Bifidobacterium longum subsp.infantis capable of improving physiological activity of vitamin A and application thereof

Abstract

The invention discloses application of bifidobacterium longum subsp.infantis capable of improving physiological activity of vitamin A in xerophthalmia, and belongs to the field of microorganisms. According to the bifidobacterium longum subsp. Infantis CCFM1426 disclosed by the invention, vitamin A can be subjected to external metabolism to generate retinoic acid; the infiltration of ulcerative colitis in mice is relieved; the RA level of the mouse colon with ulcerative colitis is improved; the levels of IFABP and DAO in serum of mice with ulcerative colitis are reduced; corneal injury and conjunctival goblet cell injury of mice with xerophthalmia are relieved; the RA level in serum of the dry-eye mouse is improved; the levels of IL-6 and IL-17 in serum of mice with xerophthalmia are reduced. Therefore, the bifidobacterium longum subsp. Infantis CCFM1426 has a huge application prospect in preparation of products for preventing and / or relieving xerophthalmia.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium longum subsp. infantis that can enhance the physiological activity of vitamin A and its applications, belonging to the field of microbiology. Background Technology

[0002] Vitamin A is a fat-soluble vitamin that the human body cannot synthesize on its own and must obtain through diet or supplements. It has various physiological functions, including maintaining vision, participating in tissue growth and cell differentiation, maintaining epithelial integrity, and regulating immune responses. Retinoic acid is the physiologically active form of vitamin A and plays an important role in maintaining the integrity of the epithelial barrier and regulating mucosal immunity.

[0003] Ulcerative colitis is a chronic inflammatory disease of the gastrointestinal system, one of the main forms of inflammatory bowel disease, primarily affecting the colon and rectum. Ulcerative colitis severely damages the intestinal barrier, intestinal immunity, and gut microbiota, affecting the absorption of nutrients and easily inducing vitamin deficiencies. Consequently, patients with ulcerative colitis often face vitamin A deficiency and restricted retinoic acid signal transduction, leading to the ineffective absorption and conversion of vitamin A into retinoic acid, triggering a vicious cycle. Dry eye syndrome is a common ocular surface disease, primarily affecting the cornea and conjunctiva. Dry eye patients have a reduced number of conjunctival goblet cells, resulting in decreased retinoic acid secretion and making the ocular epithelium more susceptible to damage. Furthermore, serum vitamin A deficiency is often closely linked to dry eye syndrome. Therefore, the need for vitamin A supplementation and enhancement of its physiological activity in the treatment of intestinal and ocular surface diseases is becoming increasingly urgent.

[0004] In recent years, with the deepening research on "gut microbiota," the correlation between gut microbiota and intestinal and eye diseases has become self-evident. Influenced by various environmental factors such as urbanization, antibiotic intake, and gastrointestinal infections, and through long-term accumulation, the balance of the gut microbiota has been disrupted, affecting the absorption of nutrients and inducing or promoting intestinal inflammation, leading to damage to the epithelial barrier of the intestine and eye. Conversely, gut microbiota and its related metabolites may play a crucial role in the body's resistance to eye and intestinal diseases and in maintaining the integrity of the epithelial barrier. In particular, the ability of gut symbiotic bacteria to metabolize vitamin A independently of the body has been confirmed in recent years. Imbalance in the gut microbiota and reduced physiological activity of vitamin A in the body are considered to have profound effects on the body. Regulating the gut microbiota with probiotics is also considered an effective approach.

[0005] Meanwhile, current methods of vitamin A supplementation for patients with enterophthalmic diseases primarily focus on the dosage of vitamin A and its combination with other nutrients, lacking consideration of vitamin A's physiological activity in the body and its conversion to retinoic acid. Therefore, there are limitations, such as drug dependence, adverse drug reactions, poor efficacy of vitamin A supplementation, and adverse reactions from excessive or prolonged vitamin A supplementation. Furthermore, current synthesis methods using retinol or retinal as raw materials through oxidation and isomerization are complex, prone to decomposition, costly, and easily generate byproducts, leading to environmental pollution.

[0006] Currently, the main challenge facing the technology is to find a probiotic that can effectively enhance the physiological activity of vitamin A in the body, that is, increase the level of retinoic acid in serum or tissues, thereby maintaining the integrity of the epithelial barrier, regulating the body's immunity, and promoting the recovery of patients' eye and intestinal functions.

[0007] In summary, there is an urgent need to screen probiotics that can enhance the physiological activity of vitamin A and increase retinoic acid levels, in order to regulate the body's immune response and alleviate the pathological characteristics of ulcerative colitis and dry eye in mice, thus providing a more effective and lasting treatment for the prevention and relief of these conditions. This approach aims to overcome the shortcomings of traditional treatments and provide an innovative and feasible therapeutic pathway, offering new insights for the effective prevention of ulcerative colitis, dry eye, or diseases related to vitamin A metabolism. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a *Bifidobacterium longum* subsp. *infantii* strain capable of enhancing vitamin A physiological activity and alleviating symptoms of ulcerative colitis and dry eye, along with its applications. The aim is to solve the current problems where vitamin A supplementation focuses primarily on dosage and supplementation combinations, neglecting consideration of enhancing vitamin A physiological activity. Furthermore, the chemical synthesis of all-trans retinoic acid is complex, easily decomposed, leading to high costs, environmental pollution, and uncertain safety. This invention involves screening strains to enhance vitamin A physiological activity. The method involves assembling the protein sequences of retinol dehydrogenase and retinal dehydrogenase from humans, mice, and *Bacillus cereus*, *Bifidobacterium bifidum*, and segmented filamentous bacteria with potential vitamin A metabolic capacity into target alignment sequences. Based on comparative genomics, the target alignment sequences are compared with the gene draft information of strains in the Jiangnan University strain bank to obtain strains with potential vitamin A metabolic capacity. In vitro vitamin A metabolism experiments are then conducted to verify the strains' capabilities, and further screening is performed to obtain potential target strains. Ultimately, based on animal experiments to verify the efficacy of potential target strains, strains that enhance the physiological activity of vitamin A were screened.

[0009] The first technical solution provided by this invention is a strain of Bifidobacterium longum subsp. infantis CCFM1426, which was deposited on September 20, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:65152, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The *Bifidobacterium longum* subsp. *infantis* CCFM1426 was obtained from the strain bank of Jiangnan University. Sequencing analysis of this strain revealed its 16S rRNA sequence as shown in SEQ ID NO:1. Alignment of this sequence with GenBank confirmed that the strain is *Bifidobacterium longum* subsp. *infantis*, and it was named *Bifidobacterium longum* subsp. *infantis* CCFM1426.

[0011] This invention provides a microbial preparation containing the Bifidobacterium longum subsp. infantis CCFM1426 described in the first technical solution.

[0012] In some embodiments, the microbial preparation contains wet cells or frozen stem cells of the Bifidobacterium longum infant subsp. CCFM1426.

[0013] In some embodiments, the amount of *Bifidobacterium longum* subsp. infantis CCFM1426 added to the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0014] Furthermore, in the microbial preparation, the amount of Bifidobacterium longum subsp. infantis CCFM1426 added is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0015] In some embodiments, the preparation method of the microbial preparation is as follows: Bifidobacterium longum subsp. infantis CCFM1426 is inoculated into the culture medium at an inoculation rate of 4%, and cultured at 37°C for 30 h to obtain a culture solution; the culture solution is centrifuged and the bacterial cells are collected; the bacterial cells are washed three times with phosphate buffer at pH 7.2 and then resuspended with a lyophilization protectant to obtain a resuspended solution; the resuspended solution is lyophilized using a vacuum freeze-drying method to obtain the fermentation agent of Bifidobacterium longum subsp. infantis CCFM1426.

[0016] In some embodiments, the freeze-drying protectant is one or more of skim milk powder, glycerin, trehalose, maltodextrin, and monosodium glutamate.

[0017] The third technical solution provided by the present invention is a drug comprising the Bifidobacterium longum subsp. infantis CCFM1426 described in the first technical solution or the microbial preparation described in the second technical solution.

[0018] In some embodiments, the viable count of *Bifidobacterium longum* subsp. infantis CCFM1426 in the drug is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0019] Furthermore, the viable count of Bifidobacterium longum subsp. infantis CCFM1426 in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0020] In some embodiments, the pharmaceutical product also contains a drug carrier and / or pharmaceutical excipients.

[0021] In some embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0022] In some embodiments, the pharmaceutical excipient includes excipients and additives.

[0023] In some embodiments, the pharmaceutical excipient comprises one or more of the following: stabilizer, flavoring agent, coloring agent, filler, binder, wetting agent, disintegrant, and lubricant.

[0024] In some embodiments, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0025] The fourth technical solution provided by this invention is the application of Bifidobacterium longum subsp. infantis CCFM1426 described in the first technical solution or the microbial preparation described in the second technical solution in the fermentation of vitamin A to produce retinoic acid.

[0026] In some embodiments, the specific method for fermenting vitamin A to produce retinoic acid is as follows: the Bifidobacterium longum subsp. infantis CCFM1426 described in the first technical solution or the microbial preparation described in the second technical solution is inoculated into a fermentation system containing vitamin A to produce retinoic acid through fermentation.

[0027] Furthermore, in the fermentation system, the amount of *Bifidobacterium longum* subsp. infantis CCFM1426 added is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0028] Furthermore, the fermentation temperature is 37℃, and the fermentation time is no less than 1.5 hours.

[0029] The fifth technical solution provided by this invention is the use of the Bifidobacterium longum subsp. infantis CCFM1426 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a medicine for relieving and / or treating ulcerative colitis.

[0030] In some embodiments, the viable count of *Bifidobacterium longum* subsp. infantis CCFM1426 in the drug is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0031] Furthermore, the viable count of Bifidobacterium longum subsp. infantis CCFM1426 in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0032] In some embodiments, the pharmaceutical product also contains a drug carrier and / or pharmaceutical excipients.

[0033] In some embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0034] In some embodiments, the pharmaceutical excipient includes excipients and additives.

[0035] In some embodiments, the pharmaceutical excipient comprises one or more of the following: stabilizer, flavoring agent, coloring agent, filler, binder, wetting agent, disintegrant, and lubricant.

[0036] In some embodiments, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0037] In some implementations, the application has at least one of the following functions:

[0038] (1) Reduce colonic damage in individuals with ulcerative colitis;

[0039] (2) Increase colonic RA levels in individuals with ulcerative colitis;

[0040] (3) Downregulate serum IFABP levels in individuals with ulcerative colitis;

[0041] (4) Downregulate the serum DAO level in individuals with ulcerative colitis.

[0042] The sixth technical solution provided by the present invention is the application of the Bifidobacterium longum subsp. infantis CCFM1426 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of products for relieving and / or treating dry eye syndrome.

[0043] In some implementations, the application includes at least one of the following functions:

[0044] (1) Reduce corneal damage in individuals with dry eye syndrome.

[0045] (2) Reduce conjunctival goblet cell damage in individuals with dry eye syndrome.

[0046] (3) Increase the serum RA level in individuals with dry eye.

[0047] (4) Downregulate the serum IL-6 level in individuals with dry eye.

[0048] (5) Downregulates the serum IL-17 level in individuals with dry eye.

[0049] In some embodiments, the viable count of *Bifidobacterium longum* subsp. infantis CCFM1426 in the drug is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0050] Furthermore, the viable count of Bifidobacterium longum subsp. infantis CCFM1426 in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0051] In some embodiments, the pharmaceutical product also contains a drug carrier and / or pharmaceutical excipients.

[0052] In some embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0053] In some embodiments, the pharmaceutical excipient includes excipients and additives.

[0054] In some embodiments, the pharmaceutical excipient comprises one or more of the following: stabilizer, flavoring agent, coloring agent, filler, binder, wetting agent, disintegrant, and lubricant.

[0055] In some embodiments, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0056] The technical effects of this invention are as follows:

[0057] This invention provides a strain of *Bifidobacterium longum* subsp. *infant* CCFM1426 that enhances the physiological activity of vitamin A, thereby preventing and / or alleviating ulcerative colitis induced by sodium dextran sulfate. Specifically, it manifests in the following ways: (2) reducing colonic inflammatory infiltration in mice with ulcerative colitis; (3) increasing retinoic acid levels in the colon of mice with ulcerative colitis by 24.43%; (4) decreasing serum IFABP levels in mice with ulcerative colitis by 27.87%; (5) decreasing serum DAO levels in mice with ulcerative colitis by 23.70%; (6) reducing corneal epithelial damage and basal vacuolation in individuals with dry eye; (7) reducing conjunctival goblet cell damage in individuals with dry eye; (8) increasing serum RA levels in mice with dry eye by 17.68%; (9) decreasing serum IL-6 levels in mice with dry eye by 29.89%; and (10) decreasing serum IL-17 levels in mice with dry eye by 14.78%.

[0058] Therefore, Bifidobacterium longum infantis subsp. CCFM1426 enhances the physiological activity of vitamin A and has great application potential in the preparation of products (such as food, medicine or health food) for the prevention and / or relief of ulcerative colitis and dry eye syndrome.

[0059] Biological Preservation

[0060] A strain of Bifidobacterium longum subsp. infantis (CCFM1426) was deposited on September 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:65152, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0061] Figure 1 This is the result of the BLAST alignment.

[0062] Figure 2 The level of RA produced by the strain during vitamin A fermentation.

[0063] Figure 3 Comparison of histopathological sections of colon tissue from different groups of experimental mice.

[0064] Figure 4 The colonic RA level in mice from different experimental groups.

[0065] Figure 5 The levels of IFABP in the serum of mice in different experimental groups.

[0066] Figure 6 The DAO levels in the serum of mice in different experimental groups.

[0067] Figure 7 The corneal fluorescein staining of mice in different groups.

[0068] Figure 8 Comparison of corneal tissue pathological sections from different groups of experimental mice.

[0069] Figure 9 Comparison of conjunctival tissue pathological sections from different groups of experimental mice.

[0070] Figure 10 The levels of RA in the serum of mice in different experimental groups.

[0071] Figure 11 The levels of IL-6 in the serum of mice in different experimental groups.

[0072] Figure 12 The levels of IL-17 in the serum of mice in different experimental groups. Detailed Implementation

[0073] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0074] Test method:

[0075] 1. Method for detecting viable bacteria count: The national standard GB 4789.35-2016, "National Food Safety Standard - Microbiological Testing of Food - Lactic Acid Bacteria Detection", was adopted.

[0076] 2. Acidity testing method: GB 431334-2010 is adopted.

[0077] 3. Preparation of pathological sections from mouse colon: Colon tissue was extracted and immediately fixed in 10% neutral buffered formaldehyde solution for 24-48 hours. Next, the tissue was dehydrated with a gradient of alcohols, cleared with xylene, and then embedded in paraffin. The embedded tissue was cut into 4-5 micrometer thick sections and stained with hematoxylin and eosin (H&E) under a microscope. Finally, the tissue structure and pathological changes of the sections were observed and analyzed under a microscope.

[0078] 4. Preparation of Pathological Sections from Mouse Corneas: Eyeball tissue was removed and immediately fixed in 10% neutral buffered formaldehyde solution for 24-48 hours. Following graded alcohol dehydration and xylene clearing, the tissue was embedded in paraffin. The embedded tissue was cut into 4-5 micrometer thick sections and stained with hematoxylin and eosin (H&E) under a microscope. Finally, the tissue structure and pathological changes of the sections were observed and analyzed under a microscope.

[0079] 5. Preparation of Pathological Sections from Mouse Conjunctiva: Conjunctival tissue was removed and immediately fixed in 10% neutral buffered formaldehyde solution for 24-48 hours. Following graded alcohol dehydration and xylene clearing, the tissue was embedded in paraffin. The embedded tissue was cut into 4-5 micrometer thick sections and stained with hematoxylin and eosin (H&E) under a microscope. Finally, the tissue structure and pathological changes of the sections were observed and analyzed under a microscope.

[0080] The experimental results were statistically analyzed using Graphpad Prism. Unless otherwise specified, the results were compared with the model group. **** represents P < 0.0001, *** represents P < 0.001, ** represents P < 0.01, and * represents P < 0.05.

[0081] Raw materials used in the examples:

[0082] The SPF-grade C57BL / 6J male mice used in the following examples were purchased from Vital River Pharmaceuticals in Beijing.

[0083] The culture media involved in the following examples are as follows:

[0084] Each liter of MRS medium contains: 10g peptone, 10g beef extract, 20g glucose, 2g sodium acetate, 5g yeast extract, 2g diammonium hydrogen citrate, 2.6g K₂PO₄·3H₂O, 0.1g MgSO₄·7H₂O, 0.05g MnSO₄, 1mL Tween 80, and 0.5g cysteine. All reagents used to prepare the medium were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0085] MRS solid culture medium formula (1L): peptone 10g, beef extract 10g, glucose 20g, sodium acetate 2g, yeast extract 5g, diammonium hydrogen citrate 2g, K₂PO₄·3H₂O 2.6g, MgSO₄·7H₂O 0.1g, MnSO₄ 0.05g, Tween 80 1mL, cysteine ​​phosphate 0.5g, agar 20g. All reagents used to prepare the culture medium were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0086] Example 1: Screening and Identification of Strains

[0087] (1) Screening and identification of strains

[0088] A gene alignment file was constructed by collecting retinaldehyde dehydrogenase protein sequences from human, mouse, and strains reported to possess VA metabolic capabilities. This file was then compared with the gene draft information of strains in the Jiangnan University bacterial bank. Bioedit's BLAST algorithm was used to identify potential functional probiotics that could enhance VA physiological activity. The sequence alignment files are shown in Table 1, and the alignment results are shown in [Table 1]. Figure 1 .

[0089] Table 1 Sequence alignment files

[0090]

[0091] Figure 1 The results showed that Bifidobacterium longum infantis subspecies CCFM1426 had a score (bits) of 867 and an E.Value of 0, indicating that it has the potential to metabolize vitamin A.

[0092] The target strain was obtained from the bacterial bank of Jiangnan University and inoculated into MRS solid medium. After anaerobic incubation at 37°C for 24 hours, single colonies were picked and transferred to MRS liquid medium, then incubated in an anaerobic environment at 37°C for 24 hours. The resulting purified culture was mixed, centrifuged, and the supernatant was discarded to obtain bacterial cells. The bacterial cells were sent to the company for genome sequencing. The 16S rDNA sequence obtained from the sequencing was compared with the nucleic acid sequence in GenBank, and the results showed that it was *Bifidobacterium longum* subsp. *infantitidis*.

[0093] (2) Cultivation of bacterial strains and preparation of bacterial suspension

[0094] After inoculating the strain into MRS solid medium and culturing at 37°C for 48 hours, its colonies were observed and the bacterial cells were observed under a microscope. It was found that the colonies were milky white, round and raised, with a smooth surface. The bacterial cells were straight rods with neat edges and round ends, usually existing singly, in pairs, or in a V-shape.

[0095] Bifidobacterium longum subsp. infantis CCFM1426 was inoculated into MRS liquid medium and cultured at 37°C for 24 h. Then, it was transferred to fresh MRS liquid medium at an inoculum volume of 4% and cultured under the same conditions for 24 h. After centrifugation at 8000×g for 15 min, the bacterial cells were collected. The bacterial cells were washed with 0.9% physiological saline and centrifuged again at 8000×g for 15 min. The supernatant was discarded and the bacterial cells were collected. The cells were resuspended in 30% glycerol solution (containing 0.05% L-cysteine) to prepare a resuspended solution, which was then frozen at -80°C for later use.

[0096] When using Bifidobacterium longum subsp. infantis CCFM1426 for gavage in mice, the sample was removed from -80℃, centrifuged at 8000×g for 15 min, the supernatant was discarded, and the sample was resuspended in sterile physiological saline to obtain a gavage concentration of 1×10⁻⁶. 9 CFU / mL bacterial suspension.

[0097] Example 2: In vitro vitamin A metabolism experiment by bacterial strain

[0098] After activation and identification, the strain was cultured in an anaerobic environment at 37°C, and subcultured every 24-48 hours. The culture was then transferred to fresh MRS liquid medium at a 4% inoculum size. After three subcultures, the experiment began after 13 hours of culture. Vitamin A was added to a concentration of 100 μM, and the culture was incubated at 37°C for 1.5 hours. The bacterial culture was then centrifuged, and 200 μL of the supernatant was collected. The concentration change of retinoic acid, a vitamin A metabolite, in the supernatant was detected using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.). The specific experimental groups for in vitro vitamin A metabolism are shown in Table 2.

[0099] Table 2 In vitro experiments

[0100]

[0101] The specific method is as follows:

[0102] Blank group: Take 200 μL of sterile MRS liquid culture medium from the laminar flow hood and put it into a centrifuge tube.

[0103] Control group: 200 μL of sterile MRS liquid culture medium was taken from the laminar flow hood and vitamin A was added to bring the concentration to 100 μM before being placed in a centrifuge tube.

[0104] Co-culture group: After the strain was cultured for 13 hours, VA was added to 100 μM in a clean bench, shaken to mix, and then 200 μL was taken out and placed into a centrifuge tube.

[0105] After culturing in an anaerobic environment at 37℃ for 1.5 h, the samples were transferred to centrifuge tubes and centrifuged. The supernatant was then transferred to new centrifuge tubes and measured using a retinoic acid ELISA kit (Tianjin Kevino Biotechnology Co., Ltd.) according to the kit instructions.

[0106] Depend on Figure 2 It can be seen that Bifidobacterium longum subsp. infantis CCFM1426 metabolizes vitamin A to produce retinoic acid in vitro. The retinoic acid content in the blank group and the control group did not change significantly. The retinoic acid production in the co-culture group reached 361.2±69.93 pg / mL within 1.5 hours.

[0107] Example 3: The alleviating effect of Bifidobacterium longum infantis subsp. CCFM1426 on colonic inflammation in mice with ulcerative colitis.

[0108] Six-week-old SPF-grade male C57BL / 6J mice were randomly divided into four groups: a normal control group, a model group, a vitamin A treatment group, and a CCFM1426 experimental group, with six mice in each group. The animals were housed at the Experimental Animal Center of Jiangnan University, fed standard feed, maintained at a constant temperature of 20-26℃ and humidity of 40-70%, and kept under a 12-hour light / dark cycle. All procedures in this animal experiment were performed in accordance with the "Jiangnan University Experimental Animal Management Regulations," and animal ethics were approved by the Experimental Animal Welfare and Ethics Committee of Jiangnan University.

[0109] The experiment lasted 21 days. From day 8 to day 14, mice were anesthetized and administered 0.2 mL of a 3% sodium dextran sulfate solution via gavage to induce ulcerative colitis. From day 15 to day 21, the experimental group was administered 0.2 mL of a solution containing 1 × 10⁻⁶ live bacteria via gavage daily. 9 The mice were given a bacterial suspension at CFU / mL. The normal group and the model group were administered an equal volume of sterile saline via gavage as a control. All groups had free access to water and food. Mice were weighed after gavage each day until they were sacrificed on day 21.

[0110] The grouping and treatment methods for experimental animals are shown in Table 3:

[0111] Table 3 Grouping of experimental animals

[0112]

[0113] Mice were sacrificed on day 21, and their colons were collected to prepare pathological sections for histopathological analysis.

[0114] Depend on Figure 3 It was found that, compared with the colon tissue of the blank control group mice, some crypts in the colon of the model group mice were destroyed, the crypts shifted upwards and the crypt structure was twisted and branched; there was inflammatory cell infiltration, increased basal plasma cells, lymphocyte aggregation and hemorrhage. After treatment with Bifidobacterium longum subsp. infantis CCFM1426, the inflammation was significantly relieved, the inflammatory cell infiltration in the colon tissue of mice was significantly reduced, the crypt damage was restored, the crypt morphology was more intact, and there was no obvious bleeding.

[0115] The above results indicate that the function of Bifidobacterium longum infantis subspecies CCFM1426 in alleviating intestinal inflammation in mice with intestinal damage is superior to that of vitamin A.

[0116] Example 4: Effect of Bifidobacterium longum infantis subspecies CCFM1426 on colonic retinoic acid levels in mice with ulcerative colitis. The mouse grouping and modeling methods were the same as in Example 3.

[0117] Mice were sacrificed on day 21 to obtain colon tissue. The colon tissue was homogenized, and the supernatant was collected at 12000 rpm for 15 minutes. The retinoic acid level in the supernatant was detected using an ELISA kit (Tianjin Kevino Biotechnology Co., Ltd.). The results are as follows: Figure 4 As shown.

[0118] Depend on Figure 4It was found that the retinoic acid level in the intestines of the model group mice was significantly lower than that of normal mice (P < 0.01). Compared with the model group mice, the retinoic acid level in the intestines of mice with intestinal damage significantly increased after intervention with vitamin A or Bifidobacterium longum subsp. infantis CCFM1426 (P < 0.05). Notably, after intervention with Bifidobacterium longum subsp. infantis CCFM1426, the retinoic acid level in the intestines of mice with intestinal damage returned to normal levels, and increased by 24.43% compared with the colonic retinoic acid level in the model group mice (95.58 ± 6.92 pg / mgprot), reaching 118.93 ± 5.29 pg / mgprot.

[0119] The results showed that *Bifidobacterium longum* subsp. infantis CCFM1426 increased retinoic acid levels in the colonic tissue of mice with ulcerative colitis. Example 5: Effect of *Bifidobacterium longum* subsp. infantis CCFM1426 on serum intestinal fatty acid-binding protein levels in mice with ulcerative colitis.

[0120] The mouse grouping and modeling methods were the same as in Example 3.

[0121] Mice were sacrificed on day 21, and blood was collected by removing the eyeballs. The blood was centrifuged at 3000 rpm for 15 min to obtain the supernatant, which was then used to detect the serum intestinal fatty acid binding protein level using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0122] Depend on Figure 5 It was found that the serum intestinal fatty acid-binding protein level in the model group (637.26±38.86 pg / mL) was significantly increased compared with that in the normal group (320.72±45.34 pg / mL) (P<0.0001); while the serum intestinal fatty acid-binding protein level in mice administered Bifidobacterium longum subsp. infantis CCFM1426 via gavage was significantly decreased by 27.87% (P<0.05) compared with the model group, decreasing to 459.65±35.66 pg / mL.

[0123] The above experimental results indicate that Bifidobacterium longum infant subspecies CCFM1426 can effectively reduce the level of intestinal fatty acid-binding protein, a protein related to intestinal barrier damage, in the serum of mice with ulcerative colitis, which indirectly suggests that Bifidobacterium longum infant subspecies CCFM1426 can alleviate / reduce intestinal barrier damage in mice with ulcerative colitis.

[0124] Example 6: Effect of Bifidobacterium longum infantis subsp. CCFM1426 on serum diamine oxidase levels in mice with ulcerative colitis

[0125] The mouse grouping and modeling methods were the same as in Example 3.

[0126] Mice were sacrificed on day 21, and blood was collected by removing the eyeballs. The blood was centrifuged at 3000 rpm for 15 min to obtain the supernatant, and the serum diamine oxidase level was detected using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0127] Depend on Figure 6 It was found that the serum diamine oxidase level in the model group (3876.86±109.73 pg / mL) was significantly increased compared with that in the normal group (2806.67±214.91 pg / mL) (P<0.05); while the serum intestinal fatty acid binding protein level in mice administered Bifidobacterium longum subsp. infantis CCFM1426 via gavage was significantly decreased by 23.70% (P<0.05) compared with the model group, decreasing to 2958.17±231.84 pg / mL.

[0128] The above experimental results indicate that Bifidobacterium longum infant subspecies CCFM1426 can effectively reduce the level of diamine oxidase, a protein related to intestinal barrier damage, in the serum of mice with ulcerative colitis, which indirectly suggests that Bifidobacterium longum infant subspecies CCFM1426 can alleviate / reduce intestinal barrier damage in mice.

[0129] Example 7: Alleviating effect of Bifidobacterium longum infantis subspecies CCFM1426 on corneal damage in mice with dry eye syndrome

[0130] Six-week-old SPF-grade male C57BL / 6J mice were randomly divided into four groups: a normal control group, a model group, a vitamin A treatment group, and a CCFM1426 experimental group, with six mice in each group. The animals were housed at the Experimental Animal Center of Jiangnan University, fed standard feed, maintained at a constant temperature of 20-26℃ and humidity of 40-70%, and kept under a 12-hour light / dark cycle. All procedures in this animal experiment were performed in accordance with the "Jiangnan University Experimental Animal Management Regulations," and animal ethics were approved by the Experimental Animal Welfare and Ethics Committee of Jiangnan University.

[0131] The experiment lasted 21 days. From day 8 to day 14, mice were anesthetized and induced with dry eye by twice-daily eye drops of 5 μL of 0.2% benzalkonium chloride solution. From day 15 to day 21, the experimental group was administered 0.2 mL of a live bacteria solution (1 × 10⁻⁶) via gavage daily. 9 The mice were given a bacterial suspension at CFU / mL. The normal and model groups were administered an equal volume of sterile saline via gavage as a control. All groups had free access to water and food until sacrifice on day 21. Before sacrifice on day 21, the mice's corneas were observed using a slit lamp with a cobalt blue lens. The grouping and treatment methods for the experimental animals are shown in Table 4.

[0132] Table 4 Grouping of experimental animals

[0133]

[0134] Mice were sacrificed on day 21, and their eyeballs were collected to prepare pathological sections for histopathological analysis.

[0135] Depend on Figure 7 It was observed that, compared to the corneal tissue of the blank control group mice, the cornea of ​​the model group mice showed obvious patchy sodium fluorescein staining. After intervention with vitamin A or CCFM1426, the dry eye symptoms of the mice were significantly relieved, but obvious punctate and slight patchy staining still existed. Figure 8 It was found that, compared with the control group mice, the corneal layer of the model group mice was significantly thinner, and sparse gaps and vacuoles appeared in the corneal stroma. After vitamin A intervention, the corneal thickness was restored, and the symptoms of sparse gaps and vacuoles in the corneal stroma were alleviated. Gavage administration of Bifidobacterium longum infantis subsp. CCFM14 alleviated the significant thinning of the corneal layer, but the symptoms of sparse gaps and vacuoles in the corneal stroma still existed.

[0136] The above results indicate that Bifidobacterium longum infant subspecies CCFM1426 has the function of alleviating corneal damage in mice with dry eye syndrome.

[0137] Example 8: Alleviating effect of Bifidobacterium longum infantis subspecies CCFM1426 on conjunctival goblet cell damage in mice with dry eye syndrome. The mouse grouping and modeling methods were the same as in Example 7.

[0138] Mice were sacrificed on day 21, and conjunctiva was obtained to prepare pathological sections for histopathological analysis.

[0139] Depend on Figure 9 It was found that, compared with the corneal tissue of the blank control group mice, the conjunctival goblet cells of the model group mice were atrophied and reduced. After intervention with vitamin A or CCFM1426, the number of conjunctival goblet cells in the mice recovered to some extent.

[0140] The above results indicate that Bifidobacterium longum infant subspecies CCFM1426 has the function of alleviating conjunctival goblet cell damage in mice with dry eye syndrome.

[0141] Example 9: Effect of Bifidobacterium longum infantis subsp. CCFM1426 on serum retinoic acid levels in mice with dry eye syndrome

[0142] The mouse grouping and modeling methods were the same as in Example 7.

[0143] Mice were sacrificed on day 21, and blood was collected by removing the eyeballs. The blood was centrifuged at 3000 rpm for 15 min to obtain the supernatant, and the serum retinoic acid level was detected using an ELISA kit (Tianjin Kevino Biotechnology Co., Ltd.).

[0144] Depend on Figure 10It was found that the serum retinoic acid level in the model group (292.78±7.02 pg / mL) was significantly lower than that in the normal group (362.54±6.75 pg / mL) (P<0.01); while the serum retinoic acid level in mice administered Bifidobacterium longum subsp. infantis CCFM1426 via gavage was significantly increased by 17.68% (P<0.05) compared to the model group, reaching 344.54±22.38 pg / mL.

[0145] The results showed that Bifidobacterium longum infant subspecies CCFM1426 could improve serum retinoic acid levels in mice with dry eye syndrome.

[0146] Example 10: Effect of Bifidobacterium longum infantis subsp. CCFM1426 on serum IL-6 levels in mice with dry eye syndrome

[0147] The mouse grouping and modeling methods were the same as in Example 7.

[0148] Mice were sacrificed on day 21, and blood was collected by removing the eyeballs. The blood was centrifuged at 3000 rpm for 15 min to obtain the supernatant, and the serum IL-6 level was detected using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0149] Depend on Figure 11 It was found that the serum IL-6 level in the model group (71.32±4.74 pg / mL) was significantly increased compared with that in the normal group (45.35±2.95 pg / mL) (P<0.05); while the serum IL-6 level in mice administered Bifidobacterium longum subsp. infantis CCFM1426 via gavage was significantly decreased by 29.89% compared with that in the model group (P<0.05), decreasing to 50.00±6.70 pg / mL.

[0150] The above experimental results indicate that Bifidobacterium longum infant subspecies CCFM1426 has the function of downregulating the level of the inflammatory factor IL-6 in the serum of mice with dry eye syndrome.

[0151] Example 11: Effect of Bifidobacterium longum infantis subsp. CCFM1426 on serum IL-17 levels in mice with dry eye syndrome

[0152] The mouse grouping and modeling methods were the same as in Example 7.

[0153] Mice were sacrificed on day 21, and blood was collected by removing the eyeballs. The blood was centrifuged at 3000 rpm for 15 min to obtain the supernatant, and the serum IL-17 level was detected using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0154] Depend on Figure 12It was found that the serum IL-17 level in the model group (137.55±2.19 pg / mL) was higher than that in the normal group (125.99±2.71 pg / mL); while the serum IL-17 level in mice administered Bifidobacterium longum subsp. infantis CCFM1426 via gavage was significantly lower than that in the model group by 14.78% (P<0.05), decreasing to 117.22±2.86 pg / mL.

[0155] The above experimental results indicate that Bifidobacterium longum infant subspecies CCFM1426 has the function of downregulating the level of the inflammatory factor IL-17 in the serum of mice with dry eye syndrome.

[0156] Example 12: Application of Bifidobacterium longum subsp. infantis

[0157] The Bifidobacterium longum infantis subsp. CCFM1426 described in this invention is used to prepare pharmaceutical compositions and fermented foods that alleviate ulcerative colitis and dry eye syndrome, including increasing retinoic acid levels in the body, reducing intestinal oxidative stress, and regulating immune disorders in the body, and has very broad application prospects.

[0158] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Bifidobacterium longum* subsp. *infantis* CCFM1426, characterized in that, The described Bifidobacterium longum subspecies CCFM1426 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 20, 2024, with accession number GDMCC No:65152.

2. A microbial preparation, characterized in that, The microbial preparation contains the Bifidobacterium longum infant subspecies CCFM1426 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, In the microbial preparation, the amount of Bifidobacterium longum subsp. infantis CCFM1426 added is not less than 1×10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

4. The use of the Bifidobacterium longum subsp. infantis CCFM1426 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in the fermentation of vitamin A to produce retinoic acid.

5. The application according to claim 4, characterized in that, The specific method for fermenting vitamin A to produce retinoic acid is as follows: the Bifidobacterium longum subsp. infantis CCFM1426 described in claim 1 or the microbial preparation described in claim 2 or 3 is inoculated into a fermentation system containing vitamin A to produce retinoic acid through fermentation.

6. The use of the *Bifidobacterium longum* subsp. infantis CCFM1426 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in the preparation of a medicament for relieving and / or treating ulcerative colitis, characterized in that... The application has at least one of the following functions: (1) Reduce colonic damage in individuals with ulcerative colitis; (2) Increase colonic RA levels in individuals with ulcerative colitis; (3) Downregulate serum IFABP levels in individuals with ulcerative colitis; (4) Downregulate the serum DAO level in individuals with ulcerative colitis.

7. The use of the Bifidobacterium longum subsp. infantis CCFM1426 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in the preparation of a medicament for relieving and / or treating dry eye syndrome.

8. The application according to claim 7, characterized in that, The application includes at least one of the following functions: (1) Reduce corneal damage in individuals with dry eye syndrome. (2) Reduce conjunctival goblet cell damage in individuals with dry eye syndrome. (3) Increase the serum RA level in individuals with dry eye. (4) Downregulate the serum IL-6 level in individuals with dry eye. (5) Downregulates the serum IL-17 level in individuals with dry eye.

9. The application according to claim 7, characterized in that, In the aforementioned drug, the viable count of Bifidobacterium longum subsp. infantis CCFM1426 is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

10. The application according to claim 7, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients.

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