Lactobacillus reuteri subsp. reuteri LR1349 isolate and its uses

Abstract

This invention discloses a strain of *Lactobacillus reuteri* subsp. reuteri LR1349, deposited with accession number BCRC 911241 at the Bioresource Conservation and Research Center (BCRC) of the Food Industry Development Institute (FIRDI). This isolate of *Lactobacillus reuteri* subsp. reuteri LR1349 can be used to improve blue light-induced retinal damage.

Description

Technical Field

[0001] This invention relates to an isolate of *Lactobacillus reuteri* subsp. *reuteri* LR1349, deposited under accession number BCRC 911241 at the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI), and under accession number DSM 35135 at the German Microbiology and Cell Culture Collection Center GmbH (DSMZ). This invention also relates to the use of this *Lactobacillus reuteri* subsp. *reuteri* LR1349 isolate in improving blue light-induced retinal damage. Prior Technology

[0002] Blue light is a type of high-energy visible light. When the eyes are exposed to excessive blue light, it can lead to retinal damage.

[0003] Currently, there are no effective methods in clinical practice to improve blue light-induced retinal damage. The only options are to reduce exposure to blue light sources (such as electronic devices) and supplement with eye-healthy products like lutein and fish oil. Therefore, researchers in this field are dedicated to developing drugs that can effectively improve blue light-induced retinal damage.

[0004] Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) and are well-known and widely used probiotics. They have been found to inhibit the growth of pathogenic bacteria in the gastrointestinal tract, alleviate lactose intolerance, have anti-cancer effects, and lower blood pressure. Currently, there are many types of lactic acid bacteria that can be used as probiotics, such as Lactobacillus (sometimes also known as Limosilactobacillus), Lactococcus, Pediococcus, Enterococcus, Streptococcus, and Bifidobacterium.

[0005] To the best of the applicant's knowledge, no literature or patent has yet revealed that *Lactobacillus reuteri* subsp. reuteri (also known as *Lactobacillus reuteri*) can be used to improve blue light-induced retinal damage. Summary of the Invention

[0006] [Invention Summary]

[0007] In this invention, the applicant unexpectedly isolated a strain of *Lactobacillus reuteri* subsp. *reuteriisolate*, which the applicant named "*Lactobacillus reuteri* subsp. *reuteriisolate* LR1349" and has been deposited with domestic and international storage facilities. *Lactobacillus reuteri* subsp. *reuteri* LR1349 has been demonstrated through in vivo experiments to effectively improve visual acuity loss, retinal dysfunction, and retinal diseases caused by blue light exposure, thus possessing high potential to be developed into a drug for improving blue light-induced retinal damage.

[0008] Therefore, in the first aspect, the present invention provides Lactobacillus reuteri subspecies LR1349, which is registered under registration number BCRC 911241 at the Center for Biological Resources Preservation and Research of the Food Industry Development Institute Foundation, and under registration number DSM 35135 at the German Center for the Collection of Microorganisms and Cell Cultures GmbH.

[0009] In a second aspect, the present invention provides the use of the Lactobacillus reuteri subsp. reuteri LR1349 as described above for the preparation of a composition for improving blue light-induced retinal damage.

[0010] In a third aspect, the present invention provides a method for improving blue light-induced retinal damage in an individual, comprising administering to an individual in need a Lactobacillus reuteri subsp. reuteri LR1349 as described above. Simple Explanation of the Diagram

[0011] The above and other objects, features and advantages of the present invention will become apparent upon reference to the following detailed description and preferred embodiments and the accompanying drawings, wherein: Figure 1 shows the gene fingerprints of *Lactobacillus reuteri* subsp. *reuteri* LR1349 and the known *Lactobacillus reuteri* subsp. *DSM* 20016 T obtained by RAPD analysis using 10-mer primers RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F, and RAPD-G, respectively. Path M represents the DNA ladder marker (100-3000 bp); and paths 1 and 2 represent the amplification products of *Lactobacillus reuteri* subsp. *reuteri* DSM 20016 T and LR1349, respectively. Figure 2 shows the visual acuity thresholds of the mice in Example 2 at the end of weeks 8 and 16 after the start of blue light exposure. In the same number of weeks, data from different groups are labeled with different letters to indicate statistical differences between them. Figure 3 shows the changes in retinal potentials measured at the end of weeks 8 and 16 after the mice in each group of Example 2 began exposure to blue light. Among the same ERG waves (i.e., a wave and b wave) measured at the same week number, the data from different groups are labeled with different letters, which indicates that there are statistical differences between these data. Figure 4 shows the results of hematoxylin-eosin staining of retinal tissue sections from each group of mice in Example 2 at the end of week 16 after the start of blue light exposure. In the figures, “GCL” represents the ganglion cell layer; “INL” represents the inner nuclear layer; “ONL” represents the outer nuclear layer; “IS / OS” represents the inner segment / outer segment layer; and “RPE” represents retinal pigment epithelium. Figure 5 shows the mean ONL thickness of retinal tissue sections from mice in each group of Example 2 at the end of week 16 after the start of blue light exposure. Data from different groups are labeled with different letters to indicate statistical differences between them. Figure 6 shows the mean IS / OS thickness of retinal tissue sections from each group of mice in Example 2 at the end of week 16 after the start of blue light exposure. The data from different groups are labeled with different letters to indicate statistical differences between them. Figure 7 shows the number of ONL cell nuclei measured in retinal tissue sections of mice in Example 2 at the end of week 16 after the start of blue light exposure. Data from different groups are labeled with different letters to indicate statistically significant differences. Figure 8 shows the relative fluorescence intensity of rhodopsin in the retinal tissue of mice in each group of Example 2 at the end of week 16 after the start of blue light exposure. The data from different groups are labeled with different letters to indicate statistical differences between them. Implementation

[0012] [Detailed Description of the Invention]

[0013] It should be understood that if any prior publication is cited herein, such prior publication does not constitute an admission that it forms part of the common general knowledge in the art in Taiwan or any other country.

[0014] For the purposes of this instruction manual, it will be clearly understood that the word “comprising” means “including but not limited to”, and the word “comprises” has a corresponding meaning.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. One skilled in the art will recognize many similar or equivalent methods and materials that can be used to practice this invention. Of course, this invention is by no means limited to the methods and materials described.

[0016] This invention provides a *Lactobacillus reuteri* subsp. *reuteri* LR1349, which is registered under registration number BCRC 911241 at the Bioresource Conservation and Research Center of the Food Industry Development Foundation (BCRC of FIRDI) and under registration number DSM 35135 at the German Microbial and Cell Culture Collection Center GmbH (DSMZ).

[0017] The present invention also provides the use of the Lactobacillus reuteri subsp. reuteri LR1349 as described above for the preparation of a composition for improving blue light-induced retinal damage. Preferably, the improvement of blue light-induced retinal damage includes at least one of the following: improved visual acuity, improved retinal function, and improved retinal lesions.

[0018] According to the present invention, *Lactobacillus reuteri* subsp. reuteri LR1349 can be live or dead, concentrated or non-concentrated, liquid, paste, semi-solid, or solid [e.g., pellet, granule, or powder], and can be heat-inactivated, frozen, dried, or freeze-dried [e.g., in freeze-dried or spray / fluid bed dried form]. In a preferred embodiment of the present invention, *Lactobacillus reuteri* subsp. reuteri LR1349 is present in live form.

[0019] According to the present invention, *Lactobacillus reuteri* subsp. reuteri LR1349 can be prepared as a bacterial suspension with a bacterial concentration of 1.0 × 10⁶ to 1.0 × 10⁹ CFU / mL. In a preferred embodiment of the present invention, the bacterial suspension has a bacterial concentration of 3.3 × 10⁸ CFU / mL.

[0020] The culture medium suitable for culturing Lactobacillus reuteri subsp. reuteri LR1349 can be prepared by those skilled in the art or is a commercially available product, including, but not limited to: MRS broth, MRS broth supplemented with cysteine, and mineral medium supplemented with glucose and soy peptone. In a preferred embodiment of the invention, the culture medium is MRS broth.

[0021] As used in this article, the terms “culturing,” “fermentation,” and “cultivation” may be used interchangeably.

[0022] The procedures and parameters for training fall within the scope of the professional competence and routine techniques of those familiar with this technology.

[0023] According to the present invention, the culture can be carried out at a temperature within the range of 30°C to 37°C for 16 to 18 hours. In a preferred embodiment of the present invention, the culture is carried out at 37°C for 16 to 18 hours.

[0024] According to the present invention, the composition can be a food composition, for example, in the form of a food additive, which can be added to an edible material to prepare a food product for human or animal consumption. According to the present invention, the types of food products may include, but are not limited to: milk powder, fermented milk, yogurt, butter, beverages (e.g., tea, coffee), functional beverages, flour products, baked foods, confectionery, candies, fermented foods, animal feeds, health foods, and dietary supplements.

[0025] According to the present invention, the composition may be a pharmaceutical composition.

[0026] According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more reagents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these reagents fall within the scope of professional competence and routine practice of those skilled in the art.

[0027] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration or parenteral administration using techniques known to those skilled in the art.

[0028] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques known to those skilled in the art, including, but not limited to: sterile powders, tablets, troche, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0029] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for non-enteric administration [including injection, for example, sterile aqueous solution or dispersion] using techniques known to those skilled in the art, and administered via a route selected from the group consisting of: intralesional injection and sublingual administration.

[0030] The present invention also provides a method for improving blue light-induced retinal damage in an individual, comprising administering to an individual in need a Lactobacillus reuteri subsp. reuteri LR1349 as described above.

[0031] As used herein, the terms “administering” and “administration” may be used interchangeably and mean introducing, providing, or delivering a predetermined active ingredient to an individual by any suitable means to perform its intended effect.

[0032] As used in this article, the term "subject" refers to any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0033] In certain specific cases, the individual is not a person with dry eye syndrome.

[0034] According to the present invention, the dosage and frequency of administration of *Lactobacillus reuteri* subsp. *reuteri* LR1349 will vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition, and response of the individual to be treated. Generally, *Lactobacillus reuteri* subsp. *reuteri* LR1349 according to the present invention can be administered orally or non-enterally in a single dose or in several doses. [Detailed Description of Preferred Embodiments]

[0035] The present invention will be further described with reference to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the implementation of the present invention. [Example] [, General experimental materials: , ] 1. Laboratory animals:

[0036] The male ICR mice (8 weeks old) used in the following examples were purchased from BioLasco Taiwan Co., Ltd. All experimental animals were individually housed in an animal room with 12 hours of light and 12 hours of darkness, a room temperature maintained at 22±2°C, and a relative humidity maintained at 60±10%. Sufficient water and feed were provided, and all animals were pre-acclimatized for 2 weeks prior to the experiment. All experimental procedures involving the experimental animals were approved by the Institutional Animal Care and Use Committees in the National Taiwan Normal University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). [, General experimental methods: , ] [, , ] 1. Statistical analysis:

[0037] The experimental data obtained in the following examples are expressed as mean ± standard deviation (SD). All data were analyzed using one-way analysis of variance (one-way ANOVA) combined with post-hoc tests to assess differences between groups. A p-value < 0.05 was considered statistically significant. [Example] [1.] [Lactobacillus isolate] [( , Limosilactobacillus , isolates) [LR1349] [Screening and Feature Identification] [A、] [Lactobacillus isolate] [LR1349] [The origin and separation of]

[0038] The applicant collected fecal samples from healthy humans over 85 years of age who had not used antibiotics for 3 months for the isolation and screening of lactobacilli. First, the feces were mixed with an appropriate amount of saline solution [containing 0.05% L-cysteine], and then homogenized using a Vortex-Genie 2 homogenizer (Scientific Industries). Next, the homogenate was serially diluted 10-fold to prepare dilutions with different dilution factors (10⁻¹–10⁻⁶). Then, 0.1 mL of each dilution was taken and evenly spread onto Difco Lactobacilli MRS agar, and subsequently anaerobically cultured at 37°C for 5–7 days. After colonies formed, the colonies on the MRS agar medium were selected to obtain a lactobacillus isolate LR1349. [B、] [Preliminary test:]

[0039] Gram staining of Lactobacillus isolate LR1349 showed that it was a Gram-positive bacillus. [C、] [Genome] [DNA] [Extraction] [(extraction of genomic DNA)] [:]

[0040] Genomic DNA extraction of Lactobacillus isolate LR1349 was generally performed according to the method described in Zhu, H et al. (1993), Nucleic Acids Res., 21(22):5279-5280, using an extraction buffer (containing 100 mM Tris and 40 mM EDTA, pH 9.0), 0.1 mm Mini-BeadBeater glass mill beads (BioSpec Products, Cat. No. 11079101), and 500 µL of a phenol-chloroform-isoamyl alcohol mixture (25:24:1, v / v / v). [D、] [16S rDNA] [Sequence Analysis:]

[0041] First, using the genomic DNA obtained in section C above as a template, a polymerase chain reaction (PCR) was performed using a primer pair designed for bacterial 16S rDNA genes, 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 conditions are shown in Table 1 below. Forward Intro 27F 5'-agagtttgatcctggctcag-3' (Sequence identification number: 1) Reverse primer 1492R 5'-ggttaccttgttacgact-3' (Sequence identification number: 2) Table 1. PCR reaction conditions Contents Volume (µL) Genomic DNA (10 ng / µL) 1 Forward inlet F1 (10 µM) 0.5 Reverse primer R1 (10 µM) 0.5 Fast-Run TM2X Tag Master Mix (Protech, Cat. No. SA-TMM228) 10 Deionized water 8 Operating conditions: Denaturation was performed at 95°C for 5 minutes, followed by 30 cycles of the following: 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.

[0042] After PCR was completed, sequencing analysis was performed by Genomics Biosci & Tech Co., Ltd., resulting in a partial 16S rDNA sequence (sequence identification number: 3) of the Lactobacillus isolate LR1349. Comparison with the NCBI gene database revealed that the partial 16S rDNA sequence of Lactobacillus isolate LR1349 shared over 97% identity with a partial 16S rDNA sequence of Lactobacillus reuteri subsp. reuteri.

[0043] Based on the above experimental results, the Lactobacillus isolate LR1349 obtained in this invention was preliminarily identified as *Lactobacillus reuteri* subsp. *reuteri*. To confirm whether *Lactobacillus reuteri* subsp. *reuteri* LR1349 is a novel isolate, it was further analyzed in section E below. [E、] [Polymorphism of random amplification] [DNA (Random Amplified Polymorphic DNA, RAPD)] [analyze:]

[0044] The RAPD analysis was largely performed with reference to Akopyanz N. et al. (1992), Nucleic Acids Res., 20:5137-5142. In short, the genomic DNA obtained in section C above was used as a template, and PCR reactions were performed using the reaction conditions shown in Table 3, employing eight 10-mer primers (RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F, and RAPD-G) with the nucleotide sequences shown in Table 2 below. Table 2. Primer pairs used for RAPD Introduction Nucleotide sequence (5'→3') RAPD-1 ctcaggtcgc (Sequence identification number: 4) RAPD-A ccgcagccaa (Sequence identification number: 5) RAPD-B aacgcgcaac (Sequence Identification Number: 6) RAPD-C gcggaaatag (Sequence identification number: 7) RAPD-D gaggacaaag (Sequence identification number: 8) RAPD-E ggcgtcggtt (Sequence identification number: 9) RAPD-F ggccacggaa (Sequence identification number: 10) RAPD-G cgaactagac (Sequence ID: 11) Table 3. PCR reaction conditions Contents Volume (µL) Genomic DNA (50-100 ng) 1 10-member primer (10 µM) 4 2X Taq Master Mix (Ampliqon, 5200300-1250) 12.5 MgCl₂ (25 mM) 2 Deionized water 5.5 Operating conditions: Denaturation reaction was performed at 94°C for 2 minutes, followed by 6 cycles as follows: denaturation reaction at 94°C for 30 seconds, initiator bonding reaction at 36°C for 60 seconds, and extension reaction at 72°C for 90 seconds; followed by 30 cycles as follows: denaturation reaction at 94°C for 20 seconds, initiator bonding reaction at 36°C for 30 seconds, and extension reaction at 72°C for 90 seconds; finally, extension reaction was performed at 72°C for 3 minutes.

[0045] After PCR was completed, the PCR amplification products were subjected to electrophoresis on 1.5% agarose gel, stained, and then observed and photographed under ultraviolet light.

[0046] In addition, for comparison, the applicant used the known Lactobacillus reuteri subsp. reuteri DSM 20016 T (corresponding to BCRC14625 T) as a comparison strain for comparative analysis. The obtained electrophoresis results are shown in Figure 1.

[0047] Figure 1 shows the gene fingerprints of *Lactobacillus reuteri* subsp. *reuteri* LR1349 and the known *Lactobacillus reuteri* subsp. *reuteri* DSM 20016 T obtained by RAPD analysis using 10-member primers RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F, and RAPD-G, respectively. As can be seen from Figure 1, the gene fingerprints of the two isolates obtained by RAPD analysis using different 10-member primers are different from each other.

[0048] Based on the above characteristic identification results, the applicant believes that: Lactobacillus reuteri subsp. reuteri LR1349 of the present invention is a novel Lactobacillus reuteri subsp. 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) in Taiwan (No. 331, Food Rd., Hsinchu City, Taiwan 300), and on September 9, 2024, with the registration number DSM 35135 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). [Example] [2.] Lactobacillus reuteri subsp. reuteri [LR1349] [Improving blue light] [-] [Induced retinal damage] [(blue light-induced retinal damage)] [Utility assessment] [, Experimental materials: , ] [, , ] 1. Preparation of test bacterial suspension of Lactobacillus reuteri subsp. reuteri LR1349:

[0049] Lactobacillus reuteri subsp. reuteri LR1349 was inoculated into MRS broth medium (BD Difco™, catalog number 288130) and cultured at 37°C for 16-18 hours to activate the strain. The resulting culture was then centrifuged at 5,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The cells were then washed with an appropriate amount of phosphate-buffered saline (PBS), and the supernatant was used to adjust the bacterial concentration to 3.3 × 10⁸ CFU / mL, thus obtaining the test bacterial suspension of Lactobacillus reuteri subsp. reuteri LR1349. [, , ] [, Experimental methods and results: , ] [, , ] [A、] [The Induction of Retinal Damage Through Bacterial Drug Administration:]

[0050] First, male ICR mice were randomly divided into three groups (n=10 per group): one normal control group, one pathological control group, and one experimental group. The experimental group mice were then administered the test bacterial suspension of *Lactobacillus reuteri* subsp. reuteri LR1349 (dose: 1×10⁸ CFU / mouse) obtained in item 1 of the "Experimental Materials" section above via oral gavage. The pathological and normal control groups were administered the drug via an equal volume of MRS broth. The drug was administered once daily for a total of 17 weeks.

[0051] Starting from the second week after the start of drug administration, mice in the pathological control group and the experimental group were irradiated with blue light with a wavelength of 465±10 nm and an illuminance of 108 lux (44.8 µW / cm 2). The irradiation was carried out twice a day for 3 hours each time, and the total irradiation time lasted for 16 weeks. Mice in the normal control group were not treated in any way. [B、] [Visual acuity] (visual acuity) [Detection:]

[0052] At the end of weeks 8 and 16 after the start of blue light exposure, visual acuity in mice was assessed, largely following the method described in Liou JC et al. (2018), J. Funct. Foods, 49:314-323. Mice were placed individually on a cylindrical platform in the center of a dark chamber with screens on all four sides. Spatial frequencies of 0.437, 0.328, 0.164, 0.082, 0.055, and 0.033 cpd were sequentially played on the screens. Each spatial frequency lasted for 2 minutes, during which the grating rotated at 12° / s, alternating between clockwise and counterclockwise directions. The visual acuity threshold was then determined by observing the spatial frequencies at which the mouse's head moved more than 70% accurately in response to the grating's movement.

[0053] Then, analyze the obtained experimental data according to the method described in item 1 of the "General Experimental Methods" above.

[0054] Figure 2 shows the visual acuity thresholds of mice in each group at the end of weeks 8 and 16 after the start of blue light exposure. As can be seen from Figure 2, at week 8 after the start of blue light exposure, compared with the normal control group, the visual acuity threshold of the pathological control group was significantly reduced after blue light exposure, indicating that blue light successfully induced a decrease in visual acuity in the mice. In contrast, the visual acuity threshold of the experimental group was significantly increased compared with the pathological control group, and was approximately the same as that of the normal control group. Similar results were observed at week 16 after the start of blue light exposure. These experimental results demonstrate that *Lactobacillus reuteri* subsp. reuteri LR1349 of the present invention can effectively improve the decrease in visual acuity caused by blue light exposure. [C、] [Electroretinogram] [(electroretinogram, ERG)] [Analysis:]

[0055] At the end of weeks 8 and 16 after the start of blue light exposure, mice in each group were placed in a light-protected environment for 12 hours. They were then anesthetized with xylazine (9.75 mg / kg) and Zoltil 100 (75 mg / kg). Next, 1-2 drops of 0.5% tropicamide were instilled into the cornea of ​​each group of mice to dilate their pupils. Retinal potential analysis was then performed using a BPM-300 electrodiagnostic system (software version 7.0.0.7, RetinoGraphics): First, a reference electrode and an indifferent electrode were attached to the outer corner of the eye and forehead of the mice, respectively. A recording electrode was attached to the corneal surface, and the resistance values ​​of the recording electrode and the reference electrode were maintained at 2.1. The resistance of the reference electrode was maintained at 12.1 ± 3.025 kΩ. Then, at a distance of about 3-5 mm from the mouse's cornea, the retina was stimulated with white light with a brightness of 3 cd s / m² and a flashing frequency of 1 time per second to produce potential changes, and the alpha wave (a-wave) and b-wave (b-wave) were recorded.

[0056] Then, analyze the obtained experimental data according to the method described in item 1 of the "General Experimental Methods" above.

[0057] Figure 3 shows the changes in retinal potential (RP) measured at the end of weeks 8 and 16 after the start of blue light exposure in each group of mice. As can be seen from Figure 3, at week 8 after the start of blue light exposure, compared with the normal control group, the RP of the pathological control group showed a significant decrease after blue light exposure, both in a-wave and b-wave, indicating that blue light successfully induced retinal dysfunction in the mice. In contrast, the RP of the experimental group showed a significant increase compared with the pathological control group, and there was no significant difference between the experimental and normal control groups; in fact, the RP was slightly higher. Similar results were observed at week 16 after the start of blue light exposure. These experimental results demonstrate that *Lactobacillus reuteri* subsp. reuteri LR1349 of this invention can effectively improve retinal dysfunction caused by blue light exposure. [D、] [Histopathological Analysis] [(histopathological analysis)] [:]

[0058] At the end of week 16 after the start of blue light exposure, mice in each group were anesthetized with 3-4% isoflurane. The mice were then sacrificed, and their eyeballs were removed and fixed overnight with a fixative solution containing 347 mL ddH₂O, 111 mL 100% acetic acid, 320 mL 99.5% ethanol, and 222 mL 10% formaldehyde. The eyes were then dehydrated with ethanol, embedded in paraffin, and sectioned to obtain retinal tissue sections with a thickness of 4 μm.

[0059] Subsequently, a portion of the tissue sections were stained with hematoxylin-eosin using techniques familiar and commonly used by those skilled in the art. They were then observed and photographed using an optical microscope (model CX33, brand Olympus) at 40x magnification. ViewPoint software was then used to measure the average thickness of the outer nuclear layer (ONL) and the inner segment / outer segment layer (IS / OS) of the retina, and to count the number of ONL cell nuclei.

[0060] Then, analyze the obtained experimental data according to the method described in item 1 of the "General Experimental Methods" above.

[0061] Figure 4 shows the results of hematoxylin-eosin staining of retinal tissue sections from each group of mice at the end of week 16 after the start of blue light exposure. Figures 5 and 6 show the mean ONL thickness and mean IS / OS thickness of retinal tissue sections from each group of mice at the end of week 16 after the start of blue light exposure, respectively.

[0062] As shown in Figures 4 to 6, compared with the normal control group, the average ONL thickness and IS / OS thickness of the pathological control group were significantly reduced after blue light irradiation, indicating that blue light successfully induced retinal lesions in mice. In contrast, compared with the pathological control group, the average ONL thickness and IS / OS thickness of the experimental group were significantly increased, and were similar to those of the normal control group.

[0063] Figure 7 shows the number of ONL cell nuclei measured in retinal tissue sections from mice in each group at the end of week 16 after blue light exposure. As can be seen from Figure 7, compared to the normal control group, the number of ONL cell nuclei in the pathological control group was significantly reduced after blue light exposure, indicating that blue light successfully induced retinopathy in the mice. In contrast, compared to the pathological control group, the number of ONL cell nuclei in the experimental group was significantly increased, and approximately the same as that in the normal control group.

[0064] These experimental results show that the Lactobacillus reuteri subsp. reuteri LR1349 of the present invention can effectively improve retinal lesions caused by blue light exposure and slow down the loss of retinal tissue. [E、] [Immunofluorescence staining analysis] [(immunofluorescence staining assay)] [:]

[0065] The tissue sections obtained from item D above were collected, and then immunofluorescence staining analysis was performed using anti-rhodopsin antibody (catalog number ab98887, manufacturer: Abcam) as the primary antibody and Alexa Fluor® 488-conjugated goat anti-mouse IgG antibody (catalog number ab150113, manufacturer: Abcam) as the secondary antibody, following the manufacturer's instructions. The stained tissue sections were observed and photographed using an imaging system (EVOS®FL Auto Imaging System, manufacturer: Thermo Fischer Scientific) at a magnification of 20x, and then the fluorescence intensity was quantified using ImageJ software. The relative fluorescence intensity was calculated by substituting the measured fluorescence intensity of each group into the following formula (1): [formula] [(1)] [:] [A] [=] [B / C] Where: A = relative fluorescence intensity B = Fluorescence intensity of each group C = fluorescence intensity of the normal control group

[0066] Then, analyze the obtained experimental data according to the method described in item 1 of the "General Experimental Methods" above.

[0067] Figure 8 shows the relative fluorescence intensity of rhodopsin in the retinal tissue of mice in each group at the end of week 16 after the start of blue light exposure. As can be seen from Figure 8, compared with the normal control group, the rhodopsin in the pathological control group was significantly reduced after blue light exposure, indicating that blue light successfully caused a large amount of photosensitive pigment breakdown in the mouse retina. In contrast, the rhodopsin in the experimental group was significantly increased compared with the pathological control group, and was similar to that of the normal control group.

[0068] The experimental results show that the Lactobacillus reuteri subsp. reuteri LR1349 of the present invention can effectively improve retinal lesions caused by blue light exposure and slow down the decomposition of photosensitive pigments.

[0069] Based on the above experimental results, it can be concluded that the Lactobacillus reuteri subsp. reuteri LR1349 of the present invention has the effect of improving blue light-induced retinal damage.

[0070] All patents and documents cited in this specification are incorporated herein by reference in their entirety. In the event of any conflict, the detailed description herein (including its definitions) shall prevail.

[0071] Although the present invention has been described with reference to the specific examples described above, it is evident that many modifications and variations can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the invention be limited only to those shown in the appended claims.

[0072] none

[0073] Republic of China, Taiwan; Bioresource Conservation and Research Center, Food Industry Development Institute (BCRC of FIRDI); 2024 / 08 / 28; BCRC 911241.

[0074] TW202619740A_114111859_SEQL.xml

Claims

1. A subsp. reuteri of Lactobacillus reuteri, LR1349, which is deposited with the registration number BCRC 911241 at the Center for Biological Resources Conservation and Research, Food Industry Development Institute.

2. Use of Lactobacillus reuteri subsp. reuteri LR1349 for the preparation of a composition for improving blue light-induced retinal damage, wherein, Lactobacillus reuteri subsp. reuteri LR1349 is deposited with the Center for Biological Resources Conservation and Research at the Food Industry Development Institute under the registration number BCRC 911241.

3. The use as described in claim 2, wherein the composition is a food composition.

4. The use as described in claim 2, wherein the composition is a pharmaceutical composition.

5. The use as described in claim 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

6. The use as described in claim 4, wherein the pharmaceutical composition is in a dosage form selected from the following routes of administration: oral administration and non-enteric administration.

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