Bifidobacterium bifidum and application thereof in ophthalmic products or products for regulating intestinal flora

Abstract

This invention belongs to the field of biomedicine, specifically relating to a strain of Bifidobacterium bifidum and its application in eye health products. The Bifidobacterium bifidum (… Bifidobacterium bifidum Bifidobacterium bifidum BB00, with accession number CGMCC No. 34389, is a product of this invention. It can improve the clinical symptoms of dry eye syndrome, increase tear secretion, prolong tear film breakup time, reduce ocular surface disease index, alleviate corneal epithelial damage, and regulate palpebral fissure opening. It can also regulate the composition of the intestinal flora, increase flora diversity, and inhibit the growth of pathogenic intestinal bacteria. Bifidobacterium bifidum BB00 not only provides an innovative solution to the clinical pain point of dry eye syndrome, achieving synergistic intervention between intestinal regulation and eye health, but also possesses scientific innovation, clinical effectiveness, and industrialization potential. It has significant value for the application of bacteria and bacterial agents in the biomedical field and for the treatment of ocular surface-intestinal related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a strain of Bifidobacterium bifidum and its application in ophthalmic products or products for regulating intestinal flora. Background Technology

[0002] For understanding the technical content of this invention:

[0003] Myopia, the most common refractive error, causes light entering the eye to focus in front of the retina, resulting in blurred vision. Besides blurred vision, myopia increases the incidence of eye diseases and, in severe cases, can even lead to blindness. For the sake of education, employment, and improving appearance, refractive surgery for myopia has become increasingly popular. Since the beginning of modern refractive surgery, the effectiveness and safety of femtosecond laser-assisted in situ keratomileusis (FS-LASIK) have been widely recognized. As one of the current mainstream surgeries, FS-LASIK has better applicability than another mainstream surgery, femtosecond laser small incision lenticule extraction (SMILE), in patients with high myopia and thin corneas. However, postoperative dry eye and glare often reduce the postoperative experience and seriously affect patients' quality of life. Reducing postoperative complications is currently a hot research topic.

[0004] Currently, some studies are considering using probiotics to treat dry eye syndrome.

[0005] Relevant patent documents retrieved:

[0006] The document, published in China (CN118045108A) on May 17, 2024, discloses the application of Bifidobacterium longum (accession number CCTCC NO:M 2022306) in the preparation of drugs for treating dry eye disease. The Bifidobacterium longum can significantly enhance tear gland secretion and alleviate symptoms of dry eye disease caused by factors such as blue light from screens.

[0007] Relevant non-patent literature retrieved:

[0008] The journal or book title is *Nutrients*, and the article title is "Probiotic LB101 alleviates dry eyein mice by suppressing matrix metalloproteinase-9 expression through the regulation of gut microbiota-involved NF-κB signaling," volume number 2017 Oct 25;9(11):1166. Published on October 25, 2017, this article discloses an IRT5, a probiotic mixture composed of *Lactobacillus reuteri*, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Streptococcus haemolyticus*, and *Bifidobacterium bifidum*. Ingestion of IRT5 was shown to effectively restore tear secretion in NOD. In prophylactic settings, IRT5 administration was still shown to effectively increase tear production, but ocular inflammatory markers were not improved, indicating that more research is needed to establish optimal intervention protocols and elucidate the mechanism of action.

[0009] The prior art represented by the aforementioned literature has at least the following unresolved technical problems or defects:

[0010] In patent document CN118045108A, only the amount of tear secretion was measured to alleviate the symptoms of dry eye disease. Other important symptoms, such as the degree of ocular surface damage and changes in the level of inflammatory factors, were not tested, making it difficult to comprehensively evaluate the therapeutic effect of this strain.

[0011] The non-patent literature study, "Probiotic LB101 alleviates dry eye in mice by suppressing matrix metalloproteinase-9 expression through the regulation of gut microbiota-involved NF-κB signaling," only involved three weeks of IRT-5 probiotic treatment and observation in mice, without evaluating the long-term efficacy and safety of IRT-5 probiotics. For example, questions such as whether long-term use would lead to gut microbiota imbalance, whether other adverse reactions would occur, and whether its immunomodulatory effects would be sustained were not addressed. This is insufficient for assessing the clinical application potential of IRT-5 probiotics.

[0012] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles:

[0013] Screening a specific strain (Bifidobacterium bifidum BB00) that can effectively improve postoperative dry eye from a large number of Bifidobacterium strains presents challenges: (1) The pathological mechanism of postoperative dry eye is complex (involving multiple aspects such as tear film stability, ocular surface inflammation, and immune regulation). It is necessary to screen strains that can play a role through gut-eye axis regulation and can also be stably colonized in vivo. There are many screening indicators and the correlation is difficult to verify; (2) Different strains of Bifidobacterium bifidum have significantly different metabolic capabilities. It is necessary to screen a large number of strains through in vitro and in vivo experiments. During the process, screening may fail due to poor strain stability (functional degradation after passage) or insufficient in vivo colonization ability. Summary of the Invention

[0014] The purpose of this invention is to provide:

[0015] A strain of Bifidobacterium bifidum and its application in ophthalmic products or products for regulating gut microbiota, and related technologies to address technical problems such as providing effective interventions to improve postoperative dry eye, or combinations thereof.

[0016] Terminology Explanation:

[0017] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0018] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0019] The definition of the standard chemical term can be found in the reference "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy", Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01.

[0020] Unless otherwise specified, conventional methods within the scope of the art, such as growth curves, hemolysis characteristics determination, cell adhesion ability, acid resistance test, bile salt resistance test, antioxidant determination, and pathogenic bacteria inhibition ability test, shall be used.

[0021] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0022] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: For example, a product includes any one or more of the following: (1) a health product for relieving visual fatigue; (2) a medicine for the prevention, treatment or adjunctive treatment of dry eye syndrome. means: the product includes “a health product for relieving visual fatigue and a medicine for the prevention, treatment or adjunctive treatment of dry eye syndrome”; or the product includes “a health product for relieving visual fatigue”; or the product includes “a medicine for the prevention, treatment or adjunctive treatment of dry eye syndrome”.

[0023] The term "Bifidobacterium bifidum" as used in this article refers to a Gram-positive, anaerobic bacterium of the genus Bifidobacterium. It is rod-shaped, often exhibiting a forked "Y" or "V" shape, and is part of the normal flora of the human gut and oral cavity. It plays a vital role in regulating gut microbiota balance, promoting nutrient absorption, and enhancing immune function. It is widely present in the gut of healthy individuals and is one of the common strains used in probiotic preparations, holding significant application value in the biomedical field.

[0024] The term "culture medium" as used in this article refers to an artificially prepared nutrient substrate suitable for the growth, reproduction, or accumulation of metabolic products by microorganisms (such as bacteria, fungi, and viruses). Its components typically include carbon sources, nitrogen sources, inorganic salts, vitamins, and water, and the formulation can be adjusted according to the needs of the cultured organism (e.g., solid culture media require the addition of agar, and anaerobic microbial culture requires a special anaerobic environment). Culture media are indispensable basic materials in experiments and industrial production involving microbial isolation, identification, cultivation, and fermentation.

[0025] The term "probiotic preparation" as used in this article refers to products made primarily of live microorganisms (such as Bifidobacteria, Lactobacillus, and yeast), which, through processing, possess specific physiological functions such as regulating the host's intestinal flora, improving intestinal function, and enhancing immunity. These products can be in the forms of powders, tablets, capsules, and liquids, and can be used in the fields of health supplements or pharmaceuticals. By supplementing beneficial microorganisms, they help maintain the host's microecological balance and improve health.

[0026] The term "dry eye syndrome" as used in this article refers to an ocular surface disease caused by insufficient tear secretion, excessive tear evaporation, or abnormal tear composition, resulting in insufficient lubrication of the ocular surface and damage to the corneal and conjunctival epithelium. Main symptoms include dry eyes, a foreign body sensation, burning sensation, photophobia, and blurred vision; if left untreated, it may affect vision. Common causes include dry environments, prolonged use of electronic devices, aging, eye surgery, and autoimmune diseases. Treatment typically includes artificial tears, improving eye habits, and anti-inflammatory therapy.

[0027] On the one hand, the present invention provides a Bifidobacterium bifidum ( Bifidobacterium bifidum The Bifidobacterium bifidum mentioned is Bifidobacterium bifidum BB00, with accession number CGMCC No.34389.

[0028] On the other hand, the present invention provides a microbial agent comprising the aforementioned Bifidobacterium bifidum.

[0029] Specifically, the bacterial agent includes one or more of the following: Bifidobacterium bifidum cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0030] Specifically, the microbial agent also includes nutritionally acceptable nutrient additives.

[0031] Preferably, the nutritional additives include any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0032] In another aspect, the present invention provides the application of the above-mentioned Bifidobacterium bifidum or bacterial agent in the preparation of ophthalmic products or products for regulating intestinal flora.

[0033] Specifically, the ophthalmic products include any one or more of the following:

[0034] (1) Products that increase tear secretion

[0035] (2) Products that prolong tear film breakup time;

[0036] (3) Products that reduce the ocular surface disease index;

[0037] (4) Products that reduce corneal epithelial damage;

[0038] (5) Products that adjust the degree of palpebral fissure opening;

[0039] (6) Products with antioxidant properties.

[0040] Preferably, the ophthalmic product includes any one or more of the following:

[0041] (1) In the preparation of products for the prevention or treatment of dry eye syndrome;

[0042] (2) Products for the prevention or treatment of conjunctivitis;

[0043] (3) Products for the prevention or treatment of keratitis;

[0044] (4) Products for the prevention or treatment of blepharitis;

[0045] (5) Products for the prevention or treatment of meibomian gland dysfunction;

[0046] (6) Products for the prevention or treatment of corneal epithelial damage;

[0047] (7) Products for the prevention or treatment of eyelid scar contracture;

[0048] (8) Products that relieve eye fatigue;

[0049] (9) Products for the prevention or treatment of cataracts.

[0050] More preferably, the dry eye syndrome is characterized by any one or more of the following: reduced tear secretion, shortened tear film breakup time, ocular surface epithelial damage, and increased ocular surface disease index.

[0051] The conjunctivitis described herein is characterized by any one or more of the following: shortened tear film breakup time, increased ocular surface disease index, and increased conjunctival oxidative stress level;

[0052] The keratitis described herein is characterized by any one or more of the following: shortened tear film breakup time, increased ocular surface disease index, corneal epithelial defects, and increased corneal oxidative stress levels.

[0053] The blepharitis described herein is characterized by any one or more of the following: shortened tear film breakup time, increased ocular surface disease index, and increased eyelid margin oxidative stress level;

[0054] The aforementioned meibomian gland dysfunction is characterized by decreased tear secretion, shortened tear film breakup time, increased ocular surface disease index, and increased level of oxidative damage to the meibomian gland epithelium.

[0055] The corneal epithelial damage described herein is manifested as any one or more of the following: shortened membrane rupture time, increased ocular surface disease index, corneal epithelial defects, and increased corneal oxidative stress level;

[0056] The aforementioned eyelid scar contracture manifests as any one or more of the following: incomplete eyelid closure, entropion, and ectropion;

[0057] The aforementioned eye fatigue is manifested by any one or more of the following: decreased tear secretion and increased ocular surface disease index.

[0058] The aforementioned cataracts are characterized by an increased level of oxidative stress in the lens.

[0059] The Bifidobacterium bifidum BB00 of this invention can effectively increase tear secretion, prolong tear film breakup time, reduce ocular surface disease index, and alleviate corneal epithelial damage. It also has antioxidant effects and can be used to prepare products described "specifically" above that increase tear secretion, prolong tear film breakup time, reduce ocular surface disease index, alleviate corneal epithelial damage, regulate palpebral fissure opening, and have antioxidant effects. The tear secretion-increasing effect of Bifidobacterium bifidum BB00 directly meets the demand for "products that increase tear secretion," the tear film breakup time-prolonging effect is suitable for "products that prolong tear film breakup time," the ocular surface disease index-reducing effect corresponds to "products that reduce ocular surface disease index," and the corneal epithelial damage-alleviating effect matches "products that alleviate corneal epithelial damage." Furthermore, its antioxidant effects, achieved by scavenging DPPH free radicals and hydroxyl free radicals, can be directly used to prepare "products with antioxidant effects." Simultaneously, the overall effect of this strain in improving ocular surface health can assist in regulating palpebral fissure opening, meeting the demand for "products that regulate palpebral fissure opening."

[0060] The Bifidobacterium bifidum BB00 of this invention can effectively increase tear secretion, prolong tear film breakup time, reduce ocular surface disease index, and alleviate corneal epithelial damage. It also has antioxidant effects and can be used to prepare ophthalmic products for the prevention or treatment of dry eye, conjunctivitis, keratitis, blepharitis, meibomian gland dysfunction, corneal epithelial damage, eyelid scar contracture, eye fatigue, and cataracts. The reason for this is that the efficacy of this strain is highly matched with the core needs of various ophthalmic products and the pathological characteristics of the corresponding eye diseases. The core symptoms of dry eye syndrome, such as reduced tear secretion and shortened tear film breakup time, can be improved by this strain through increasing tear secretion and prolonging tear film breakup time. Problems such as conjunctivitis, keratitis, blepharitis, meibomian gland dysfunction, corneal epithelial damage accompanied by tear film instability, elevated ocular surface disease index, and increased oxidative stress levels can be alleviated by this strain through prolonging tear film breakup time, reducing ocular surface disease index, reducing corneal epithelial damage, and its antioxidant effects. Abnormal eyelid closure due to eyelid scar contracture can be improved by its ability to regulate the degree of eyelid opening. Problems such as reduced tear secretion and increased ocular surface disease index due to eye fatigue can be alleviated by its ability to increase tear secretion and reduce ocular surface disease index. Increased oxidative stress levels in the lens of cataracts can be inhibited by its antioxidant effects, thereby achieving the prevention or treatment of the above-mentioned eye diseases.

[0061] Specifically, the gut microbiota regulating products include any one or more of the following:

[0062] (1) Products that improve gut microbiota beta diversity;

[0063] (2) Products that increase the abundance of ASV in the gut;

[0064] (3) Products that enhance the adhesion and colonization of intestinal epithelial cells;

[0065] (4) Products that inhibit the growth of intestinal pathogens.

[0066] Preferably, the gut microbiota regulating product includes any one or more of the following:

[0067] (1) Products for the prevention or treatment of irritable bowel syndrome;

[0068] (2) Products for the prevention or treatment of functional dyspepsia

[0069] (3) Products for the prevention or treatment of inflammatory bowel disease;

[0070] (4) Products for the prevention or treatment of diarrhea;

[0071] (4) Products for the prevention or treatment of lactose intolerance.

[0072] More preferably, the irritable bowel syndrome is characterized by any one or more of the following: decreased gut microbiota Beta diversity, decreased gut ASV richness, insufficient adhesion and colonization of beneficial bacteria to the intestinal epithelium, and proliferation of intestinal pathogenic bacteria.

[0073] The aforementioned functional dyspepsia is characterized by any one or more of the following: gut microbiota beta diversity disorder, decreased gut ASV abundance, insufficient adhesion and colonization of beneficial bacteria to the intestinal epithelium, and proliferation of intestinal pathogenic bacteria.

[0074] The inflammatory bowel disease described herein is characterized by any one or more of the following: decreased gut microbiota beta diversity, decreased gut ASV abundance, impaired intestinal epithelial cell adhesion and colonization, and proliferation of intestinal pathogenic bacteria.

[0075] The diarrhea described is characterized by any one or more of the following: decreased gut microbiota Beta diversity, decreased gut ASV abundance, impaired intestinal epithelial cell adhesion and colonization, and proliferation of intestinal pathogens.

[0076] The lactose intolerance is caused by any one or more of the following: reduced gut microbiota beta diversity, decreased gut ASV richness, insufficient adhesion and colonization of beneficial bacteria to the intestinal epithelium, and proliferation of intestinal pathogenic bacteria.

[0077] The Bifidobacterium bifidum BB00 of the present invention can be used to prepare products that improve the diversity of intestinal microbes, increase the abundance of intestinal ASV, enhance the adhesion and colonization of intestinal epithelial cells, and inhibit the growth of intestinal pathogenic bacteria as described above. Bifidobacterium bifidum BB00 can reverse the dysbiosis caused by surgery or disease, restoring the gut microbiota beta diversity to a healthy state (e.g., the gut microbiota in the intervention group after FS-LASIK was closer to the pre-operative healthy state, forming a significant spatial separation from the placebo group), thus meeting the demand for "products that improve gut microbiota beta diversity"; it can increase the abundance of intestinal ASVs (the ASV abundance in the intervention group was significantly higher than that in the placebo group), making it suitable for "products that increase the abundance of intestinal ASVs"; its in vitro experiments have demonstrated strong adhesion to intestinal epithelial cells, and it is acid-resistant (survives in pH=2-5 environment) and bile salt-resistant (tolerant to 0.1%-0.3% bile salt environment), allowing it to stably colonize the intestine, meeting the demand for "products that enhance intestinal epithelial cell adhesion and colonization"; its fermentation supernatant has antibacterial activity against 7 kinds of intestinal pathogens, including Escherichia coli (O157, K12), Staphylococcus aureus, and Shigella flexneri (a clear inhibition zone can be seen by the Oxford cup method), and can be directly used to prepare "products that inhibit the growth of intestinal pathogens".

[0078] The *Bifidobacterium bifidum* BB00 of this invention can be used to prepare the intestinal flora regulation products described in the "preferred" description for the prevention or treatment of irritable bowel syndrome (IBS), functional dyspepsia, inflammatory bowel disease (IBD), diarrhea, and lactose intolerance. The intestinal microecological regulation function of this strain is highly consistent with the core objectives of various intestinal flora regulation products and the pathological mechanisms of corresponding intestinal diseases. The core pathological characteristics of IBS, functional dyspepsia, IBD, diarrhea, and lactose intolerance all include reduced intestinal microbial Beta diversity, decreased ASV richness, insufficient colonization capacity of beneficial bacteria, and proliferation of intestinal pathogenic bacteria. *Bifidobacterium bifidum* BB00 can specifically improve these problems by restoring flora diversity, increasing the abundance of beneficial bacteria (such as *Bifidobacterium*), inhibiting the proliferation of pathogenic bacteria, enhancing the adhesion and colonization capacity of intestinal epithelial cells, and repairing the intestinal microecological balance and intestinal barrier function, thereby achieving the prevention or treatment of the aforementioned intestinal diseases.

[0079] In another aspect, the present invention provides an ophthalmic product or a product for regulating intestinal flora, wherein the ophthalmic product or product for regulating intestinal flora contains the above-mentioned Bifidobacterium bifidum or bacterial agent.

[0080] Specifically, the ophthalmic product or gut microbiota regulating product contains at least 1×10 6 Bifidobacterium bifidum CFU / mL

[0081] Preferably, the ophthalmic product or gut microbiota regulating product contains 1×109 -1×10 12 Bifidobacterium bifidum CFU / mL

[0082] More preferably, the ophthalmic product or gut microbiota regulating product contains 1×10 9 -1×10 10 1×10 10 -1×10 11 1×10 11 -1×10 12 Bifidobacterium bifidum CFU / mL

[0083] More preferably, the ophthalmic product or gut microbiota regulating product contains 1×10 9 Bifidobacterium bifidum CFU / mL

[0084] Specifically, the dosage forms of the ophthalmic products or products for regulating gut microbiota include solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

[0085] Specifically, the ophthalmic products or products for regulating gut microbiota also include excipients.

[0086] Preferably, the excipients include any one or more of the following: diluent, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, and buffer.

[0087] The beneficial effects of this invention are as follows:

[0088] The present invention relates to Bifidobacterium bifidum BB00 (accession number CGMCC No. 34389) and its bacterial agents, ophthalmic products, and products for regulating intestinal flora. These products can effectively increase tear secretion, prolong tear film breakup time, reduce ocular surface disease index, and alleviate corneal epithelial damage. Simultaneously, they can improve intestinal microbial beta diversity, increase intestinal ASV richness, enhance intestinal epithelial cell adhesion and colonization ability, and inhibit the growth of intestinal pathogenic bacteria such as Escherichia coli O157 / K12 and Staphylococcus aureus. Furthermore, they are non-hemolytically toxic, have controllable drug resistance, and cause no adverse reactions in humans. They can be used to improve dry eye after FS-LASIK surgery and to prevent and treat eye diseases such as conjunctivitis and keratitis. They can also prevent and treat intestinal diseases such as irritable bowel syndrome and functional dyspepsia. Moreover, they can be formulated into various dosage forms such as solids, semi-solids, and liquids, demonstrating significant industrialization potential.

[0089] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions:

[0090] A method for improving eye health or regulating gut microbiota, the method comprising using any of the above-described Bifidobacterium, probiotics, eye products, or gut microbiota regulating products.

[0091] The technical features include administering an effective amount of Bifidobacterium bifidum, bacterial agents, eye products, or products that regulate gut microbiota to the subject.

[0092] Among them, the technical characteristics of the subjects included mammals.

[0093] Among them, the preferred subjects for technical characteristics are human beings.

[0094] Preservation Instructions

[0095] Preserved strain: Bifidobacterium bifidum BB00;

[0096] Classification and nomenclature: Bifidobacterium bifidum Bifidobacterium bifidum ;

[0097] Accession number: CGMCC No. 34389;

[0098] Preservation period: April 28, 2025;

[0099] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0100] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0101] Figure 1 Evaluation of the probiotic properties of Bifidobacterium bifidum BB00; A in the figure is the growth curve; B is the plateau phase colony count (1 represents a dilution factor of 10). 1 ;2 represents a rare multiple of 10 2 ;3 represents a rare multiple of 10 3 ;4 represents a rare multiple of 10 4 ;5 represents a rare multiple of 10 5 ; 6 represents a rare multiple of 10 6 ;7 represents a rare multiple of 10 7 ;8 represents a rare multiple of 10 8 C represents hemolysis test; D represents cell adhesion test; E represents acid resistance test; F represents bile salt resistance test; G represents antioxidant capacity test; H represents antibacterial capacity test; and I represents drug resistance test.

[0102] Figure 2 The effect of Bifidobacterium bifidum BB00 on a mouse model of refractive intervention; A in the figure represents the animal experimental design; B represents tear secretion; C represents palpebral fissure size; D represents corneal lesion grading; "M" in the figure represents the model group; "P" represents the probiotic group; "There was a significant difference between the representative groups, p < 0.001."

[0103] Figure 3 Flowchart for patient recruitment and randomization.

[0104] Figure 4 Bifidobacterium bifidum BB00 has a beneficial effect on the incidence and symptoms of dry eye after FS-LASIK surgery; Figure A shows a schematic diagram of corneal nerve transection during FS-LASIK surgery; B shows the proportion of dry eye before and after surgery; C shows the ocular surface disease index (OSDI Score); D shows tear secretion (Schirmer I); and E shows tear film breakup time (FBUT). Statistical analysis was performed using the Mann-Whitneyu test, with a significance level of [missing value]. p<0.05; p<0.01; p<0.001.

[0105] Figure 5 Bifidobacterium bifidum BB00 can improve the disordered gut microbiota after refractive surgery; A in the figure is PCoA Beta diversity analysis; B is ASV richness analysis; C is LEfSe analysis.

[0106] Figure 6 Bifidobacterium bifidum BB00 altered the composition of the gut microbiota in patients undergoing laser refractive surgery; Figure A shows the phylum-level fecal microbiota analysis; B shows the changes in actinomycete abundance; C shows the genus-level fecal microbiota analysis; D shows the changes in bifidobacterium abundance; E shows the relative abundance of fecal cocci; F shows the relative abundance of Prevotella; G is a species composition heatmap; "ns" in the figure indicates no significant difference; "There was a significant difference between the representative groups, p < 0.05." "There was a significant difference between the representative groups, p < 0.01." "There was a significant difference between the representative groups, p < 0.001."

[0107] Figure 7 The relationship between horizontal gut microbiota composition and postoperative dry eye symptoms; "There was a significant difference between the representative groups, p < 0.05." "There was a significant difference between the representative groups, p < 0.01." "There was a significant difference between the representative groups, p < 0.001." Detailed Implementation

[0108] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0109] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0110] Example 1

[0111] Bifidobacterium strains were isolated from healthy breast milk samples. The samples were spread onto MRS anaerobic selective medium containing 0.5% cysteine ​​using a serial dilution method and anaerobically cultured at 37°C for 48 hours. Single colonies of Gram-positive bacilli exhibiting a forked "Y" or "V" shape were then picked and preliminarily identified as Bifidobacterium strains. Subsequently, targeted acclimatization was carried out.

[0112] For oxygen tolerance acclimatization, 100 μL of the isolated original strain was spread onto solid MRS medium and cultured at 37°C with an oxygen concentration of 0.1% for 48 h. Strains with larger colonies were selected and cultured again on MRS liquid medium at 37°C for 48 h. This process of spreading, culturing at 1% oxygen and 37°C, and selecting larger colonies was repeated approximately 30 times, with the oxygen concentration gradually increased to 0.5%, 1%, and 3%. The results of oxygen tolerance acclimatization are shown in Table 1. The final strains were then subjected to further gastric juice tolerance acclimatization.

[0113] Table 1

[0114]

[0115] Gastric juice acclimation: After centrifugation, the supernatant of the acclimated oxygen-tolerant strains was discarded. The cells were washed once with PBS buffer and then resuspended in artificial gastric juice (0.1g NaCl, 0.175g pepsin, 50mL water, fully dissolved, pH adjusted to 3 with dilute hydrochloric acid, mixed well, and filtered through a 0.22μm filter membrane under sterile conditions). After standing at 37℃ for 1.5h, 100μL of the bacterial suspension was spread onto solid MRS medium and incubated at 37℃ for 48h. Strains with larger colonies were selected.

[0116] Screening for resistance to intestinal fluid: The obtained gastric fluid-resistant strains were further screened. After activation for three generations, the strains were centrifuged and the supernatant was discarded. The bacterial cells were washed once with PBS buffer and then resuspended in artificial intestinal fluid (0.68 g potassium dihydrogen phosphate, 1 g trypsin, and 100 ml water were mixed and the pH was adjusted to 8. The mixture was filtered through a 0.22 μm filter under sterile conditions). After standing at 37°C for 2 h, 100 μL of the bacterial suspension was spread onto solid MRS medium and incubated at 37°C for 48 h. Strains with larger colonies were selected.

[0117] The results of gastric juice tolerance and intestinal juice tolerance screening are shown in Table 2:

[0118] Table 2

[0119]

[0120] Low-temperature cryopreservation: Superior strains acclimatized to oxygen, gastric juice, and intestinal juice were activated for three generations, frozen at -80℃ for 2 hours, thawed at room temperature, and then frozen again at -80℃, repeated three times. The final thawed bacterial suspension was inoculated at 1% onto MRS liquid medium and cultured at 37℃ with an oxygen concentration of 3% for 24 hours; this constitutes one acclimatization generation. After 30 generations of acclimatization, 100 μL of the bacterial suspension was spread onto solid MRS medium and cultured at 37℃ with an oxygen concentration of 3% for 48 hours. Strains with larger colonies were selected. Bacterial powder was prepared from the original strain and acclimatized strain under the same conditions and stored at 37℃ for one month to test survival rates.

[0121] Table 3

[0122]

[0123] As can be seen from Table 3 above, the survival rate of the strain after 30 generations of domestication is much higher than that of the original strain.

[0124] Finally, through the above domestication and screening, a strain with high oxygen tolerance, high intestinal tolerance, and high freeze resistance was obtained. It was identified as Bifidobacterium bifidum by 16S rDNA sequence and named Bifidobacterium bifidum BB00. It was sent to the China General Microbiological Culture Collection Center for preservation, with the preservation number CGMCC No. 34389.

[0125] Example 2

[0126] This invention provides *Bifidobacterium bifidum* BB00, with accession number CGMCC No. 34389. The following properties were identified in *Bifidobacterium bifidum* BB00.

[0127] 1. Growth curve

[0128] The growth curve was determined using a turbidimetric method. Since the concentration of the bacterial suspension is directly proportional to its turbidity, the concentration of the bacterial suspension can be inferred by measuring the optical density of the bacterial suspension using a spectrophotometer. Bifidobacterium bifidum BB00 strain was activated overnight, and 1 mL (1 x 10⁻⁶) was taken. 9 (CFU / mL) was inoculated into 100 mL of MRS liquid medium and cultured at 37℃ and 150 rpm. The optical density was measured at 600 nm at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. A growth curve was plotted with time on the x-axis and optical density value on the y-axis.

[0129] Figure 1 In the figure, A represents the growth curve of Bifidobacterium bifidum BB00. As the culture time increases, the OD value increases, showing the proliferation pattern of the strain. Figure 1 B in the figure represents the plateau phase colony count, which visually presents the distribution of viable Bifidobacterium bifidum BB00 and reflects its growth stability.

[0130] 2. Hemolytic characteristics determination

[0131] Collect blood from healthy rabbits and place it in an Erlenmeyer flask containing glass beads. Shake for 10 minutes, or stir the blood with a glass rod to remove fibrinogen and obtain defibrinated blood. Add approximately 10 times the volume of 0.9% sodium chloride solution, shake well, and centrifuge at 1000-1500 rpm for 15 minutes. Remove the supernatant. Wash the precipitated red blood cells 2-3 times with 0.9% sodium chloride solution as described above, until the supernatant is no longer red. Prepare a 4% suspension of the obtained red blood cells with 0.9% sodium chloride solution and add it to broth agar medium to prepare blood plates. Inoculate 1 x 102 9 Observe whether hemolysis occurs around the colony after 48 hours of CFU-Bifidobacterium bifidum BB00.

[0132] Figure 1 C in the figure represents the hemolysis test result. There was no hemolysis around the Bifidobacterium bifidum BB00 colony, indicating that Bifidobacterium bifidum BB00 has no hemolytic toxicity and good safety.

[0133] 3. Cell adhesion ability

[0134] Bifidobacterium bifidum BB00 was inoculated into MRS liquid medium and cultured at 37°C in an aerobic incubator until the OD value reached 0.6, at which point the culture was terminated, yielding a bacterial culture solution (1x10⁻⁶). 9 (CFU / mL).

[0135] Wash the intestinal epithelial cell culture plate with a 30% coverage on the coverslip once with sterile PBS buffer. Take 1 mL of the above bacterial culture medium, mix it with 1 mL of intestinal epithelial cell culture medium containing a small amount of intestinal epithelial cells, add it to a six-well plate, and incubate at 37°C in a cell culture incubator.

[0136] After 2 hours of culture, the six-well cell culture plate was removed, the culture medium was aspirated, and the cells were washed repeatedly with PBS buffer 5 times. The cells were then fixed with methanol, Gram stained, and observed under an oil immersion microscope.

[0137] Figure 1 D in the figure represents cell adhesion ability. Adhesion can be observed under Gram staining oil immersion microscope, reflecting the adhesion ability of Bifidobacterium bifidum BB00 to intestinal epithelial cells, which is beneficial for colonization and function.

[0138] 4. Acid resistance test

[0139] Bifidobacterium bifidum BB00 was cultured overnight in MRS liquid medium at 37°C in an aerobic incubator. 100 µL of the culture was then diluted with PBS buffer. 1 10 3 10 5 Centrifuge at 4000 rpm for 3 min and discard the supernatant. Add PBS buffer (pH 2, 3, 4, 5, 7), incubate for 4 h, mix well, and spread 10 μL onto a plate. Incubate in an anaerobic incubator at 37 °C for 36 h and count the viable bacteria.

[0140] Figure 1 E in the figure represents the results of the acid resistance test, which shows that Bifidobacterium bifidum BB00 is adaptable to acidic environments.

[0141] 5. Bile salt tolerance test

[0142] Bifidobacterium bifidum BB00 was cultured in MRS liquid medium containing 0.1%–0.3% bovine bile salt (without bile salt, with 0.1% bile salt, with 0.2% bile salt, or with 0.3% bile salt) at 37°C for 12 hours. After mixing, 10 μL was plated and cultured at 37°C for 36 hours. Viable bacteria were counted, and experimental records were made.

[0143] Figure 1 F in the figure represents the bile salt tolerance test. The results show that Bifidobacterium bifidum BB00 has a good tolerance to the intestinal bile salt environment.

[0144] 6. Determination of antioxidant properties

[0145] Bifidobacterium bifidum BB00 was cultured in MRS liquid medium at a concentration of 1 × 10⁻⁶. 9 Centrifuge CFU of Bifidobacterium bifidum BB00 at 10000 rpm for 10 min to precipitate the bacterial cells, collect the supernatant and store it in a refrigerator for later use.

[0146] 6.1 Determination of DPPH free radical scavenging ability

[0147] Take 2 mL of bacterial culture supernatant, add 2 mL of 0.2 mmol / L DPPH methanol solution and 2 mL of deionized water, react in the dark at room temperature for 30 min, take the supernatant and measure the OD value at 517 nm:

[0148] A blank = 2 mL deionized water + 2 mL DPPH methanol solution;

[0149] A1 = 2 mL bacterial culture supernatant + 2 mL DPPH methanol solution;

[0150] A2 = 2 mL bacterial culture supernatant + 2 mL deionized water.

[0151] DPPH free radical scavenging rate = [Aempt - (A1 - A2) / Aempt] × 100%.

[0152] 6.2 Determination of hydroxyl radical scavenging ability

[0153] In a test tube, add 1 mL of 2 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2, and 1 mL of bacterial culture supernatant sequentially. Let stand for 10 min, then add 2 mL of 6 mmol / L salicylic acid. Let stand at room temperature for 30 min, then measure the absorbance at 510 nm and calculate the hydroxyl radical scavenging rate.

[0154] A blank = blank solvent (1 mL FeSO4 solution, 1 mL H2O2) + 2 mL salicylic acid;

[0155] A1 = 1 mL bacterial supernatant + 2 mL salicylic acid;

[0156] A2 = 1 mL bacterial supernatant + blank solvent (1 mL FeSO4 solution, 1 mL H2O2).

[0157] Hydroxyl radical scavenging rate = [Aempt - (A1 - A2) / Aempt] × 100%.

[0158] 6.3 Determination of superoxide radical scavenging capacity

[0159] Add 2 mL of 150 mmol / L Tris-HCl solution (pH=8.0) and 1 mL of 1.2 mmol / L pyrogallol solution to 0.5 mL of bacterial culture medium, shake quickly, and measure the absorbance at 330 nm. Measure the absorbance every 30 s until 150 s, and calculate the rate.

[0160] A0 = The self-oxidation rate is obtained by taking the slope of the straight line fitted with the absorbance value of pyrogallol every 30s.

[0161] A1 = is the oxidation rate obtained by taking the slope of the straight line fitted with the absorbance value of the mixture of pyrogallol and bacterial supernatant every 30 seconds.

[0162] Superoxide radical scavenging rate = (A0-A1) / A0×100%.

[0163] 6.4 Fe 2+ Determination of chelating ability

[0164] Add 0.1 mL of 0.4% ferrous sulfate solution to 0.5 mL of supernatant, gently invert and mix thoroughly. Then add 0.1 mL of 1% vitamin C and 1 mL of 0.2 mol / L NaOH solution. React at room temperature for 20 min. Then add 1 mL of 10% trichloroacetic acid, centrifuge at 6000 rpm for 10 min at 4°C to remove protein. Take 0.4 mL of the above solution and add 4 mL of 0.1% o-phenanthroline. React at room temperature for 10 min and measure the absorbance at 536 nm.

[0165] A0 = the absorbance after replacing the bacterial culture medium in the reaction system with 0.5 mL of deionized water;

[0166] A1 = Absorbance of 0.5 mL of sample solution after reaction;

[0167] A2 = absorbance after replacing the ferrous sulfate solution in the reaction system with 0.1 mL of deionized water.

[0168] Fe 2+ Chelating capacity = [A0-(A1-A2)] / A0×100%.

[0169] 6.5 Determination of Total Reducing Power

[0170] Take 1 mL of the supernatant, add 1 mL of phosphate buffer (pH 6.6) and 1 mL of 1% K3[Fe(CN)6] solution, mix thoroughly by inverting, and incubate at 50℃ for 2 min. Add 1 mL of 10% trichloroacetic acid solution, shake to mix, take 1 mL of the mixture, add 4 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution, and let stand for 10 min. Then, using deionized water as a blank, measure the absorbance at 700 nm. The higher the absorbance value, the stronger the reducing power of the analyte.

[0171] A1 = Supernatant + Phosphate buffer + K3[Fe(CN)6] + Trichloroacetic acid solution + Deionized water + FeCl3 solution;

[0172] A0 = 1 mL deionized water phosphate buffer + K3[Fe(CN)6] + trichloroacetic acid solution + deionized water + FeCl3 solution;

[0173] A2 = Supernatant + Phosphate Buffer + Deionized Water.

[0174] Total reducing power = [A0-(A1-A2)] / A0×100%.

[0175] 6.6 Experimental Results

[0176] Figure 1 The G in the figure represents the antioxidant capacity of Bifidobacterium bifidum BB00. Bifidobacterium bifidum BB00 can scavenge various free radicals and chelate metal ions, thus possessing antioxidant activity.

[0177] 7. Experiment on ability to inhibit pathogenic bacteria

[0178] Bifidobacterium bifidum BB00 was inoculated into MRS liquid medium and cultured at 37°C in an aerobic incubator for 36 hours. After centrifugation at 6000 rpm for 10 minutes, the supernatant was collected, which is the bacterial culture supernatant.

[0179] 1x10 9 CFU of *Escherichia coli* O157, *Escherichia coli* K12, *Staphylococcus aureus*, *Shigella flexneri*, *Staphylococcus epidermidis*, *Clostridium perfringens*, or *Enterococcus faecalis* were plated separately on LB agar. Oxford cups were gently placed on the plates, and 250 μL of bacterial culture supernatant was aspirated into each cup (two replicates per group). The plates were incubated at 37°C, and the size of the inhibition zone was observed every 2 hours. The diameter of the inhibition zone was measured after 8 hours.

[0180] Figure 1 H in the figure represents the antibacterial ability of Bifidobacterium bifidum BB00. The results showed that Bifidobacterium bifidum BB00 had a good inhibitory effect on Escherichia coli O157, Escherichia coli K12, Staphylococcus aureus, Shigella flexneri, Staphylococcus epidermidis, Clostridium perfringens, and Enterococcus faecalis.

[0181] 8. Evaluation of drug resistance

[0182] Fresh colonies of BB00 that showed good growth on the culture medium were picked and placed into a culture medium containing LSM-L (2.11 g of ISO-Sensitest broth, 0.552 g of MRS broth, 0.03 g of L-cysteine ​​hydrochloride, 50 mL of water, stirred and mixed, then adjusted to pH 6.8-7.0, and autoclaved at 121℃ for 15 min) to prepare a bacterial suspension. The absorbance (OD value) was measured at 620 nm using a microplate reader, ensuring the OD value ranged from 0.085 to 0.095. The suspension was then diluted 500 times with LSM-L solution or concentrated MRS broth and added sequentially to microdilution plates for antimicrobial agents, 50 μL per well. The plates were incubated anaerobically at 37℃ for 48 h, and the results were observed. Antimicrobial resistance susceptibility was determined according to the European Food Safety Authority's "EFSA 5206-2018 Guidelines on the Microbiological Characteristics of Feed Additives or Fermented Products 2.2.1 Antimicrobial Susceptibility".

[0183] 9. Pathogenicity evaluation

[0184] The pathogenicity test method of food-grade bacteria in Appendix A of GB 31615.2-2025 "National Food Safety Standard - Procedure for Safety Evaluation of Food-grade Microbial Strains" was used to test Bifidobacterium bifidum BB00. The test animals showed no abnormalities or deaths, and their body weight was not statistically significant compared with the control group (p>0.05), indicating that the strain was non-pathogenic.

[0185] Example 3

[0186] 1. Laboratory animals and their care

[0187] Sixteen 6-8 week old female BALB / c mice were housed for one week in a specific pathogen-free (SPF) laboratory animal center for adaptation. Eight mice were kept in each cage under cyclical light and dark conditions, with free access to food and water. The mice were given a 7-day acclimatization period before the experiment. Temperature, humidity, and noise levels in the animal room were controlled within specified ranges, and the room was cleaned regularly each day.

[0188] 2. Collection of gut microbiota from patients with postoperative dry eye

[0189] Patients who had previously undergone femtosecond laser-assisted in situ keratomileusis (FS-LASIK) at the Ophthalmology Refractive Surgery Center of the Second Affiliated Hospital of Nanchang University were selected. One month post-surgery, patients were grouped according to their postoperative feelings and clinical manifestations, and stool samples were collected for examination. Inclusion criteria were required, and exclusion criteria were not met.

[0190] Inclusion criteria:

[0191] (1) One month or more after FS-LASIK surgery, with regular medication and follow-up visits after surgery.

[0192] (2) Age 18-45 years old, gender not limited.

[0193] (3) The patient and their family members obtain informed consent and sign the informed consent form.

[0194] Note: Students who meet all three of the above criteria will be included in this study.

[0195] Exclusion criteria:

[0196] (1) Postoperative medication did not reach the corresponding course of treatment.

[0197] (2) Has a history of eye disease or trauma.

[0198] (3) Those with serious organ diseases.

[0199] (4) Individuals with immune system deficiencies.

[0200] (5) Do not agree to participate in this study and sign the informed consent form.

[0201] Note: If any of the above criteria are met, the application will be excluded.

[0202] After the informed consent form is signed, each participant in the clinical study and their subsequent biological samples are assigned a corresponding anonymous participant identification number. These numbers and the participants' names are matched in a securely stored linked file, which records the participants' general information and relevant clinical characteristics, including name, age, gender, family contact information, postoperative complications, etc.

[0203] A total of 50 patients with dry eye were included. The criteria were: patients who underwent a follow-up examination one month after FS-LASIK surgery, and who were found to have dry eye symptoms and signs by patient reports and slit-lamp examination, and who had no other postoperative complications (epithelial ingrowth, interlaminar opacity, and surgical site fibrosis, etc.).

[0204] Fecal samples were collected from the patients. Half of the samples were aliquoted into 1.5 mL EP tubes containing glycerol for subsequent fecal microbiota transplantation, and the other half were aliquoted into EP tubes containing cysteine ​​and stored at -80°C for microbial sequencing.

[0205] 3. Animal modeling and processing

[0206] 3.1 Nerve-transection dry eye mouse model

[0207] All surgeries were performed under an operating microscope. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. To simulate the transection of corneal nerves in FS-LASIK, a 2.5mm diameter circular punch was used to sever the stromal nerves of the cornea. The punch was applied to the corneal surface and then twisted five times with slight pressure until the stroma was cut. After treatment, tobramycin eye drops were instilled onto the ocular surface. Both eyes were treated simultaneously.

[0208] 3.2 Grouping and Processing

[0209] All animals underwent a series of treatments: 1 week of acclimatization, 1 week of antibiotic gavage to eliminate the original gut microbiota (Abx antibiotic cocktail mixed in drinking water), and 1 week of transplantation of gut microbiota from postoperative dry eye patients (10g of feces was filtered, and the bacterial suspension was dissolved in 10mL of gelatin saline to prepare a 200μL suspension, which was administered by gavage three times a week). Following this, the animals were divided into groups.

[0210] Model group (M group, n=8): A mouse model of dry eye with nerve transection was established. The mice were then given 200 μL of gelatin saline by gavage three times a week for 2 weeks.

[0211] Probiotic group (P group, n=8): A mouse model of dry eye with nerve transection was established, and the mice were then administered 200 μL of Bifidobacterium bifidum BB00 by gavage at a concentration of 1×10⁻⁶. 9 CFU / mL, three times a week for two weeks. See the experimental procedure below. Figure 2 A in the middle.

[0212] 3.3 Specimen processing

[0213] Two weeks after intervention, mice were euthanized by CO2 inhalation. The eyeballs were removed and photographed under a microscope. The corneal and lacrimal gland tissues were then completely extracted for subsequent experiments.

[0214] 4. Observation Indicators

[0215] 4.1 Corneal fluorescein staining

[0216] Used for quantification of corneal epithelial damage. Corneal epithelial damage was assessed using sodium fluorescein dye (3% lysine green B; Sigma-Aldrich). Mice were anesthetized, and one drop of sodium fluorescein dye was applied to the cornea. After the mice blinked three times, excess droplets were absorbed by the soft tissue. Photographs were taken using cobalt blue light under a slit-lamp microscope. An observer scored the cornea according to observations from 0 to 3 (0 = no fluorescence, 1 = fluorescence resembling sparse dots, 2 = dense dotted pattern, 3 = very dense dotted fluorescence), with the mean score taken from both eyes. This assessment was performed one day before surgery and two weeks post-surgery.

[0217] 4.2 Degree of palpebral fissure opening

[0218] The metric used to measure dry eye was the ratio of eyelid closure (palpebral opening), which consisted of the height of the gap between the upper and lower eyelids and the distance between the two eyelids. Mice were placed on an elevated platform and allowed to acclimate for 2 minutes. A camera was mounted at the same height as the elevated platform, and the mice were recorded for 5 minutes after acclimatization. Snapshots taken during video playback showed the mouse's eyes perpendicular to the camera. Measurements were performed using ImageJ software to determine the distance (y) between the upper and lower eyelids and the distance (x) between the inner eyelids. Based on these values, the y / x ratio was calculated to determine the degree of palpebral fissure opening, with the mean value taken for both eyes. This assessment was performed one day before surgery and two weeks post-surgery.

[0219] 4.3 Tear secretion

[0220] Tear secretion in mice was measured using the phenol red cotton thread test. Under non-anesthesia conditions, mice were weighed, and a phenol red-soaked cotton thread (Tianjin Jingming New Technology Development Co., Ltd., Tianjin, China) was placed below the outer canthus of the mouse for 15 seconds. The length of the wet thread (in millimeters) was measured using calipers to determine tear secretion (accurate to 0.1 mm), with the average value taken from both eyes. This assessment was performed one day before surgery and two weeks post-surgery.

[0221] 5. Experimental Results

[0222] tear secretion as Figure 2 As shown in B, the size of the palpebral fissure is as follows: Figure 2 As shown in C, the corneal punctate grading is as follows: Figure 2 As shown in D in the figure. The results showed that, compared with the model group (group M), mice in the probiotic group (group P) had significantly reduced tear secretion, increased palpebral fissure opening, and decreased corneal punctation grade after treatment with Bifidobacterium bifidum BB00. These results indicate that Bifidobacterium bifidum BB00 can improve dry eye-related symptoms in a mouse model of nerve transection-induced dry eye, suggesting that Bifidobacterium bifidum BB00 has a certain alleviating effect on nerve transection-induced dry eye.

[0223] Example 4

[0224] 1. Research Subjects

[0225] In this embodiment, all cases were patients who were scheduled to undergo femtosecond laser-assisted lenticule extraction (FS-LASIK) at the Ophthalmology Refractive Surgery Center of the Second Affiliated Hospital of Nanchang University between September 2023 and April 2024 and met the inclusion and exclusion criteria of this study. The surgical indications, contraindications, and procedures for FS-LASIK were strictly in accordance with the "Expert Consensus on the Standardized Procedures for Femtosecond Laser Small Incision Lens Removal Surgery in my country (2018)" issued by the Ophthalmology and Optometry Group of the Chinese Medical Association.

[0226] 1.1 Inclusion criteria:

[0227] (1) Meets the indications for FS-LASIK surgery, with a refractive error (D) not exceeding 10.00 spherical myopia, astigmatism not exceeding 5.00D, significant refractive stability in the past 2 years (the spherical or cylindrical refractive error increases by less than 0.50D per year), and intraocular pressure ≤21mmHg.

[0228] (2) Age 18-45 years old, gender not limited.

[0229] (3) During the treatment period, patients can actively cooperate with routine surgical medication and probiotic treatment according to the treatment course.

[0230] (4) The patient and their family members obtain informed consent and sign the informed consent form.

[0231] Note: All four criteria must be met for inclusion in this study.

[0232] 1.2 Exclusion criteria:

[0233] (1) Meets the absolute and relative contraindications for FS-LASIK surgery, including severe dry eye, significant corneal scarring, corneal ectasia, residual stromal thickness <250μm after laser ablation, active ocular or systemic diseases, glaucoma or retinal diseases, severe systemic diseases, history of ocular surgery, pregnancy or lactation.

[0234] (2) Those with serious organ diseases.

[0235] (3) Individuals with immune system deficiencies.

[0236] (4) Individuals allergic to the drugs used in this study.

[0237] (5) Patients who resist related research work and have poor compliance.

[0238] Note: If any of the above criteria are met, the application will be excluded.

[0239] 1.3 Exclusion Criteria:

[0240] (1) Patients who were enrolled but did not ultimately choose to undergo FS-LASIK surgery.

[0241] (2) Those who are unwilling to cooperate with postoperative examinations and probiotic treatment during the trial period.

[0242] (3) Those who have physical problems or abnormal vital signs due to special reasons during treatment.

[0243] (4) There are other factors that affect the safety assessment or efficacy.

[0244] (5) The subject requests to withdraw from the clinical trial for personal reasons.

[0245] 1.4 Shedding Criteria:

[0246] (1) Those who were enrolled in this study and received at least one treatment as specified in this study, but did not complete the entire study of treatment and withdrew from treatment during the trial.

[0247] (2) Lost to follow-up.

[0248] 1.5 Termination Criteria:

[0249] (1) If a serious adverse reaction or other unexpected event occurs during the treatment period of this study, the patient should not continue to receive the treatment of this study;

[0250] (2) Serious complications occurred during the study, accompanied by related diseases.

[0251] 1.6 Management of Dropout and Discontinued Cases

[0252] (1) If a subject drops out, the observing physician should contact the subject as soon as possible to inquire about the reason, record the treatment time and details, and complete all assessment items of this study.

[0253] (2) Fill in the “Clinical Case Completion Status” in the Clinical Case Report Form in the Appendix.

[0254] (3) If a participant withdraws from the study due to adverse reactions, the “Adverse Reactions” section of the Clinical Observation Form should be completed.

[0255] (4) For cases that are withdrawn due to adverse reactions or other reasons, the observing physician shall take appropriate treatment measures according to the actual situation.

[0256] (5) If a patient withdraws from the study midway through the clinical observation period, but has completed 1 / 2 of the treatment course, the patient should be included in the final efficacy statistics.

[0257] 2. Research Content

[0258] In this embodiment, a total of 100 eligible patients were randomly assigned to either the probiotic intervention group (PB group, n=50) or the placebo intervention group (PC group, n=50). The final analysis included 44 participants in the PB group (6 were excluded due to failure to return for follow-up or concurrent use of medication / probiotics) and 45 participants in the PC group (5 were excluded for the same reason), see [link to relevant documentation]. Figure 3 During the 90-day follow-up period post-surgery, no subjects reported adverse gastrointestinal symptoms or ocular complications. Baseline demographic and clinical characteristics of the patients showed similarities between the two groups in terms of age. gender corneal morphology There were no significant differences in refractive error measurement, surgical parameters, and dry eye symptoms (all p>0.05), supporting the data for further comparative analysis.

[0259] 3. Bifidobacterium bifidum BB00 can reduce the incidence and severity of postoperative dry eye in FS-LASIK patients.

[0260] In FS-LASIK surgery, the formation of the corneal flap damages a significant portion of the corneal nerves, leading to a high incidence of postoperative dry eye syndrome. Figure 4 (A) No dry eye occurred in either group preoperatively. In the PB group, the incidence of dry eye was 77.27% (34 / 44) at 7, 30, and 90 days postoperatively. The incidence of dry eye in the PC group was 20.45% (9 / 44) and 15.90% (7 / 44). The incidence of dry eye in the PC group was 75.56% (34 / 45), 40.00% (18 / 45), and 31.11% (14 / 45). At 30 days post-surgery, the incidence of dry eye in the PB group was significantly lower than that in the PC group (20.45% vs 40.00%, p = 0.045). Figure 4 (B in the text). At 30 days post-surgery, compared with the PC group, treatment with Bifidobacterium bifidum BB00 significantly improved the postoperative OSDI score (5.21 vs 14.58, P = 0.029). Figure 4 (C) Tear film breakup time (FBUT) was 8.97 vs 5.98, P < 0.001. Figure 4 D), Schirmer value in tear secretion test (11.82 vs 9.31, P = 0.003); Figure 4 (E in the text).

[0261] 4. Bifidobacterium bifidum BB00 can improve the disordered intestinal flora in patients undergoing refractive surgery.

[0262] The floristic composition of feces from patients in the PB and PC groups and the pre-operative (BI) group was analyzed using 16S rDNA amplification and sequencing to explore the role of probiotics from a microbiological perspective. Beta diversity analysis based on Bray-Curtisdissimilarity principal coordinate analysis (PCoA) showed partial overlap among the three experimental cohorts, with the PB and BI groups being more similar, while the PC group exhibited significant spatial separation. Figure 5 (A in the original text). Furthermore, after rigorous quality screening, 3931 high-confidence ASVs were identified, of which 581 core ASVs were shared across all groups. The hierarchical distribution of ASV richness followed the order PB group > BI group > PC group (…). Figure 5 (B in the text). Furthermore, LEfSe further identified differentially enriched groups (LDA score > 2.0) in different cohorts. Figure 5The C value suggests that Bifidobacterium affects the structure and diversity of the gut microbiota in patients. Supplementation with Bifidobacterium can restore postoperative gut microbiota imbalance in patients undergoing laser refractive surgery.

[0263] 5. Bifidobacterium bifidum BB00 alters the composition of the gut microbiota in patients undergoing laser refractive surgery.

[0264] At the phylum level, the main characteristic of the fecal microbiota is Firmicutes (…). Firmicutes ) Phylum Actinobacteria Actinobacteria ) Proteobacteria ( Proteobacteria ) and Bacteroidetes ( Bacteroidetes () Figure 6 (A) Among them, the changes in actinomycetes were the most significant, with the actinomycetes in the PB group being significantly higher than those in the PC group (p<0.001). Figure 6 (B in the original text). Further analysis at the genus level revealed the top 15 gut microbiota with the highest relative abundance. Fecal microbiota are mainly composed of... Blautia, Faecalibacterium Bifidobacterium and Coprococcus composition( Figure 6 (C) The abundance of Bifidobacteria in the PC group without BB00 intervention was significantly lower than that before surgery (p=0.006), suggesting that perioperative management reduced the abundance of intestinal Bifidobacteria, while BB00 intervention significantly reversed this downward trend (p<0.001). Figure 6 (D in the text). The relative abundance of fecal cocci in group PB increased slightly, but this change did not reach statistical significance (p>0.05). Figure 6 Similarly, compared with BB00 intervention, Prevotella associated with inflammatory diseases was significantly increased after surgery (p = 0.008). Figure 6 The species composition heatmap further suggests that perioperative interventions may disrupt the patient's gut microbiota, and BB00 supplementation appears to restore this imbalance (F in the figure). Figure 6 (G in the text). In summary, these findings suggest that surgical interventions and perioperative pharmacological treatments may disrupt gut microbiota diversity, and B. bifidum The supplementary components of BB00 restored the ecological balance.

[0265] 6. Correlation analysis between postoperative dry eye symptoms and gut microbiota

[0266] Spearman correlation analysis was used to elucidate the relationship between genus-level gut microbiota composition and postoperative dry eye symptoms. Figure 7Bifidobacteria were negatively correlated with OSDI (p<0.05) and positively correlated with Schirmer secretion (p<0.001). Furthermore, *Codonopsis* had the potential to promote Schirmer secretion (p<0.05). Conversely, the abundance of *Femtobacter* and *Prevotella* was negatively correlated with tear secretion (p<0.05). Therefore, these data suggest that postoperative gastrointestinal symptoms in patients are related to gut microbiota dysbiosis.

[0267] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A type of Bifidobacterium ( Bifidobacterium bifidum ), characterized in that, The aforementioned Bifidobacterium bifidum is Bifidobacterium bifidum BB00, with accession number CGMCC No. 34389.

2. A microbial agent, characterized in that, The bacterial agent comprises the Bifidobacterium bifidum as described in claim 1.

3. The application of the Bifidobacterium bifidum of claim 1 or the bacterial agent of claim 2 in the preparation of the product, characterized in that, The products mentioned include any one or more of the following: (1) Medications for the prevention or treatment of dry eye syndrome; (2) Medications for the prevention or treatment of corneal epithelial damage; (3) Health products or medicines that relieve visual fatigue, wherein the visual fatigue is manifested as a decrease in tear secretion.

4. The application according to claim 3, characterized in that, The product described includes any one or more of the following functions: (1) Increase tear secretion; (2) Prolonging tear film breakup time; (3) Reduce the ocular surface disease index; (4) Reduce corneal epithelial damage; (5) Adjust the degree of palpebral fissure opening.

5. The use of the Bifidobacterium bifidum of claim 1 or the bacterial agent of claim 2 in the preparation of health products or pharmaceuticals that help regulate intestinal flora.

6. The application according to claim 5, characterized in that, The health products or medicines that help regulate gut microbiota include any one or more of the following effects: (1) Improve gut microbiota beta diversity; (2) Increase the abundance of ASV in the gut; (3) Enhances intestinal epithelial cell adhesion and colonization; (4) Inhibit the growth of intestinal pathogens.

7. A product characterized in that, The product comprises the Bifidobacterium bifidum of claim 1 or the bacterial agent of claim 2, and the product includes any one or more of the following: (1) Medications for the prevention or treatment of dry eye syndrome; (2) Medications for the prevention or treatment of corneal epithelial damage; (3) Health products or medicines that relieve visual fatigue, wherein the visual fatigue is manifested as a decrease in tear secretion.

8. A health product or medicine that helps regulate intestinal flora, characterized in that, The aforementioned health products or medicines that help regulate gut microbiota It contains the Bifidobacterium bifidum as described in claim 1 or the bacterial agent as described in claim 2.

9. The product according to claim 7 or the health product or medicine that helps regulate intestinal flora according to claim 8, characterized in that, The product or health supplement or medicine that helps regulate gut microbiota contains at least 1×10 6 CFU contains Bifidobacterium bifidum.

10. The product according to claim 7 or the health product or medicine that helps regulate intestinal flora according to claim 8, characterized in that, The dosage forms of the products or health products or medicines that help regulate gut microbiota include solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

11. The product according to claim 7 or the health product or medicine that helps regulate intestinal flora according to claim 8, characterized in that, The product or health product or medicine that helps regulate intestinal flora also includes excipients, which include any one or more of the following: diluents, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, and buffers.

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