ability of Lactobacillus acidophilus NHNK-620 to nasal epithelial cells RPMI-2650 in Example 10 of the present invention; Figure 4 This is a graph showing the experimental results of measuring the adhesion ability of Lactobacillus acidophilus NHNK-620 to human intestinal epithelial cells Caco-2 in Example 11 of the present invention.

[0018] The embodiments of the present invention will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0019] The present invention provides Lactobacillus acidophilus and its application for improving senile retinal degeneration. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change 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 realize and apply the technology of the present invention.

[0020] The Lactobacillus acidophilus NHNK-620 of the present invention is derived from naturally fermented shrimp paste and is identified as Lactobacillus acidophilus (NHNK-620) by 16S rDNA. Lactobacillus acidophilus ). This strain is Gram-positive, short rod-shaped under a microscope, and the end of the rod is round; it grows on MRS plates and forms round colonies with rough surfaces, curled edges, and opaque; it grows evenly in MRS liquid culture medium and has a white precipitate after long-term storage. The optimal growth temperature is 37°C. Please refer to the sequence table for the specific sequence of 16S rDNA.

[0021] Lactobacillus acidophilus ( Lactobacillus acidophilus )NHNK-620, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: August 9, 2024, deposit number: CCTCC NO: M 20241762.

[0022] Furthermore, the Lactobacillus acidophilus NHNK-620 provided by the present invention exists in the form of sterilized inactivated bacteria, or in the form of fermentation / secretion products (i.e., supernatant), and is not sterilized live bacteria. The derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation products, and inactivated bacteria.

[0023] Lactobacillus acidophilus ( Lactobacillus acidophilus ) The preparation method of live bacteria is as follows: the Lactobacillus acidophilus (Lactobacillus acidophilus ) was inoculated into the culture medium, cultured at 37°C for 48 hours, fermentation liquid was obtained, precipitated by centrifugation, and the precipitate was washed to obtain live bacteria; The method for preparing the fermentation / secretion product is as follows: Lactobacillus acidophilus ( Lactobacillus acidophilus ) was inoculated into the culture medium and cultured at 37°C for 48 h to obtain the fermentation broth. The supernatant was centrifuged and filtered at a precision of 0.22 μm. The filtrate obtained was the fermentation / secretion product. The preparation method of inactivated bacteria is as follows: Lactobacillus acidophilus ) was inoculated into the culture medium, cultured at 37°C for 48 hours to obtain a fermentation broth, centrifuged to obtain a precipitate, washed the precipitate, and inactivated at 121°C for 30 minutes to obtain inactivated bacteria.

[0024] The reagents and consumables used in the present invention are all common commercially available products. The present invention is further described below in conjunction with the embodiments: Example 1 Separation of NHNK-620 The naturally fermented shrimp paste from Qingdao was shaken and mixed, then diluted 10 times with physiological saline. After mixing again, 100 μL of the dilution was spread on MRS culture medium and placed in an anaerobic bag. After constant temperature culture at 37°C for 48 hours, white colonies were picked and repeatedly streaked for purification until a single colony with regular and uniform shape was obtained, which was named NHNK-620.

[0025] Gram staining microscopy: NHNK-620 is Gram staining positive, short rod-shaped under the microscope, and the end of the rod is round; it grows on MRS plates and can form round colonies with rough surfaces, curled edges, and opaque; it grows evenly in MRS liquid culture medium and has a white precipitate after long-term storage. The optimal growth temperature is 37°C. Figure 1 shown.

[0026] Example 2 Nucleic acid identification of NHNK-620 1. 16S rDNA gene sequence analysis: Pick a single colony and place it in MRS liquid medium. After culturing overnight at 37°C, centrifuge at 12,000 rpm for 1 min to collect the bacteria and follow the steps of the DNA extraction kit. Primers used were bacterial universal primers 27F and 1492R. The PCR amplification system was a 50 μL system, with 95°C pre-denaturation for 5 min; 94°C for 15 s, 57°C for 15 s, and 72°C for 40 s, for 35 cycles; and 72°C extension for 10 min.

[0027] 2. Sequence analysis results: The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). It was concluded that the NHNK-620 strain was Lactobacillus acidophilus ( Lactobacillus acidophilus ).

[0028] Example 3 NHNK-620 improves the survival rate of oxidatively damaged human retinal pigment epithelial cells ARPE-19 1. Preparation of NHNK-620 Pick a single colony of NHNK-620 and place it in fresh MRS medium and culture it at 37°C for 48 hours. Adjust the OD to 0. 600 =0.5, 5000rpm, take the supernatant, and then filter with a 0.22μm filter membrane to obtain the fermentation product. Collect the centrifugal precipitated bacteria, wash twice with sterile PBS, resuspend the bacteria with DMEM medium and adjust the OD 600 =0.5, and a live bacterial suspension was obtained. Part of the live bacterial precipitate was washed twice with sterile PBS and then sterilized by high pressure at 121°C for 15 min. The precipitate was resuspended in DMEM medium and adjusted to OD 600 =0.5, and inactivated bacteria were obtained.

[0029] 2. Cultivation 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 subcultured or plated after the cells were 80-90% confluent.

[0030] 3. NHNK-620 improves the relative survival rate of ARPE-19 cells damaged by H2O2 ARPE-19 cells were seeded in a 96-well cell culture plate at 1×10^4 cells / well and cultured for 12 hours until the cells adhered. The concentration of H2O2 was adjusted to 1.5mmol / L using DMEM medium. After the culture was completed, the culture medium was removed, and 100μL of DMEM medium containing 1.5mmol / L H2O2 was added to the control group and NHNK-620 group (including fermentation products, live bacterial suspension and inactivated bacteria), and incubated at 37°C for 3 hours to establish an oxidative damage model. Subsequently, the original culture medium was removed, and 100μL of DMEM medium containing 1% (v / v) NHNK-620 live bacteria / fermentation products / inactivated bacterial suspension was added to the NHNK-620 group, and an equal volume of DMEM medium was added to the control group, with 3 parallels set up for each group. Culture was carried out at 37°C and 5% CO2 for 24 hours. Figure 2 As shown. Then, 10 μL CCK-8 solution was added to each well and incubated at 37°C for 3 hours. After incubation, the absorbance at 450 nm was measured with an ELISA reader, and the calculation formula was: relative survival rate (%) = NHNK-620 group / control group*100%. The results are shown in Table 1: Table 1: NHNK-620 improves the relative survival rate of ARPE-19 cells damaged by H2O2

[0031] The results showed that NHNK-620 could increase the survival rate of human retinal pigment epithelial cells ARPE-19 under H2O2 oxidative damage and enhance the antioxidant capacity of human retinal pigment epithelial cells.

[0032] Example 4 NHNK-620 increases glutathione GSH secretion in human retinal cells ARPE-19 1. Preparation of NHNK-620 The preparation method of fermentation product and live bacterial suspension is as described in Example 3.

[0033] 2. Culture of human retinal pigment epithelial ARPE-19 cells The cell culture method is as in Example 3.

[0034] 4. NHNK-620 increases the glutathione GSH content in ARPE-19 cells ARPE-19 cells were inoculated in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The concentration of H2O2 was adjusted to 1.5mmol / L using DMEM medium. After the culture was completed, the medium was removed, and 2mL of DMEM medium containing 1.5mmol / L H2O2 was added to the control group and NHNK-620 group (including fermentation products and live bacterial suspension), and incubated at 37℃ for 3h to establish the oxidative damage model. Subsequently, the original medium was removed, 1.9mL of DMEM medium and 100μL of NHNK-620 live bacteria or fermentation products were added to the NHNK-620 group, and an equal volume of DMEM medium was added to the control group. Cultured for 24h at 37℃ and 5% CO2, the cell culture supernatant was retained after the culture was completed, and the secretion of glutathione (GSH) of the cells was detected according to the reagent instructions. The calculation formula was: GSH relative growth rate (%) = (NHNK-620 group-control group) / control group*100%, and the results are shown in Table 2: Table 2: NHNK-620 increases GSH secretion in ARPE-19 cells

[0035] The results showed that NHNK-620 could increase the glutathione GSH content secreted by human retinal pigment epithelium ARPE-19, with a relative growth rate of 14.08%~25.07%, thereby improving the antioxidant capacity of cells.

[0036] Example 5 NHNK-620 regulates the expression of inflammation, barrier and antioxidant related genes in human retinal ARPE-19 cells induced by oxidative damage 1. Preparation of NHNK-620 The preparation methods of fermentation product, live bacterial suspension and inactivated bacterial bodies are as described in Example 3.

[0037] 2. Cultivation of human retinal pigment epithelial cells ARPE-19 The cell culture method is as in Example 3.

[0038] 3. NHNK-620 regulates the expression of inflammation, barrier and antioxidant related genes in ARPE-19 cells ARPE-19 cells were seeded in 6-well cell culture plates at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The concentration of H2O2 was adjusted to 1.5mmol / L using DMEM medium. After the culture was completed, the medium was removed, and 2mL of DMEM medium containing 1.5mmol / L H2O2 was added to the control group and NHNK-620 group (including fermentation products, live bacterial suspension and inactivated bacteria), and incubated at 37℃ for 3h to establish the oxidative damage model. Then the original medium was removed, 1.9mL of DMEM medium and 100μL of NHNK-620 live bacteria / fermentation products / inactivated bacterial suspension were added to the NHNK-620 group, and an equal volume of DMEM medium was added to the control group. The cells were cultured at 37℃ and 5% CO2 for 24h. After the culture was completed, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then 1mL of cell RNA extraction reagent was added to each well, and total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After extraction, reverse transcription was performed into cDNA, and qPCR was used to determine the inflammatory gene using GAPDH as the internal reference gene. 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 group gene is F=1, using 2 -ΔΔCT The F value of each sample is calculated by the formula: F=2-△△CT, where △CT 实验 =CT 实验 -CT 内参（实验） ; △CT 对照 =CT 对照 -CT 内参（对照） ; △△CT=△C T实验 -△CT 对照 The results are shown in Table 3, Table 4 and Table 5: Table 3: NHNK-620 live bacteria regulates gene expression in ARPE-19 cells induced by oxidative damage

[0039] Table 4: NHNK-620 fermentation products regulate the gene expression of ARPE-19 cells induced by oxidative damage

[0040] Table 5: NHNK-620 inactivated bacteria regulates gene expression in ARPE-19 cells induced by oxidative damage

[0041] The results showed that NHNK-620 could regulate the gene expression of ARPE-19 cells induced by oxidative damage and downregulate the genes related to pro-inflammatory factors. TNF-α, IL-8, IL-6 and IL-1β ; Apoptosis-related genes P53 and P21 ; Angiogenesis-related genes VEGF Upregulation of barrier-related genes ZO-1 and CLD4 and antioxidant-related genes NRF2 and HO-1 It regulates the expression of retinal plexus, alleviates the inflammatory response caused by oxidative damage, strengthens the ocular barrier and reduces ocular neovascularization, thereby preventing macular degeneration.

[0042] Example 6 NHNK-620 improves the survival rate of oxidatively damaged Caco-2 cells 1. Preparation of NHNK-620 The preparation methods of fermentation product, live bacterial suspension and inactivated bacterial bodies are as described in Example 3.

[0043] 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 subcultured or plated after the cells were 80-90% confluent.

[0044] 3. NHNK-620 increases the relative survival rate of Caco-2 cells damaged by H2O2 Caco-2 cells were seeded in 96-well cell culture plates at 1×10^4 cells / well and cultured for 12 hours until the cells adhered to the wall. The concentration of H2O2 was adjusted to 1.5mmol / L using DMEM medium. After the culture was completed, the medium was removed, and 100μL of DMEM medium containing 1.5mmol / L H2O2 was added to the control group and NHNK-620 group (including fermentation products, live bacterial suspension and inactivated bacteria), and incubated at 37℃ for 3 hours to establish the oxidative damage model. Subsequently, the original medium was removed, and 100 μL of DMEM medium containing 1% (v / v) NHNK-620 live bacteria / fermentation products / inactivated bacterial suspension was added to the NHNK-620 group, and an equal volume of DMEM medium was added to the control group, with 3 parallels set for each group. After culturing for 24 hours at 37℃ and 5% CO2, 10μL of CCK-8 solution was added to each well and incubated at 37℃ for 3 hours. After the incubation was completed, the absorbance at 450 nm was measured with an ELISA instrument, and the calculation formula was: relative survival rate (%) = NHNK-620 group / control group*100%. The results are shown in Table 6: Table 6: NHNK-620 improves the relative survival rate of Caco-2 cells damaged by H2O2

[0045] The results showed that NHNK-620 intervention could increase the survival rate of Caco-2 cells after H2O2 oxidative damage. Compared with the model group, the relative survival rate of the NHNK-620 group was between 111.18% and 125.93%.

[0046] Example 7 NHNK-620 increases glutathione GSH secretion in Caco-2 cells 1. Preparation of NHNK-620 The preparation method of NHNK-620 fermentation product and live bacterial suspension is as described in Example 3.

[0047] 2. Cultivation of human intestinal epithelial cells Caco-2 The Caco-2 cell culture method refers to Example 6.

[0048] 4. NHNK-620 increases the glutathione GSH content in Caco-2 cells Caco-2 cells were inoculated in 6-well cell culture plates at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The concentration of H2O2 was adjusted to 1.5mmol / L using DMEM medium. After the culture was completed, the medium was removed, and 2mL of DMEM medium containing 1.5mmol / L H2O2 was added to the control group and NHNK-620 group, and the oxidative damage model was established at 37℃ for 3h. The original medium was then removed, and 1.9mL of DMEM medium and 100μL of NHNK-620 fermentation product or live bacterial suspension were added to the NHNK-620 group, and an equal volume of DMEM medium was added to the control group. The cells were cultured at 37℃ and 5% CO2 for 24h. After the culture was completed, the cell culture supernatant was retained, and the secretion of glutathione (GSH) of the cells was detected according to the instructions of the kit. The calculation formula was: GSH relative growth rate (%) = (NHNK-620 group-control group) / control group*100%. The results are shown in Table 7: Table 7: NHNK-620 increases GSH secretion in Caco-2 cells

[0049] The results showed that NHNK-620 live bacteria and fermentation products could increase the GSH content of Caco-2 cells, with a relative growth rate between 18.18% and 27.27%, thereby improving the antioxidant properties of cells.

[0050] Example 8 NHNK-620 regulates oxidative damage-induced Caco-2 cell barrier gene expression 1. Preparation of NHNK-620 The preparation method of fermentation product and live bacterial suspension is as described in Example 3.

[0051] 2. Cultivation of human intestinal epithelial cells Caco-2 The Caco-2 cell culture method refers to Example 6.

[0052] 4. NHNK-620 regulates the expression of barrier-related genes in Caco-2 cells damaged by oxidative damage Caco-2 cells were inoculated in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The concentration of H2O2 was adjusted to 1.5mmol / L using DMEM medium. After the culture was completed, the culture medium was removed, and 2mL of DMEM medium containing 1.5mmol / L H2O2 was added to the control group and the NHNK-620 group, and incubated at 37°C for 3 hours to establish an oxidative damage model. Subsequently, the original culture medium was removed, 1.9mL of DMEM medium and 100μL of NHNK-620 fermentation product or live bacterial suspension were added to the NHNK-620 group, and an equal volume of DMEM medium was added to the control group. Cultured at 37°C and 5% CO2 for 24 hours, the supernatant was discarded, washed twice with sterile PBS, and then 1mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After extraction, it was reverse transcribed into cDNA, and GAPDH was used as the internal reference gene to determine the barrier gene by qPCR. ZO-1、 OCLD , CLD4 The relative expression of the gene is F=1, using 2 -ΔΔCT The F value of each sample was calculated by the method. The results are shown in Table 8 and Table 9: Table 8: NHNK-620 fermentation products regulate oxidative damage-induced Caco-2 cell gene expression

[0053] Table 9: NHNK-620 live bacteria regulates oxidative damage-induced Caco-2 cell gene expression

[0054] The results showed that NHNK-620 could upregulate Caco-2 cell barrier genes ZO-1, OCLD and CLD4 expression, thereby maintaining the integrity of the intestinal barrier. Example 9 NHNK-620 forms biofilm 1. Formation of NHNK-620 biofilm A single colony of NHNK-620 was selected and placed in MRS liquid medium, cultured at 37°C for 24 h, and the OD was adjusted to 0. 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then cultured at 37 degrees for 24 hours.

[0055] 2. Crystal violet staining After the incubation, the supernatant was discarded, 100 μL of sterile PBS was added to each well for washing twice, and then 100 μL of 4% paraformaldehyde fixative was added to each well for fixing at room temperature for 30 minutes. The fixative was discarded, 100 μL of crystal violet was added to each well, and staining was performed at room temperature for 30 minutes. After staining, the cells were washed twice with sterile PBS and dried, and 100 μL of anhydrous ethanol was added to each well. After standing for 1 minute, the absorbance at 600 nm was measured. The results are shown in Table 10: Table 10: NHNK-620 biofilm formation

[0056] The results showed that NHNK-620 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0057] Example 10 NHNK-620 biofilm improves colonization ability of nasal epithelial cells RPMI-2650 1. Preparation of NHNK-620 live bacterial suspension Pick a single colony of NHNK-620 in fresh MRS medium and culture at 37°C for 24 hours. Centrifuge at 5000 rpm for 10 minutes to collect the cells, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5 to obtain a live bacterial suspension.

[0058] 2. Preparation of NHNK-620 biofilm bacterial suspension Pick a single colony of NHNK-620 in MRS liquid medium, culture it at 37℃ for 24h, and adjust the OD to 0. 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, the bottom biofilm was resuspended after washing twice with PBS, and the bacteria were collected by centrifugation at 5000rpm for 10min. The bacteria were resuspended in DMEM culture medium and the OD was adjusted. 600 =0.5 to obtain biofilm bacterial suspension.

[0059] 2. NHNK-620 biofilm improves the colonization ability of bacteria on RPMI-2650 cells RPMI-2650 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the degree of confluence reached 80%, the cells were digested with trypsin and collected, and inoculated into a 6-well plate containing a cell slide for overnight culture. After the culture, the supernatant was discarded, washed twice with sterile PBS, and 1mL of DMEM medium without FBS serum and 1mL of live bacterial suspension or biofilm bacterial suspension were added, and the incubation continued for 2h. After the incubation, wash twice with PBS to remove non-adherent bacteria. The cell slide was immersed in 4% paraformaldehyde for 15 minutes, followed by Gram staining and photography. Figure 3 shown.

[0060] The results showed that NHNK-620 biofilm could increase the adsorption of bacteria to nasal epithelial cells.

[0061] Example 11 NHNK-620 biofilm improves bacterial colonization ability in intestinal epithelial cells Caco-2 1. Preparation of NHNK-620 live bacteria and biofilm bacterial suspension The preparation method is similar to that of Example 10.

[0062] 2. NHNK-620 biofilm improves the colonization ability of bacteria on Caco-2 cells Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the degree of confluence reached 80%, the cells were digested with trypsin and collected, and inoculated into a 6-well plate containing a cell slide for overnight culture. After the culture, the supernatant was discarded, and the plate was washed twice with sterile PBS. 1mL of DMEM medium without FBS serum and 1mL of live bacterial suspension or biofilm bacterial suspension were added, and the incubation continued for 2h. After the incubation, it was washed twice with PBS to remove non-adherent bacteria. The slide was fixed in 4% paraformaldehyde for 15 minutes, followed by Gram staining and photography. Figure 4 shown.

[0063] The results showed that NHNK-620 biofilm could increase the adsorption of bacteria to intestinal epithelial cells.

[0064] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Lactobacillus acidophilus ( Lactobacillus acidophilus ), which is Lactobacillus acidophilus ( Lactobacillus acidophilus ) NHNK-620 was deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCC NO: M 20241762.

2. The Lactobacillus acidophilus according to claim 1 ( Lactobacillus acidophilus ) is characterized in that The product is Lactobacillus acidophilus ( Lactobacillus acidophilus ) of live bacteria, inactivated bacteria or fermentation products.

3. The Lactobacillus acidophilus according to claim 1 ( Lactobacillus acidophilus ) is characterized in that The product also includes auxiliary materials and / or adjuvants.

4. The Lactobacillus acidophilus according to claim 1 ( Lactobacillus acidophilus ) or the Lactobacillus acidophilus according to claim 2 ( Lactobacillus acidophilus ) Use of the prepared product in the preparation of products for improving senile retinal degeneration.

5. The use according to claim 3, characterized in that: The improvement of senile retinal degeneration includes at least one of improving retinal antioxidant capacity, increasing retinal barrier, reducing retinal apoptosis, reducing retinal neovascularization and reducing retinal inflammation.

6. The use according to claim 3, characterized in that: The improvement of age-related retinal degeneration includes at least one of the following a)-e): a) Improve the antioxidant capacity of the retina, increase the secretion of glutathione GSH in human retinal pigment epithelial cells; increase the survival rate of oxidatively damaged human retinal pigment epithelial cells; upregulate the antioxidant-related nuclear factor E2-related factor 2 gene in oxidatively damaged human retinal pigment epithelial cells NRF2 and heme oxygenase 1 gene HO-1 Expression; b) Increase retinal barrier and upregulate barrier-related zonula closure protein-1 gene in oxidatively damaged human retinal pigment epithelial cells ZO-1 and claudin 4 gene CLD4 Expression; c) Reduce retinal apoptosis and downregulate the apoptosis-related tumor protein 53 gene in oxidatively damaged human retinal pigment epithelial cells P53 cyclin-dependent kinase inhibitor gene P21 Expression; d) Reduce retinal neovascularization and downregulate the vascular endothelial growth factor gene associated with neovascularization of human retinal pigment epithelial cells damaged by oxidation VEGF Expression; e) Reduce retinal inflammation and downregulate the pro-inflammatory factor-related tumor necrosis factor α gene in oxidatively damaged human retinal pigment epithelial cells TNF-α, Interleukin-8 gene IL-8 Interleukin-6 gene IL-6 and interleukin-1β gene IL-1β expression.

7. The Lactobacillus acidophilus according to claim 1 ( Lactobacillus acidophilus ) or the Lactobacillus acidophilus according to claim 2 ( Lactobacillus acidophilus ) is used in increasing the colonization ability of bacterial biofilm on nasal epithelial cells and intestinal epithelial cells.

Images