Bifidobacterium longum subsp. Longum strain and application thereof in improving blue light induced eye injury

Abstract

The invention discloses a bifidobacterium longum subsp. Longum strain and application thereof in improving blue light induced eye injury. The bifidobacterium longum subsp. Longum BL531 disclosed by the invention is preserved in the German Microorganism and Cell Culture Collection Center DSMZ, and the preservation number is DSM 35139; the bifidobacterium longum subsp. Longum BL1363 is preserved in the German Microorganism and Cell Culture Collection Center DSMZ, and the preservation number is DSM 35138. The bifidobacterium longum subsp. Longum strain disclosed by the invention can effectively improve the reduction of visual acuity, retinal dysfunction and retinopathy caused by blue light irradiation. Therefore, the bifidobacterium longum subsp. Longum strain disclosed by the invention can be used for preparing medicines, foods or food additives for improving eye injury induced by blue light.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbiology, and specifically relates to a use of a Bifidobacterium longum subsp. longum strain in improving blue light-induced eye damage. BACKGROUND

[0002] Blue light is high-energy visible light with a wavelength of 400-500 nm, which can penetrate the cornea, lens and vitreous body to reach the retina, trigger photochemical reactions and generate 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. Ultraviolet light (UV) with a wavelength below 295 nm is mostly absorbed by the cornea, and the lens also blocks UVA (315-400 nm) and UVB (280-315 nm) from penetrating. Only the shorter wavelength blue light and a small amount of ultraviolet light in visible light can cause photochemical damage to the retina, and thus is considered as harmful light.

[0004] In recent years, electronic products using light-emitting diodes (LEDs) as light sources, such as liquid crystal televisions, computer screens, mobile phones or tablets, are widely used in daily life. It is known that blue light is the main color rendering component of cool-white LED light, which has gradually replaced traditional incandescent or fluorescent lamps to become the light source for modern lighting or screen display. Blue light hazard has become an important public health issue that cannot be ignored, and blue light has become a new emerging environmental pollution source.

[0005] From the in vivo and in vitro studies of blue light damage, it was found that the irradiation of blue light or blue-rich LED light source can cause the morphological changes of the retinal tissue of experimental animals. Among them, the outer nuclear layer (ONL) and retinal pigment epithelial cells (RPE) of the three-layer structure are most severely damaged. Pathological phenomena such as thinning of ONL and RPE, reduction of ONL / RPE cell nuclei, distortion or disappearance of the boundary between layers, and photoreceptor cell nucleus pyknosis can be observed in tissue sections (Hunter, J. J., et al., The susceptibility of the retina to photochemical damage from visible light. Prog Retin 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 irradiation of monkey retinas can cause macular lesions. After 7 days of blue light irradiation in rats, retinal tissue atrophy, leakage, and retinal vascular bending were observed. High-intensity blue light can also damage the cytoskeleton and tight junction protein structure of RPE cells.

[0007] Blue light can cause a decrease in the activity of antioxidant enzymes in retinal tissue, promote the generation of superoxide anion (O2 ·- ), H2O2, -OOH, and various oxidative stress indicators such as 8-OHdG, acrolein, and nitro-tyrosine. TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) staining showed that blue light can cause apoptosis of RPE and photoreceptor cells. At the cellular and molecular level, blue light can significantly induce mitochondrial ROS generation and activate the transcription factor NF-kB, initiate the secretion of various inflammatory factors such as TNF-α and IL-1β, and activate inflammasome, initiate apoptosis and pyroptosis, and interfere with autophagy and self-repair of cells.

[0008] The average indoor light exposure of daytime workers is usually below 1000 lux. Illumination below 300 lux is defined as low intensity light source. While conventional lighting devices and electronic displays rarely cause serious damage in daily life, life patterns of long time and periodic fixation of LED screens can 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) simulated indoor lighting with 500 lux LED light source, and observed changes in retinal structure after 28 days of continuous irradiation of experimental animals. Even if the illumination of mobile phone screens can be as low as 5 lux, long time use can still cause reduction of electrophysiological function of photoreceptor cells and abnormal retinal morphology.

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

[0010] The purpose of the present application is to provide a use of Bifidobacterium longum subsp. longum strain in improving blue light-induced eye damage.

[0011] According to the exposure time and period of blue light, the light exposure mode is divided into three types:

[0012] (1) short-term light exposure mode with light exposure time less than 6h;

[0013] (2) 12h / 12h mode following the light-dark cycle of animals;

[0014] (3) 24h continuous light exposure mode without dark cycle.

[0015] The light source used in most studies has an illumination of 1000 lux or more, and its whole light exposure time is only completed in one day or one week, which is quite different from the life situation of people exposed to blue light. In addition, the phagocytosis of RPE cells to photoreceptor cells is regulated by circadian rhythm, and this metabolic process reaches a peak about 2h after the start of the light cycle.

[0016] Therefore, the present application includes the period of three hours after the start of the light cycle (AM 0600-0900) when designing animal light exposure, in order to optimize the blue light chemical effect and detect the health care effect of probiotic samples (BL531, BL1363) on blue light-induced retinal damage in mice.

[0017] Technical solution: The purpose of the present application is achieved by the following technical solutions:

[0018] The present application provides a Bifidobacterium longum subsp. longum BL531 strain, which is deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with the accession number DSM 35139.

[0019] The present application also provides a Bifidobacterium longum subsp. longum BL1363 strain, which is deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with the accession number DSM 35138.

[0020] Biological preservation information:

[0021] In accordance with the provisions of the Budapest Treaty, the Bifidobacterium longum subsp. longum BL531 strain was deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) on September 9, 2024, with the accession number DSM 35139.

[0022] In accordance with the provisions of the Budapest Treaty, the Bifidobacterium longum subsp. longum BL1363 strain was deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) on September 9, 2024, with the accession number DSM 35138.

[0023] The present application also provides the use of the above-mentioned Bifidobacterium longum subsp. longum strain in the preparation of a drug, food or food additive for improving blue light-induced eye damage.

[0024] The eye damage is a decrease in visual acuity, retinal dysfunction and retinopathy caused by blue light irradiation.

[0025] The eye damage includes macular lesions.

[0026] The present application also provides a drug, food or food additive for improving blue light-induced eye damage, which comprises the Bifidobacterium longum subsp. longum BL531 and / or Bifidobacterium longum subsp. longum BL1363 strain and a pharmaceutically acceptable excipient or a food acceptable excipient.

[0027] The drug, food or food additive improves eye damage by repairing retinal epithelial cell damage caused by blue light irradiation.

[0028] The present application also provides the use of the above-mentioned Bifidobacterium longum subsp. longum strain in the preparation of a drug, food or food additive for relieving visual fatigue.

[0029] The present application also provides a medicament, food or food additive for alleviating visual fatigue, which comprises Bifidobacterium longum subsp. longum BL531 and / or Bifidobacterium longum subsp. longum BL1363 strain and pharmaceutically acceptable excipients or food acceptable excipients.

[0030] Beneficial effects:

[0031] The Bifidobacterium longum subsp. longum BL531 and BL1363 of the present application are proved by in vivo experiments to be effective in improving visual acuity decline, retinal dysfunction and retinopathy caused by blue light irradiation. After continuous blue light exposure, the visual acuity (VA) of the pathological control group of mice decreased significantly, while the VA of the mice treated with BL531 or BL1363 increased significantly, showing that they help maintain visual recognition ability. Electroretinogram (ERG) detection also showed that BL531 or BL1363 can significantly restore the a-wave and b-wave response amplitude of the damaged blue light induced, showing that they have the effect of protecting the retinal photoreception and nerve conduction function.

[0032] Histopathological analysis results show that blue light irradiation causes a significant decrease in the thickness of the mouse retinal outer nuclear layer (ONL) and inner / outer segment layer (IS / OS), and a significant decrease in the number of ONL nuclei, showing that the retinal structure is damaged. However, both BL531 and BL1363 can effectively increase the number of ONL nuclei; at the same time, the thickness of the ONL and IS / OS layers of the mice treated with BL531 increased significantly, close to the level of the normal control group, showing that they can significantly inhibit the loss of retinal cells and structural degradation. In addition, the results of immunofluorescence staining show that blue light reduces the expression of rhodopsin in the retina, and BL531 can significantly increase the fluorescence intensity, which helps to maintain the function of photoreceptor cells. On the other hand, the oxidative stress indicator 8-OHdG is significantly increased in the pathological control group, indicating an increase in DNA damage, and the level of 8-OHdG is significantly decreased after treatment with BL531, showing that it can alleviate the oxidative damage induced by blue light.

[0033] It can be seen that the Bifidobacterium longum subsp. longum BL531 and BL1363 strains provided by the present application have a significant protective effect on slowing down the damage to the retinal tissue caused by blue light, and have a high potential to develop into a new probiotic therapeutic preparation for preventing and improving blue light-induced retinal damage. Therefore, the above-mentioned Bifidobacterium longum subsp. longum strain of the present application can be used for preparing a medicament, food or food additive for improving blue light-induced eye damage. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1Bifidobacterium longum subsp. longum BL1363 and BL531 and Bifidobacterium longum subsp. longum DSM 20219 T The gene fingerprints 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; wherein lane M represents a DNA ladder marker (100-3000 bp); and lanes 1 to 3 represent Bifidobacterium longum subsp. longum DSM 20219 T and the amplification products of Bifidobacterium longum subsp. longum BL531 and BL1363 of the present application.

[0035] Figure 2 The visual acuity threshold measured at the end of week 8 and 16 after the start of blue light exposure for each group of mice; wherein in the same week, when data from different groups are denoted with different English letters, it means that there is a statistical difference between these data.

[0036] Figure 3 The change in retinal potential measured at the end of week 8 and 16 after the start of blue light exposure for each group of mice; wherein in the same week, when data from different groups are denoted with different English letters, it means that there is a statistical difference between these data.

[0037] Figure 4 The results observed by hematoxylin-eosin staining of retinal tissue sections of each group of mice at the end of week 16 after the start of blue light exposure; wherein “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 the retinal pigment epithelium.

[0038] Figure 5 The average ONL thickness measured from retinal tissue sections of each group of mice at the end of week 16 after the start of blue light exposure; wherein when data from different groups are denoted with different English letters, it means that there is a statistical difference between these data.

[0039] Figure 6 The ONL cell nuclei number and IS / OS average thickness of each group of mice at the end of the 16th week after the start of blue light irradiation; wherein when the data of different groups are marked with different English letters, it means that there is a statistical difference between these data.

[0040] Figure 7 The relative fluorescence intensity of rhodopsin and 8-OHdG in the retinal tissue of each group of mice at the end of the 16th week after the start of blue light irradiation; wherein when the data of different groups are marked with different English letters, it means that there is a statistical difference between these data. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be described in detail below through specific examples, but the protection scope of the present application is not limited to the examples.

[0042] Probiotic samples BL531 and BL1363 were prepared and provided by Shenghe Biotechnology Co., Ltd.; the strain concentration was 1 x 10 8 / 300 μL (containing bacterial bodies and supernatant).

[0043] Experimental animals:

[0044] Rodents are the most commonly used animal species to explore blue light retinal damage. Male eight-week-old ICR (albino) mice were selected for this study, purchased from Lesko Biotechnology Co., Ltd. Experimental animals were raised in an animal house, four mice per cage, after a two-week adaptation period, the experiment began at the age of ten weeks. During the adaptation period, commercially available experimental animal granular Lab5001 diet (Ralston Purina, St. Louis, USA) was given, free access to food and water. The animal house temperature was controlled at 22±2℃, the relative humidity was 60±10%, and it was maintained in a 12h / 12h light-dark cycle, the light was turned on at six o'clock in the morning, and turned off at six o'clock in the afternoon.

[0045] Example 1 Screening and Characterization of Isolates BL1363 and BL531

[0046] (1) Source and isolation of isolates BL1363 and BL531

[0047] Feces of healthy human over 85 years old and not using antibiotics for 3 months were collected as a sample source for isolation and screening of Bifidobacterium. First, the feces were mixed with an appropriate amount of saline water (0.05% L-Cysteine was added) and then homogenized using a homogenizer (Vortex-Genie 2, Scientific Industries). Next, the obtained homogenate was subjected to 10-fold serial dilution, prepared as dilutions having different dilution folds (10 -1 -10 -6 times), and then 0.1 mL of each dilution was taken and uniformly spread on MRS agar medium, followed by anaerobic culture at 37°C for 5-7 days. After the formation of colonies, colonies on the MRS agar medium were selected to obtain two isolates, BL1363 and BL531.

[0048] (2) Preliminary test

[0049] Gram staining was performed on the isolates BL1363 and BL531, and as a result, both the isolates BL1363 and BL531 were Gram-positive bacilli.

[0050] (3) Extraction of genomic DNA

[0051] The extraction of genomic DNA of the isolates BL1363 and BL531 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 grinding beads (BioSpec Products, Cat. No. 11079101), and 500 μL of phenol-chloroform-isoamyl alcohol mixture (25:24:1, v / v / v).

[0052] (4) 16S rDNA sequence analysis

[0053] First, the genomic DNA obtained in step (3) was used as a template, and a polymerase chain reaction (PCR) was performed using a primer pair, forward primer 27F and reverse primer 1492R, designed for bacterial 16S rDNA genes, having the nucleotide sequences shown below, to amplify the 16S rDNA fragments of the isolates BL1363 and BL531. The PCR reaction system is shown in Table 1.

[0054] Forward primer 27F:

[0055] 5'-agagtttgatcctggctcag-3'

[0056] Reverse primer 1492R:

[0057] 5'-ggttaccttgttacgact-3'

[0058] The above primers were synthesized by Genomics Biosci&Tech Co., Ltd. The primer sequences are described 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.

[0059] Table 1 PCR reaction system

[0060] Content Volume (μL) Genomic DNA (10 ng / μL) 1 Forward primer Fl (10 μM) 0.5 Reverse primer Rl (10 μM) 0.5 Fast-Run™ 2X Tag Master Mix (Protech, Cat. No. SA-TMM228) 10 Deionized water 8

[0061] PCR reaction program:

[0062] Denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 60 seconds, primer annealing at 50°C for 60 seconds, and extension at 72°C for 60 seconds, and finally elongation at 72°C for 8 minutes.

[0063] After the PCR was completed, Genomics Biosci&Tech Co., Ltd. was commissioned to perform sequencing analysis, and partial 16S rDNA sequences of the isolates BL1363 and BL531 were obtained, respectively:

[0064] The sequence of the 16s rDNA fragment of isolate BL1363 is shown as SEQ ID NO. 1 and the sequence of the 16s rDNA fragment of isolate BL531 is shown as SEQ ID NO. 2.

[0065] SEQ ID NO. 1 :

[0066] CGGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGA

[0067] CCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCT

[0068] CCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCG

[0069] CGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTA

[0070] GCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACT

[0071] CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGC

[0072] AGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGG

[0073] GGAGCAAGCGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTG

[0074] CCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCG

[0075] TAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAAC

[0076] GGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGA

[0077] ATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGA

[0078] AGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGC

[0079] GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATG

[0080] TGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCC

[0081] TGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGC

[0082] ACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCT

[0083] GGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGG

[0084] TTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA

[0085] GTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGG

[0086] AACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTC

[0087] AGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTAC

[0088] AACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGT

[0089] TCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCG

[0090] CGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCC

[0091] GTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCGCACCC

[0092] GAAGCCGGTGGCCTAACCC

[0093] SEQ ID NO.2:

[0094] CTTACCATGCAAGTCGAACGGGATCCATCAAGCTTGCTTGGTGGTGAGAGTG

[0095] GCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTC

[0096] CTGGAAACGGGTGGTAATGCCGGATGTTCCAGTTGATCGCATGGTCTTCTGG

[0097] GAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGG

[0098] GTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGG

[0099] CCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG

[0100] GAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGAT

[0101] GGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGT

[0102] TTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATAC

[0103] GTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGG

[0104] TTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGG

[0105] TACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGT

[0106] GGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGG

[0107] CCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATA

[0108] CCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCA

[0109] CGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGC

[0110] CGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAG

[0111] CATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGT

[0112] TCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGGTTCACAGGTGGT

[0113] GCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG

[0114] AGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGG

[0115] GACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATG

[0116] CCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATG

[0117] CGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGC

[0118] AGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGC

[0119] AACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGG.

[0120] After comparing with the gene database in the NCBI website, it was found that the partial 16S rDNA sequences of the isolated strains BL1363 and BL531 have more than 97% identity with the partial 16S rDNA sequences of Bifidobacterium longum subsp. longum.

[0121] According to the above experimental results, the isolated strains BL1363 and BL531 isolated by the present application are preliminarily identified as Bifidobacterium longum subsp. longum.

[0122] (5) Random Amplified Polymorphic DNA (RAPD) analysis

[0123] In order to confirm whether the Bifidobacterium longum subsp. longum BL1363 and BL531 are novel Bifidobacterium longum subsp. longum isolated strains, the above-mentioned isolated strains were subjected to random amplified polymorphic DNA analysis.

[0124] The RAPD analysis was performed according to the reference method (Akopyanz N. et al. 1992, Nucleic Acids Res., 20: 5137

[0125] PCR was performed using the genomic DNA obtained in step (3) as a template and 8 10-mer primers, RAPD-1, RAPD-A, RAPD-B, RAPD-C, RAPD-D, RAPD-E, RAPD-F and RAPD-G, having the nucleotide sequences shown in Table 2, were selected. The PCR reaction system is shown in Table 3.

[0126] The 8 10-mer primers were synthesized by GenScript Corporation. The primer sequences are described 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.

[0127] Table 2 Primer sequence information

[0128] Primer Nucleotide sequence (5'→ 3') RAPD-1 ctcaggtcgc RAPD-A ccgcagccaa RAPD-B aacgcgcaac RAPD-C gcggaaatag RAPD-D gaggacaaag RAPD-E ggcgtcggtt RAPD-F ggccacggaa RAPD-G cgaactagac

[0129] Table 3 PCR reaction system

[0130]

[0131] PCR reaction procedure:

[0132] Denaturation at 94°C for 2 minutes, followed by 6 cycles of denaturation at 94°C for 30 seconds, primer annealing at 36°C for 60 seconds, and extension at 72°C for 90 seconds, followed by 30 cycles of denaturation at 94°C for 20 seconds, primer annealing at 36°C for 30 seconds, and extension at 72°C for 90 seconds, and finally extension at 72°C for 3 minutes.

[0133] After the PCR was completed, the PCR amplification products were subjected to electrophoresis in a 1.5% agarose gel, then stained, and observed and photographed under ultraviolet light.

[0134] In addition, for comparison, the known Bifidobacterium longum subsp. longum DSM 20219 T (bought from the Bioresource Collection and Research Center, BCRC, i.e., standard strain BCRC 11847 TThis strain was used as a comparison strain for analysis. The obtained electrophoresis results are shown in... Figure 1 middle.

[0135] Figure 1 This invention demonstrates the presence of *Bifidobacterium longum* subsp. *longum* BL1363 and BL531, as well as *Bifidobacterium longum* subsp. *longum* DSM20219. 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. Figure 1 It is evident that the RAPD analysis results obtained from the three isolates using different primers showed differences among them.

[0136] Based on the above characteristic identification results, both BL1363 and BL531 of *Bifidobacterium longum* subsp. *longum* are novel isolates of *Bifidobacterium longum*. They were deposited on August 28, 2024, with accession numbers BCRC 911238 and BCRC 911239 respectively, at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). They were also deposited on September 9, 2024, with accession numbers DSM 35138 and DSM 35139 respectively, at the German Collection of Microorganisms and Cell Cultures (DSMZ).

[0137] Example 2: Preparation of probiotic samples BL531 and BL1363

[0138] Bifidobacterium longum subsp. BL531 and BL1363 were inoculated into MRS broth medium (brand: BDDIfco). TM The strain was cultured at 37°C for 18 hours (product number 288130) to activate the bacteria.

[0139] The resulting cultures were then centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatant was collected. Next, the cells were 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 test bacterial solutions of Bifidobacterium longum subsp. longum BL1363 and BL531, respectively.

[0140] Example 3 Establishment of a model of low intensity blue light-induced retinal damage in mice

[0141] Animal light exposure device:

[0142] The mouse light exposure device used a blue LED light bar with a wavelength of 465 ± 10 nm detected by a fiber optic spectrometer (#USB4000, Ocean optics, USA); an illuminometer (IM-600, Topcon, Japan) and a high-frequency optical power meter (PM100A, Thorlabs, Newton, USA) were used to detect the average illuminance and average optical power at the four corners a-d and the central e position points in the irradiation cage.

[0143] After a 2-week adaptation period, the experimental animals were pre-fed with probiotic samples (test bacterial solution of B. longum subsp. longum BL1363 or BL531 prepared in Example 2) or MRS culture solution (i.e., MRS broth) for one week (tube-feeding volume 300 μL), and then subjected to blue light irradiation. Each group of experimental mice consisted of 12 mice, which were randomly divided into the following groups:

[0144] (1) Control group (Ctrl group): given MRS culture solution (300 μL) without blue light irradiation;

[0145] (2) Blue light group (BL group): given MRS culture solution (300 μL) daily and subjected to 6h blue light irradiation;

[0146] (3) BL531 group: given 1 × 10 8 CFU of probiotic strain BL531 daily and subjected to 6h blue light irradiation;

[0147] (4) BL1363 group: given 1 × 10 8 CFU of probiotic strain BL1363 daily and subjected to 6h blue light irradiation.

[0148] The light in the animal room was 150-200 lux, and the blue light exposure group was additionally subjected to blue light irradiation for a total of 6h daily during the two light cycle periods of AM 06:00-09:00 and PM 15:00-18:00 in the presence of room light, and the dark cycle period was from PM 18:00 to the next day's AM 06:00. This experiment used low-intensity long-term blue light (long-term low intensity) to simulate the living environment, and the light exposure conditions were 108 lux (44.8 μW / cm 2 After 16 weeks of continuous irradiation, the mice were sacrificed under anesthesia with 3% isoflurane, and their retinal tissues were analyzed. The temperature in the animal room was maintained at 22 ± 2°C during the experiment, and the food and water intake and body weight were recorded regularly.

[0149] Example 4 Visual acuity (VA) experiment

[0150] The visual acuity experiment was performed according to the method of Liou et al. (Liou J.C. et al. 2018, J. Funct. Foods, 49:314-323) with modifications.

[0151] First, the experimental mice were placed in a light-proof dark box. A cylindrical platform with a height of 13 cm and a diameter of 5 cm was set at the center of the bottom of the dark box for the test mice to stand and move freely. A digital screen was set 15 cm from the center platform on each side of the dark box to play a black-and-white moving stripe grating pattern to stimulate the animals to exhibit compensatory head or body movements in the direction of the stimulus, thereby evaluating the animal's optomotor response ability.

[0152] At the beginning of the experiment, the screen was first flashed for 5 seconds to stimulate the mice's response, and then different spatial frequencies (cycles per degree, cpd) of black-and-white gratings were played alternately in clockwise and counterclockwise directions at a constant speed of 12 degrees per second. The frequencies were 0.437, 0.328, 0.164, 0.082, 0.055, and 0.033 cpd, respectively, with 6 levels, and each level was tested for 2 minutes with two repetitions.

[0153] The accuracy of the mouse head swing was recorded during the experiment. The accuracy was calculated by dividing the number of correct responses by the total number of responses. When the accuracy was higher than 70%, it was determined that the mouse's visual acuity reached the threshold standard of that spatial frequency.

[0154] Figure 2 The visual acuity thresholds of the mice in each group were measured at the end of the 8th and 16th weeks after the start of blue light irradiation. From Figure 2 It can be seen that at the 8th week after the start of blue light irradiation, compared with the normal control group, the visual acuity threshold of the blue light group showed a significant decrease after blue light irradiation, indicating that blue light successfully induced a decrease in visual acuity in mice. Compared with the blue light group, the visual acuity thresholds of the BL1363 group and the BL531 group showed a significant increase and were similar to the values of the normal control group. At the 16th week after the start of blue light irradiation, similar results were observed. This experimental result shows that the long bifidobacterium longum BL1363 and BL531 of the present application can effectively improve the decrease in visual acuity caused by blue light irradiation.

[0155] Example 5 Electroretinogram (ERG) analysis

[0156] Retinal potential analysis was performed using a retinal function analyzer (BPM-300, Dioptrix, Canada) and RetinoGraphics BPM300 software (version 7.0.0.7).

[0157] Before the experiment, mice underwent a 12-hour dark acclimatization period. The experiment was conducted entirely in the dark, and electronic devices emitting electromagnetic waves were excluded from the environment. Mice were then administered Zoltil 100 (Victal, France, 75 mg / kg bw) and xylazine. After anesthesia and sedation with Bayer (9.75 mg / kg bw), the animal was placed on a 37°C constant-temperature pad. One to two drops of Better Eye Drops (Madison, 0.5% Tropicamide) were instilled onto the cornea to paralyze the ciliary muscle and dilate the pupil, increasing light intake. Reference and indifference electrodes were then attached to the outer corner of the eye and forehead, respectively, while a recording electrode was attached to the corneal surface. Throughout the experiment, the resistance of the recording and reference electrodes was maintained at 2.1 kΩ, while the indifference electrode was maintained at 12.1 kΩ or within ±25% of this value to eliminate background noise interference. Finally, a flash of light at an intensity of 3 cd.s / m² was applied to the cornea at a distance of approximately 3-5 mm. 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.

[0158] 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 potentials in the BL1363 and BL531 groups were significantly increased compared with the blue light group, and there was no significant difference between them and the normal control group; in fact, they were slightly increased. Similar results were observed at week 16 after blue light exposure. These experimental results show that *Bifidobacterium longum* subsp. *longum* BL1363 and BL531 of this invention can effectively improve retinal dysfunction caused by blue light exposure.

[0159] Example 6: Pathological Tissue Analysis of the Retinal

[0160] 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 fovea (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.

[0161] Figure 4 The results, shown, are retinal tissue sections of mice from each group observed at the end of week 16 after blue light exposure began, 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 BL1363 group and the BL531 group were significantly increased and similar to those of the normal control group.

[0162] from Figure 5 , Figure 6 As can be seen, compared with the normal control group, the average thickness of 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 ONL, IS / OS, and the number of ONL cell nuclei in the BL531 group were significantly increased, and approximately the same as the normal control group. These experimental results show that both *Bifidobacterium longum* subsp. *longum* BL1363 and BL531 of this invention can effectively improve retinopathy caused by blue light irradiation and slow down retinal tissue loss.

[0163] Example 7 Immunohistochemical staining analysis of molecules related to retinal injury

[0164] The methods of immunohistochemical staining are briefly described below:

[0165] 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 in TBS (25 mM Tris-base, 137 mM NaCl, 2.7 mM KCl), also known as TBST, was used to wash three times, to hydrophobic pen circle the tissue, and to cover the eye tissue with 1% bovine serum albumin (BSA) in TBST (Tris-buffered Saline with Tween20) at room temperature to block non-specific antigen, followed by the primary antibody (anti-visual pigment antibody, Abeam, item number ab98887), completely covering the tissue to react at 4°C. Finally, 4', 6-diamidino-2-phenylindole (DAPI) solution was used to stain the nucleus to locate, to mount with mounting medium and cover glass. After the mounting medium was dry, the FL Auto Imaging System (Thermo Fischer Scientific, USA) was used to take pictures of the fluorescence of the tissue at 20 times, and the Image J software was used to quantify the fluorescence RawIntDen (raw integrated density) intensity.

[0166] Figure 7 The relative fluorescence intensity of visual pigment and 8-OHdG in the retinal tissue of each group of mice at the end of the 16th week after starting to irradiate blue light. Among them, "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 the retinal pigment epithelium.

[0167] From Figure 7 It can be seen that, compared with the normal control group, the visual pigment of the blue light group showed a significant decrease after experiencing blue light irradiation, and 8-OHdG showed a significant increase, indicating that blue light successfully caused a large amount of decomposition of the light-sensitive pigment in the mouse retina. Compared with the blue light group, the visual pigment of the BL531 group showed a significant increase, and 8-OHdG showed a significant decrease, and was similar to the normal control group. The experimental results show that the Bifidobacterium longum subsp. longum BL531 of the present application can effectively improve the retinal lesions caused by blue light irradiation and slow down the decomposition of the light-sensitive pigment.

[0168] ​While the application has been described and illustrated with reference to specific preferred embodiments, it is not intended that it be limited to these particulars. Various changes in form and detail can be made without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. Bifidobacterium longum subsp. longum BL531 strain, characterized in that, The Bifidobacterium longum subsp. longum BL531 is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 35139.

2. Bifidobacterium longum subsp. longum BL1363 strain, characterized in that, The Bifidobacterium longum subsp. longum BL1363 is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 35138.

3. Use of a strain of Bifidobacterium longum subsp. longum according to claim 1 or 2 for the manufacture of a medicament, food or food additive for improving blue light-induced eye damage.

4. Use according to claim 3, characterized in that, The eye damage is a decrease in visual acuity, retinal dysfunction and retinopathy caused by blue light exposure.

5. Use according to claim 3, characterized in that, The eye damage comprises macular lesions.

6. A drug, food or food additive for ameliorating blue light-induced eye damage, characterized by, The medicament, food or food additive comprises a strain of Bifidobacterium longum subsp. longum BL531 and / or Bifidobacterium longum subsp. longum BL1363 and a pharmaceutically acceptable excipient or a food acceptable excipient.

7. Use according to claim 6, characterized in that, The medicament, food or food additive improves eye damage by repairing retinal epithelial cell damage caused by blue light exposure.

8. Use of a strain of Bifidobacterium longum subsp. longum according to claim 1 or 2 for the manufacture of a medicament, food or food additive for alleviating visual fatigue.

9. A medicament, food or food additive for alleviating visual fatigue, characterized by, The medicament, food or food additive comprises a strain of Bifidobacterium longum subsp. longum BL531 and / or Bifidobacterium longum subsp. longum BL1363 and a pharmaceutically acceptable excipient or a food acceptable excipient.

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