Lactobacillus reuteri LR1349 and application thereof in improving blue light induced eye injury

Abstract

The invention discloses lactobacillus reuteri LR1349 and application thereof in improving eye injury induced by blue light, and belongs to the technical field of microorganisms. The lactobacillus reuteri LR1349 disclosed by the invention is preserved in the German Microorganism and Cell Culture Collection Center (DSMZ), and the preservation number is DSM 35135. The lactobacillus reuteri LR1349 provided by the invention can effectively improve the reduction of visual acuity, retinal dysfunction and retinopathy caused by blue light irradiation. Therefore, the lactobacillus reuteri LR1349 disclosed by the invention can be used for preparing medicines, foods or food additives for improving eye injury induced by blue light or relieving visual fatigue.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Lactobacillus reuteri LR1349 and its use in improving blue light-induced eye damage. Background Technology

[0002] Blue light is high-energy visible light with a wavelength between 400 and 500 nm. It can penetrate the cornea, lens, and vitreous humor to reach the retina, stimulating photochemical reactions and generating reactive oxygen species (ROS), causing photooxidative damage to the retina. Studies have shown that blue light is significantly associated with various retinal diseases such as age-related macular degeneration (AMD) and glaucoma (Ouyang, X., et al., Mechanisms of blue light-induced eye hazard and protective measures: A review. Biomedicine & Pharmacotherapy, 2020, 130).

[0003] In nature, the sun is the main source of blue light. Most ultraviolet light (UV) with wavelengths below 295nm can be absorbed by the cornea. The lens also blocks UVA (315–400nm) and UVB (280–315nm) from penetrating. Only shorter wavelengths of blue light and a small amount of ultraviolet light in the visible light can cause photochemical damage to the retina, and are therefore considered harmful light.

[0004] In recent years, electronic products using light-emitting diodes (LEDs) as light sources, such as LCD TVs, computer screens, mobile phones, and tablets, have become widely used in daily life. Blue light is known to be the main color rendering component of cool-white LED light and has gradually replaced traditional incandescent or fluorescent lamps as the light source for modern lighting and screen displays. The hazards of blue light have become a significant public health issue, and blue light has emerged as a new source of environmental pollution.

[0005] A review of in vivo and in vitro studies on blue light damage has revealed that exposure to blue light or blue-light-rich LED light sources can cause morphological changes in the retinal tissue of experimental animals. Among these changes, the three layers of the outer nuclear layer (ONL) and retinal pigment epithelial cells (RPE) are the most severely damaged. In tissue sections, pathological phenomena such as thinning of the ONL and RPE, reduction of the number of ONL / RPE cell nuclei, distortion or disappearance of the boundaries between the layers, or pyknosis of photoreceptor cell nuclei can be observed (Hunter, JJ, et al., Thesusceptibility of the retina to photochemical damage from visible light. ProgRetin Eye Res, 2012.31(1):28-42).

[0006] Ueda et al. (Ueda, T., et al., Eye damage control by reduced blue illumination. Experimental Eye Research, 2009, 89(6): 863-868) found that high-intensity blue light exposure to the retina of monkeys leads to macular degeneration. In rats, retinal tissue atrophy, leakage, and tortuous retinal blood vessels were observed after 7 days of blue light exposure. High-intensity blue light also damages the retinal cytoskeleton and tight-binding protein structure of the retinal photoreceptor (RPE).

[0007] Blue light can cause a decrease in the activity of antioxidant enzymes in retinal tissue and promote the production of superoxide anions (O2). ·- Blue light induces the production of H2O2, -OOH, and various oxidative stress markers such as 8-OHdG, acrolein, and nitrogen-tyrosine. TUNEL (Terminal deoxynucleotidyl transferase dUTPick End Labeling) staining showed that blue light can cause reactive oxygen species (RPE) and photoreceptor cell apoptosis. At the cellular and molecular level, blue light can significantly induce mitochondrial ROS production, activate the transcription factor NF-κB, initiate the secretion of various inflammatory factors such as TNF-α and IL-1β, activate inflammasomes, initiate apoptosis and pyroptosis, and interfere with cellular autophagy and self-repair.

[0008] The average indoor light intensity for daytime workers is typically below 1000 lux. Illuminance below 300 lux is defined as low-intensity light source. While traditional lighting equipment and electronic displays rarely cause serious damage in daily life, prolonged and periodic fixed-position viewing of LED screens may still increase the risk of blue light chemical damage. Krigel et al. (Krigel, A., et al., Light-induced retinal damage using different light sources, protocols and rat strains reveals LED phototoxicity. Neuroscience, 2016, 339:296-307) used a 500 lux LED light source to simulate indoor lighting and observed retinal structural changes in experimental animals after 28 days of continuous irradiation. Even if the illuminance of a mobile phone screen is as low as 5 lux, prolonged use may still lead to reduced electrophysiological function of photoreceptor cells and retinal morphological abnormalities.

[0009] Probiotics are live microorganisms that are beneficial to the health of the host. Therefore, it is of great significance to find and develop probiotics and their microecological preparations that can improve blue light-induced eye damage. Summary of the Invention

[0010] Purpose of the invention: The purpose of this invention is to provide a *Lactobacillus reuteri* LR1349 and its use in improving blue light-induced eye damage.

[0011] Technical solution: The objective of this invention is achieved through the following technical solution:

[0012] This invention provides a *Lactobacillus reuteri* LR1349, which is deposited at the German Microbiological and Cell Culture Collection Center (DSMZ) with accession number DSM 35135.

[0013] Biological Preservation Information:

[0014] In accordance with the provisions of the Budapest Treaty, the strain of *Limosilactobacillus reuteri* LR1349 was deposited on September 9, 2024, at the German Center for Microbiology and Cell Culture (DSMZ), with accession number DSM 35135, and classified as *Limosilactobacillus reuteri*.

[0015] The present invention also provides the use of the above-mentioned *Lactobacillus reuteri* LR1349 in the preparation of pharmaceuticals, foods or food additives for improving blue light-induced eye damage.

[0016] The eye damage mentioned refers to decreased visual acuity, retinal dysfunction, and retinal diseases caused by blue light exposure.

[0017] The eye damage includes macular degeneration.

[0018] The present invention also provides the use of the above-mentioned *Lactobacillus reuteri* LR1349 in the preparation of pharmaceuticals, foods or food additives for relieving visual fatigue.

[0019] The medicine, food, or food additive improves eye damage or relieves visual fatigue by repairing retinal epithelial cell damage caused by blue light exposure.

[0020] The present invention also provides a probiotic composition comprising Lactobacillus reuteri LR1349 strain and pharmaceutically acceptable or food-acceptable excipients.

[0021] Based on the exposure time and cycle of blue light, illumination patterns are divided into three types:

[0022] (1) Short-term illumination mode with illumination time of less than 6 hours;

[0023] (2) Follow the 12h / 12h pattern of animal light-dark cycle;

[0024] (3) 24-hour continuous illumination mode without dark cycle.

[0025] Most studies use light sources with illuminance levels exceeding 1000 lux, and the entire exposure period is completed within a day or week, which is vastly different from the typical blue light exposure patterns of the general population. Furthermore, because the cytophagy of photoreceptor cells by RPE cells is regulated by circadian rhythms, this metabolic process peaks approximately 2 hours after the start of the photocycle.

[0026] Therefore, this invention incorporates a three-hour period (AM 06:00–09:00) after the start of the photocycle when designing animal illumination to optimize the chemical effects of blue light and to detect the health benefits of Lactobacillus reuteri LR1349 for blue light-induced retinal damage in mice.

[0027] Beneficial effects:

[0028] The *Lactobacillus reuteri* LR1349 of this invention has been demonstrated in in vivo to effectively improve visual acuity loss, retinal dysfunction, and retinopathy caused by blue light exposure. After continuous blue light exposure, the visual acuity (VA) of mice in the pathological control group significantly decreased, while the VA of mice treated with LR1349 significantly increased, indicating that it helps maintain visual recognition ability. Retinal potential analysis (ERG) also showed that LR1349 can significantly restore the a-wave and b-wave response amplitudes damaged under blue light, demonstrating its protective effect on retinal photosensitivity and nerve conduction function.

[0029] Histopathological analysis revealed that blue light irradiation significantly reduced the thickness of the outer nuclear layer (ONL) and inner segment / outer segment layer (IS / OS) of the mouse retina, and also significantly reduced the number of ONL cell nuclei, indicating retinal structural damage. However, LR1349 treatment effectively increased the number of ONL cell nuclei. Simultaneously, the thickness of the ONL and IS / OS layers in LR1349-treated mice significantly recovered, approaching the levels of the normal control group, demonstrating a significant inhibition of retinal cell loss and structural degeneration. Furthermore, immunofluorescence staining showed that blue light reduces rhodopsin expression in the retina, while LR1349 significantly increased its fluorescence intensity, helping to maintain photoreceptor cell function. On the other hand, the oxidative stress indicator 8-OHdG was significantly elevated in the blue light group, indicating increased DNA damage; after LR1349 treatment, 8-OHdG levels significantly decreased, showing that it can alleviate blue light-induced oxidative damage.

[0030] Therefore, the *Lactobacillus reuteri* LR1349 provided by this invention has a significant protective effect against retinal tissue damage caused by blue light, and has high potential to be developed into a novel probiotic therapeutic agent for the prevention and improvement of blue light-induced retinal damage. Therefore, the *Lactobacillus reuteri* LR1349 of this invention can be used to prepare pharmaceuticals, foods, or food additives that improve blue light-induced eye damage or relieve visual fatigue. Attached Figure Description

[0031] Figure 1 This invention relates to *Lactobacillus reuteri* LR1349 and *Lactobacillus reuteri* DSM 20016. T Gene fingerprints were obtained using RAPD analysis with primers consisting of 10 bases: RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F, and RAPD-G. In this analysis, diameter M represents a DNA ladder marker (100-3000 bp), and diameters 1 and 2 represent *Lactobacillus reuteri* DSM 20016, respectively. TAnd the amplification products of LR1349.

[0032] Figure 2 Visual acuity thresholds were measured at the end of weeks 8 and 16 after the mice began exposure to blue light. Data from different groups were labeled with different letters at the same number of weeks to indicate statistical differences between them.

[0033] Figure 3 The changes in retinal potentials were measured at the end of weeks 8 and 16 after the mice began exposure to blue light. Among the same ERG waves (i.e., a wave and b wave, where "A" represents a wave and "B" represents b wave) measured at the same week, the data from different groups were labeled with different letters, which indicates that there was a statistical difference between these data.

[0034] Figure 4 The results of retinal tissue sections from mice in each group at the end of week 16 after the start of blue light exposure were observed by hematoxylin-eosin staining; where “GCL” represents the ganglion cell layer; “INL” represents the nuclear layer; “ONL” represents the outer nuclear layer; “IS / OS” represents the inner segment / outer segment layer; and “RPE” represents the retinal pigment epithelial cells.

[0035] Figure 5 The mean ONL thickness was measured in retinal tissue sections of mice in each group at the end of week 16 after blue light exposure began; where data from different groups are labeled with different letters, it indicates that there is a statistical difference between these data.

[0036] Figure 6 The number of ONL cell nuclei and the mean IS / OS thickness were measured in retinal tissue sections of mice in each group at the end of week 16 after blue light exposure began. Data from different groups were labeled with different letters to indicate statistical differences between them.

[0037] Figure 7 The relative fluorescence intensity of rhodopsin and 8-OHdG in the retinal tissue of mice in each group was measured at the end of week 16 after blue light exposure began. Data from different groups were labeled with different letters to indicate statistical differences. Detailed Implementation

[0038] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0039] The *Lactobacillus reuteri* LR1349 strain of this invention was prepared and provided by Biostime Biotechnology Co., Ltd., with a strain concentration of 1×10⁻⁶. 8 / 300μL (containing bacterial cells and supernatant).

[0040] Laboratory animals:

[0041] Rodents are the most commonly used animal species to investigate blue light-induced retinal damage. This study used male eight-week-old ICR (albino) mice purchased from Lesco Biotechnology Co., Ltd. The experimental animals were housed in an animal facility with four mice per cage. After a two-week acclimatization period, the experiment began at ten weeks of age. During the acclimatization period, the mice were given commercially available granular Lab5001 diet (Ralston Purina, USA) with free access to food and water. The temperature in the animal facility was controlled at 22±2℃, the relative humidity at 60±10%, and a 12h / 12h light / dark cycle was maintained. Light was introduced at 6:00 AM and turned off at 6:00 PM daily.

[0042] Example 1: Screening and Characterization of Isolator LR1349

[0043] (1) Origin and isolation of isolate LR1349

[0044] Fecal samples were collected from healthy individuals over 85 years of age who had not used antibiotics for 3 months for the isolation and screening of Bifidobacteria. First, the feces were mixed with an appropriate amount of saline solution (containing 0.05% L-cysteine) and homogenized using a Vortex-Genie 2 homogenizer (Scientific Industries). Next, the homogenate was serially diluted 10-fold to prepare samples with different dilution factors (10...). -1 -10 -6 The solution was diluted (by a factor of 1), and 0.1 mL of the diluted solution was then evenly spread onto MRS agar medium. The medium was then anaerobically cultured at 37°C for 5-7 days. After colony formation, colonies on the MRS agar medium were selected to obtain a Lactobacillus isolate, LR1349.

[0045] (2) Preliminary test

[0046] Gram staining of Lactobacillus isolate LR1349 showed that Lactobacillus isolate LR1349 was a Gram-positive bacillus.

[0047] (3) Extraction of genomic DNA

[0048] Genomic DNA extraction of Lactobacillus isolate LR1349 was performed according to the literature method (Zhu, H et al. 1993, Nucleic Acids Res., 21(22): 5279-5280), using an extraction solution (containing 100 mM Tris and 40 mM EDTA, pH 9.0), 0.1 mm Mini-BeadBeater glass beads (BioSpec Products, Cat. No. 11079101), and 500 μL of phenol-chloroform-isoamyl alcohol mixture (25:24:1, v / v / v).

[0049] (4) 16S rDNA sequence analysis

[0050] First, using the genomic DNA obtained in step (3) as a template, a polymerase chain reaction (PCR) was performed using a primer pair designed for the bacterial 16S rDNA gene, with the nucleotide sequence shown below: forward primer 27F and reverse primer 1492R, to amplify the 16S rDNA fragment of the Lactobacillus isolate LR1349. The PCR reaction system is shown in Table 1.

[0051] Forward primer 27F:

[0052] 5'-agagtttgatcctggctcag-3'

[0053] Reverse primer 1492R:

[0054] 5'-ggttaccttgttacgact-3'

[0055] The primers were synthesized by Jilongmix Biotechnology Co., Ltd. Primer sequences are referenced in Frank JA, et al., 2008, Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes, Appl Environ Microbiol. 74(8):2461-70.

[0056] Table 1 PCR reaction system

[0057] Contents Volume (μL) Genomic DNA (10 ng / μL) 1 Forward primer F1 (10 μM) 0.5 Reverse primer R1 (10 μM) 0.5 Fast-RunTM2XTagMasterMix(Protech, Cat.No.SA-TMM228) 10 Deionized water 8

[0058] PCR reaction procedure:

[0059] Denaturation was performed at 95°C for 5 minutes; then 30 cycles were performed as follows: denaturation at 95°C for 60 seconds, primer annealing at 50°C for 60 seconds, extension at 72°C for 60 seconds; and finally, elongation at 72°C for 8 minutes.

[0060] After PCR was completed, sequencing analysis was performed by Genomics Biosci & Tech Co., Ltd., which yielded a partial 16S rDNA sequence of the Lactobacillus isolate LR1349:

[0061] The sequence of the 16S rDNA fragment of isolate LR1349 is shown in SEQ ID NO.1.

[0062] SEQ ID NO.1:

[0063]

[0064] After comparison with the gene database on the NCBI website, it was found that a portion of the 16S rDNA sequence of the Lactobacillus isolate LR1349 had more than 97% identity with a portion of the 16S rDNA sequence of Limosilactobacillus reuteri subsp. reuteri.

[0065] Based on the above experimental results, the lactobacillus isolate LR1349 obtained in this invention has been preliminarily identified as *Lactobacillus reuteri*.

[0066] (5) Random Amplified Polymorphic DNA (RAPD) Analysis

[0067] To confirm whether *Lactobacillus reuteri* LR1349 is a novel *Lactobacillus reuteri* isolate, the isolate was subjected to random amplification polymorphic DNA analysis.

[0068] RAPD analysis reference method (Akopyanz N. et al. 1992, Nucleic Acids Res., 20:5137)

[0069] -5142) was used. The genomic DNA obtained in step (3) was used as a template, and PCR reaction was performed using eight primers consisting of 10 bases, namely RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F and RAPD-G, which have the nucleotide sequences shown in Table 2.

[0070] The PCR reaction system is shown in Table 3.

[0071] The above eight primers, each consisting of 10 bases, were synthesized by Jilongmix Biotechnology Co., Ltd. Primer sequences are referenced in Akopyanz, N., et al., 1992, DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting, Nucleic Acids Research, 20(19), 5137-5142.

[0072] Table 2 Primer sequence information

[0073]

[0074]

[0075] Table 3 PCR reaction system

[0076]

[0077] PCR reaction procedure:

[0078] The denaturation reaction was carried out at 94°C for 2 minutes; then 6 cycles were performed as follows: denaturation reaction at 94°C for 30 seconds, primer annealing at 36°C for 60 seconds, and extension reaction at 72°C for 90 seconds; then 30 cycles were performed as follows: denaturation reaction at 94°C for 20 seconds, primer annealing at 36°C for 30 seconds, and extension reaction at 72°C for 90 seconds; finally, extension reaction was carried out at 72°C for 3 minutes.

[0079] After PCR was completed, the PCR amplification products were subjected to electrophoresis on a 1.5% agarose gel, stained, and then observed and photographed under ultraviolet light.

[0080] For comparison, this invention uses *Lactobacillus reuteri* DSM 20016. T (Purchased from the Bioresource Conservation and Research Center (BCRC), i.e., standard strain BCRC 14625) T This strain was used as a comparison strain for analysis. The obtained electrophoresis results are shown on... Figure 1 middle.

[0081] Figure 1 This invention demonstrates the presence of *Lactobacillus reuteri* LR1349 and *Lactobacillus reuteri* DSM 20016. T Gene fingerprints were obtained by RAPD analysis using primers consisting of 10 bases: RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F, and RAPD-G.

[0082] Depend on Figure 1 It is evident that the RAPD analysis results obtained from the two isolates using different primers showed differences between each other.

[0083] Based on the above characteristic identification results, the *Lactobacillus reuteri* LR1349 of the present invention is a novel *Lactobacillus reuteri* isolate, which was deposited on August 28, 2024, with the registration number BCRC 911241, at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI), Taiwan Province (No. 331, Food Rd., Hsinchu City, Taiwan 300, China).

[0084] It was also deposited on September 9, 2024, with the deposit number DSM 35135 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ).

[0085] Example 2: Preparation of probiotic sample LR1349

[0086] Lactobacillus reuteri LR1349 was inoculated into MRS broth medium (brand: BD Difco). TM The strain was cultured at 37°C for 18 hours (product number 288130) to activate the bacteria.

[0087] The obtained culture was centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatant was collected. The cells were then washed with phosphate-buffered saline (PBS), and the supernatant was used to adjust the culture concentration to 3.3 × 10⁻⁶. 8 The bacterial concentration was determined by CFU / mL, thereby obtaining the test bacterial solution of Lactobacillus reuteri LR1349.

[0088] Example 3: Establishment of a low-intensity blue light-induced retinal damage pattern in mice

[0089] Animal lighting devices:

[0090] The mouse illumination device used blue LED light strips and a fiber optic spectrometer (#USB4000, Ocean Optics, USA) to detect wavelengths of 465±10nm. An illuminance meter (IM-600, Topcon, Japan) and a high-frequency optical power generator (PM100A, Thorlabs, Newton, USA) were used to detect the average illuminance and average optical power at the four corners a to d and the center e of the illumination cage.

[0091] After a 2-week acclimatization period, the experimental animals were pre-fed with probiotic samples (test bacterial suspension of *Lactobacillus reuteri* LR1349 prepared in Example 2) or MRS culture medium (i.e., MRS broth medium) for one week (300 μL via tube feeding), followed by blue light irradiation. Twelve mice were randomly divided into the following groups:

[0092] (1) Control group (Ctrl group): given MRS culture medium (300 μL) and not exposed to blue light;

[0093] (2) Blue light group (BL group): MRS culture medium (300 μL) was given daily and blue light irradiation for 6 hours;

[0094] (3) LR1349 group: daily bacterial count 1×10 8 CFU probiotic strain LR1349 was used, and the patient was exposed to blue light for 6 hours daily.

[0095] The indoor lighting in the animal room is 150-200 lux. The blue light group receives additional blue light irradiation during two light cycle periods each day, from 06:00-09:00 AM and from 15:00-18:00 PM, in addition to the indoor lighting. The total irradiation time is 6 hours per day. The dark cycle period is from 18:00 PM to 06:00 AM the next day.

[0096] This experiment used long-term low-intensity blue light to simulate a living environment, with an illumination condition of 108 lux (44.8 μW / cm²). 2 After 16 weeks of continuous irradiation, mice were anesthetized with 3% isoflurane, and their retinal tissue was collected for analysis. The animal room temperature was maintained at 22±2℃ throughout the experiment, and food and water intake, as well as body weight, were recorded regularly.

[0097] Example 4: Visual Acuity (VA) Experiment

[0098] The visual acuity experiment was conducted according to the method of Liou et al. (Liou JC et al. 2018, J. Funct. Foods, 49:314-323) and modified.

[0099] First, the experimental mice were placed in a dark chamber. A cylindrical platform, 13 cm high and 5 cm in diameter, was placed at the center of the bottom of the chamber for the mice to stand and move freely. Digital screens were placed on all four sides of the chamber, 15 cm from the central platform, to display a black and white moving stripe pattern. This visual stimulation caused the animals to involuntarily exhibit compensatory head or body movements in the direction of the stimulus, thereby assessing the animals' optomotor response ability.

[0100] The experiment began with the screen flashing for 5 seconds to stimulate the mice's response. Then, the screen was moved alternately clockwise and counterclockwise at a constant speed of 12 degrees per second, playing black and white gratings of different spatial frequencies (cycles per degree, cpd) in turn. The frequencies were divided into six levels from high to low: 0.437, 0.328, 0.164, 0.082, 0.055, and 0.033 cpd. Each level was tested for 2 minutes, and the experiment was repeated twice.

[0101] The experiment recorded the accuracy of the mouse's head swaying. The accuracy was calculated by dividing the number of correct responses by the total number of responses. When the accuracy was higher than 70%, the mouse's visual acuity was determined to have reached the threshold standard for that spatial frequency.

[0102] Figure 2 This shows the visual acuity thresholds of mice in each group at the end of weeks 8 and 16 after blue light exposure began. Figure 2 As can be seen, at week 8 after blue light exposure, compared with the normal control group, the visual acuity threshold of the blue light group was significantly reduced after blue light exposure, indicating that blue light successfully induced a decrease in visual acuity in mice. In contrast, the visual acuity threshold of the LR1349 group was significantly increased compared with the blue light group, and was approximately the same as that of the normal control group. Similar results were observed at week 16 after blue light exposure. The experimental results indicate that the *Lactobacillus reuteri* isolate LR1349 of this invention can effectively improve the decrease in visual acuity caused by blue light exposure.

[0103] Example 5 Electroretinogram (ERG) Analysis

[0104] Retinal potential analysis was performed using a retinal function analyzer (BPM-300, Dioptri, Canada) and RetinoGraphics BPM300 software (version 7.0.0.7).

[0105] Before the experiment, mice underwent 12 hours of dark acclimatization. The experiment was conducted entirely in the dark, and electronic devices emitting electromagnetic waves were excluded from the environment. After anesthesia and sedation with Zoltil 100 (Victal, France, 75 mg / kg bw) and xylazine (9.75 mg / kg bw), the mice were placed on a 37°C constant-temperature pad. One to two drops of Better Eyedrops (Madison, 0.5% tropicamide) were instilled into the cornea to paralyze the ciliary muscle and dilate the pupil, increasing the amount of light entering the pupil. Then, a reference electrode and an indifference electrode were attached to the outer corner of the eye and forehead, respectively, and a recording electrode was attached to the corneal surface. Throughout the experiment, the resistance values ​​of the recording and reference electrodes were maintained at 2.1 kΩ, while the resistance of the indifference electrode was maintained at 12.1 kΩ or within ±25% of this value to eliminate background noise interference. Finally, apply the flash at a point approximately 3-5 mm from the animal's cornea, with a flash intensity of 3 cd.s / m. 2 White LEDs flashing at a frequency of 1 time per second stimulated the retina of mice to produce potential changes, and the changes in A-wave and B-wave in the retina were recorded.

[0106] Figure 3 This shows the changes in retinal potentials measured at the end of weeks 8 and 16 after the start of blue light exposure in each group of mice. Figure 3 As can be seen, at week 8 after blue light exposure, compared with the normal control group, the retinal potential in the blue light group, whether in the a-wave or b-wave, was significantly reduced after blue light exposure, indicating that blue light successfully induced retinal dysfunction in mice. In contrast, the retinal potential in the LR1349 group was significantly increased compared with the blue light group, and there was no significant difference between the two groups, with a slight increase observed even after blue light exposure. Similar results were observed at week 16 after blue light exposure. The experimental results indicate that the *Lactobacillus reuteri* isolate LR1349 of this invention can effectively improve retinal dysfunction caused by blue light exposure.

[0107] Example 6: Pathological Tissue Analysis of the Retinal

[0108] ICR mice were anesthetized with 3% isoflurane, perfused with physiological saline via the portal vein, and then their eyeballs were sacrificially removed. The eyeball tissue was immersed in Davidson fixative overnight, followed by gradient dehydration with different concentrations of ethanol (70%, 80%, 95%, 100%). After tissue embedding and sectioning, the retinal tissue was stained with H&E for pathological morphology observation, and the thickness and nucleus count of the retinal fossa (ONL) were analyzed. The pathological morphology of the eyeball tissue was observed using a 40x optical microscope, and quantification was performed using ViewPoint Light (Precipint, Freising, Germany) software. The average thickness of the ONL and IS / OS of the retina, as well as the number of ONL nuclei, were measured at 120 μm intervals to the left and right of the fovea centralis.

[0109] Figure 4 The results show the retinal tissue sections of mice in each group observed at the end of week 16 after the start of blue light exposure, stained with hematoxylin and eosin. Figure 4 As can be seen, compared with the normal control group, the ONL thickness and IS / OS thickness of the blue light group were significantly reduced after blue light exposure, indicating that blue light successfully induced retinal lesions in mice. In contrast, the ONL thickness and IS / OS thickness of the LR1349 group were significantly increased and similar to those of the normal control group.

[0110] Depend on Figure 5 , Figure 6 As can be seen, compared with the normal control group, the average thickness of the ONL, IS / OS, and the number of ONL cell nuclei in the blue light group were significantly reduced after blue light irradiation, indicating that blue light successfully induced retinopathy in mice. In contrast, compared with the blue light group, the average thickness of the ONL, IS / OS, and the number of ONL cell nuclei in the LR1349 group were significantly increased, and approximately the same as the normal control group. The experimental results show that the *Lactobacillus reuteri* isolate LR1349 of this invention can effectively improve retinopathy caused by blue light irradiation and slow down retinal tissue loss.

[0111] Example 7 Immunohistochemical staining analysis of molecules related to retinal injury

[0112] The methods of immunohistochemical staining are briefly described below:

[0113] After dewaxing with xylene, ocular sections were rehydrated in gradients with different concentrations of alcohol (100%, 95%, 80%, 70%), followed by antigen retrieval using an antigen retrieval apparatus (Bio SB, Inc., USA). Finally, they were immersed in an apparatus containing 0.025% xylene. X-100 TBS (25mM Tris-base, 137mM NaCl, 2.7mM KCl), also known as TBST, was used to wash the tissue three times. The tissue was then circled with a hydrophobic pen and covered with TBST (Tris-buffered Saline with Tween 20) containing 1% bovine serum albumin (BSA) at room temperature to block non-specific antigens. Next, a primary antibody (anti-rhodopsin antibody, Abcam, catalog number ab98887) was used to completely cover the tissue, and the reaction was carried out at 4°C. Finally, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution, and the slides were mounted with mounting medium and coverslips. After the mounting gel dried, the slides were used... The FLAuto Imaging System (Thermo Fischer Scientific, USA) captured tissue fluorescence images at 20x magnification, and then ImageJ software quantified the fluorescence RawIntDen (raw integrated density) intensity.

[0114] Figure 7 This shows the relative fluorescence intensity of rhodopsin and 8-OHdG in the retinal tissue of mice in each group at the end of week 16 after blue light exposure began. Here, "GCL" represents the ganglion cell layer; "INL" represents the nuclear layer; "ONL" represents the outer nuclear layer; "IS / OS" represents the inner / outer ganglion layer; and "RPE" represents retinal pigment epithelial cells.

[0115] Depend on Figure 7 As can be seen, compared with the normal control group, the rhodopsin level in the blue light group was significantly reduced and the 8-OHdG level was significantly increased after blue light exposure, indicating that blue light successfully caused a large amount of photosensitive pigment decomposition in the mouse retina. In contrast, the rhodopsin level in the LR1349 group was significantly increased and the 8-OHdG level was significantly reduced, similar to the normal control group. The experimental results show that the *Lactobacillus reuteri* isolate LR1349 of this invention can effectively improve retinopathy caused by blue light exposure and slow down the decomposition of photosensitive pigment.

[0116] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A type of *Lactobacillus reuteri* LR1349, characterized in that, The Lactobacillus reuteri LR1349 is deposited at the German Microbiology and Cell Culture Collection Center (DSMZ) with accession number DSM 35135.

2. Use of the Lactobacillus reuteri LR1349 of claim 1 in the preparation of pharmaceuticals, foods or food additives for improving blue light-induced eye damage.

3. The use according to claim 2, characterized in that, The eye damage refers to decreased visual acuity, retinal dysfunction, and retinal diseases caused by blue light exposure.

4. The use according to claim 2, characterized in that, The eye damage includes macular degeneration.

5. Use of the *Lactobacillus reuteri* LR1349 as described in claim 1 in the preparation of pharmaceuticals, foods or food additives for relieving visual fatigue.

6. The use according to claim 2 or 5, characterized in that, The medicine, food, or food additive improves eye damage or relieves visual fatigue by repairing retinal epithelial cell damage caused by blue light exposure.

7. A probiotic composition, characterized in that, The probiotic composition contains Lactobacillus reuteri LR1349 strain and pharmaceutically acceptable or food-acceptable excipients.

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