Lactobacillus rhamnosus and its postbiotic product and application for preventing and / or treating diabetic retinopathy and protecting eyesight

Abstract

The present invention provides a rhamnosus lactobacillus and its postbiotic products and applications for preventing and / or treating diabetic retinopathy and protecting vision, relating to the field of microbial technology, wherein the postbiotic products comprise inactivated bacteria and / or metabolites of rhamnosus lactobacillus ProSci-82. The present invention analyzes the changes in relevant indicators in the eye blood and feces of mice before and after using the postbiotic products, as well as the effects on vision and blood indicators of the population, and finds that the postbiotic composition can regulate the proportion of intestinal flora, reduce the expression of eye-related inflammatory cytokines, improve vision and fundus symptoms, and reduce the level of platelet-derived growth factor (PDGF-B) in serum, thereby achieving the effect of preventing and / or treating diabetic retinopathy and protecting vision.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to Lactobacillus rhamnosus and a postbiotic product and application thereof for preventing and / or treating diabetic retinopathy and protecting eyesight. Background Art

[0002] Diabetic retinopathy (DR) is a serious eye disease caused by diabetes. It is one of the most serious complications of diabetes and one of the three major eye diseases that cause blindness as announced by the World Health Organization. The disease can progress rapidly, leading to irreversible vision loss. The longer the course of diabetes, the higher the prevalence and the more severe the condition. In the early stages of the disease, patients do not experience significant changes in vision. As the disease progresses, patients often experience varying degrees of visual impairment, such as decreased vision or photopsia, fundus findings of normal or thin retinal arteries, dilated retinal veins with a beaded appearance, numerous microaneurysms at the posterior pole, retinal hemorrhages or spots, and macular degeneration.

[0003] Probiotics are believed to regulate blood sugar levels. By balancing the gut microbiota, reducing oxidative damage, and modulating immune factors, they may have a positive effect on alleviating diabetes-related symptoms. Studies in a mouse model of type 2 diabetes have shown that intermittent fasting can alter the gut microbiota composition and promote the production of the beneficial secondary bile acid tauroursodeoxycholic acid. Tauroursodeoxycholic acid may provide neuroprotection in the retina, preventing the progression of diabetic retinopathy. Compared with healthy mice, mice with type 2 diabetes have increased Verrucomicrobia and Firmicutes bacteria in their guts, while decreasing Bacteroidetes and Firmicutes bacteria. Type 2 diabetes is also associated with decreased goblet cell numbers and increased plasma peptidoglycan concentrations. Furthermore, altered gut microbiota composition in type 2 diabetes is associated with the progression of diabetic retinopathy. In a mouse model of type 1 diabetes, altered gut microbiota composition increases peptidoglycan biosynthesis. Hyperglycemia, acting on intestinal epithelial cells, disrupts the gut vascular barrier, allowing peptidoglycan from the intestinal lumen to translocate into the circulation and reach the retina. In retinal endothelial cells, peptidoglycan damages the integrity of the blood-retinal barrier and exacerbates diabetic retinopathy. By repairing the gut vascular barrier, systemic peptidoglycan levels will be reduced, reducing retinal barrier dysfunction and thus preventing the progression of diabetic retinopathy. [1] Therefore, there is an urgent need to develop probiotic products that can improve the structure of intestinal flora, thereby preventing and / or treating diabetic retinopathy and protecting vision.

[0004] Patent CN116694537B discloses that Lactobacillus rhamnosus has the following functions: lowering blood sugar levels; improving the pathological state of the liver, kidneys and pancreas; improving blood lipid levels; regulating inflammation through the NFκB-p65 pathway; and regulating sugar metabolism through the AMPK pathway, which plays an important role in preventing or treating type 2 diabetes and has broad application prospects.

[0005] Postbiotics are preparations of nonliving microorganisms and / or other components that have beneficial effects on host health. Due to their unique biological activities and potential to replace antibiotics, postbiotics have been widely used in gastrointestinal therapeutics. In particular, they can lower blood glucose levels, improve insulin sensitivity, and shorten the duration of DR. Postbiotics hold great potential as a viable adjunctive therapy.

[0006] [1]Floyd, Jason L, and Maria B Grant. "The Gut-Eye Axis: Lessons Learned from Murine Models." Ophthalmology and therapy vol.9,3(2020):499-513. Summary of the Invention

[0007] In order to solve the above problems, the present invention provides a Lactobacillus rhamnosus and a postbiotic product thereof for preventing and / or treating diabetic retinopathy and protecting eyesight.

[0008] A strain of Lactobacillus rhamnosus ProSci-82, deposited on April 24, 2024, with a deposit number of CGMCC No. 30412.

[0009] On the other hand, the present invention provides a postbiotic product for preventing and / or treating diabetic retinopathy and protecting vision, wherein the postbiotic product comprises at least one of the following: inactivated bacteria, fermentation broth, bacterial lysate, intracellular extract, and metabolites after fermentation of Lactobacillus rhamnosus ProSci-82.

[0010] Specifically, the postbiotic product can be a solid preparation or a liquid preparation.

[0011] In certain specific embodiments of the present invention, the preparation steps of the postbiotic product are:

[0012] (1) inoculating the Lactobacillus rhamnosus ProSci-82 described in claim 1 into a fermentation medium and culturing the medium to obtain a fermentation broth;

[0013] (2) sterilizing and inactivating the bacterial solution by heat treatment to obtain a fermentation liquid;

[0014] (3) The sterilized and inactivated fermentation broth was spray-dried to obtain the postbiotic Lactobacillus rhamnosus ProSci-82.

[0015] More specifically, the fermentation medium formula is: the mass concentration of defatted soy flour is 1-5%, the mass concentration of defatted milk powder is 4-8%, and the solvent is water.

[0016] In certain specific embodiments of the present invention, the fermentation medium is formulated as follows: the mass concentration of defatted soy flour is 1%, the mass concentration of defatted milk powder is 8%, and the solvent is water.

[0017] More specifically, the culture step in step (1) is maintained at 30-37°C.

[0018] In certain specific embodiments of the present invention, the culturing step in step (1) is maintained at 30°C.

[0019] Specifically, the uses of the postbiotic products provided by the present invention include but are not limited to:

[0020] (1) Regulate the proportion of intestinal flora;

[0021] (2) reduce the expression of eye-related proinflammatory factors;

[0022] (3) Increase the expression of eye-related anti-inflammatory factors;

[0023] (4) Improve visual acuity;

[0024] (5) Adjustment of fundus symptoms;

[0025] (6) Reduce the level of PDGF-B in serum.

[0026] In certain specific embodiments of the present invention, the pro-inflammatory factors include: IL-2, IL-6 and IFN-γ.

[0027] In certain specific embodiments of the present invention, the anti-inflammatory factor includes IL-10.

[0028] In another aspect, the present invention provides use of the above-mentioned postbiotic preparation in the preparation of a product for preventing, alleviating and / or treating diabetic retinopathy.

[0029] In another aspect, the present invention provides a use of the above-mentioned postbiotic preparation in the preparation of a product for reducing the level of a growth factor.

[0030] In another aspect, the present invention provides a use of the above-mentioned postbiotic preparation in the preparation of a product for increasing the level of anti-inflammatory factors.

[0031] In another aspect, the present invention provides use of the above-mentioned postbiotic preparation in the preparation of a product for alleviating eye diseases.

[0032] In another aspect, the present invention provides use of the above-mentioned postbiotic preparation in the preparation of a product for reducing PDGF-B in serum.

[0033] In another aspect, the present invention provides a use of the above-mentioned postbiotic preparation in the preparation of a product for protecting eyesight.

[0034] Specifically, the above-mentioned products include medicines.

[0035] More specifically, the drug further includes pharmaceutical excipients, which include excipients, diluents, fillers and / or absorption enhancers, and the drug is in the form of tablets, granules, powders, capsules, solutions, suspensions, or lyophilized preparations.

[0036] Preservation Instructions

[0037] Biomaterial name: ProSci-82.

[0038] Taxonomic name: Lacticaseibacillus rhamnosus.

[0039] Date of preservation: April 24, 2024.

[0040] Deposit number: CGMCC No.30412.

[0041] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms.

[0042] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0044] Example 1 Preparation of Lactobacillus rhamnosus ProSci-82 postbiotics

[0045] Seed culture: 0.1 mL of Lactobacillus rhamnosus ProSci-82 bacterial culture was inoculated into MRS liquid culture medium (purchased from Beijing Road & Bridge Technology Co., Ltd., catalog number CM187), and cultured at 30°C for 30 h to obtain ProSci-82 seed culture.

[0046] Fermentation: Lactobacillus rhamnosus ProSci-82 seed solution was inoculated into the fermentation medium until the total viable count of the fermentation medium was 2×10 6 CFU / mL, and cultured at 30°C for 30 h to obtain the fermentation broth.

[0047] Post-treatment: The fermentation broth was sterilized at 85°C for 15 min to obtain a sterilized fermentation broth; the sterilized fermentation broth was spray-dried to obtain the postbiotic Lactobacillus rhamnosus ProSci-82 (total bacterial count of 2.0×10 11 pcs / g);

[0048] Wherein, the preparation method of fermentation medium is as follows:

[0049] The fermentation medium comprises, by weight percentage, 1% defatted soy flour, 8% defatted milk powder, and the balance water. The components of the fermentation medium are mixed in proportion, dissolved at 58° C., and stirred at 150 rpm for 15 minutes to obtain a feed solution.

[0050] The feed liquid is homogenized once at 58° C., a primary pressure of 19 MPa, and a secondary pressure of 5.0 MPa to obtain a homogenized feed liquid; the homogenized feed liquid is sterilized at 93° C. for 30 min to obtain a sterilized feed liquid; the sterilized feed liquid is cooled to 35° C. to obtain a fermentation medium.

[0051] Comparative Example 1 Preparation of Lactobacillus rhamnosus LGG postbiotics

[0052] The strain LGG ATCC 53103 was used, and the postbiotic preparation steps were the same as in Example 1.

[0053] Experimental Example 1: Animal Experiment on Postbiotics Assisted in Improving Eye Diseases

[0054] 1. Experimental Methods

[0055] 1.1 The strain L. rhamnosus ProSci-82 contained in the postbiotics prepared in Example 1 was provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd.

[0056] 1.2 Experimental subjects

[0057] Eight-week-old DBA / 2J male mice were selected as experimental subjects. Two groups were set up according to experimental requirements, with 10 mice in each group.

[0058] 1.3 Experimental Grouping

[0059] Postbiotic group: oral administration of metformin + postbiotic Lactobacillus rhamnosus ProSci-82;

[0060] Placebo group: Oral administration of metformin.

[0061] 1.4 Detection Method

[0062] Twenty male mice were randomly divided into a postbiotic group and a placebo group, with 10 mice in each group. Eye blood samples and fecal samples were collected on days 0 and 90, respectively. Clinical immune factors and intestinal microbiota of the mice were analyzed. The results are shown in Tables 1 and 2.

[0063] 2. Experimental Results

[0064] 2.1 Changes in the proportion of intestinal flora

[0065] The six most represented phyla were Bacteroidetes, Firmicutes, Verrucomicrobia, Tenebrio, Actinobacteria, and Proteobacteria. Metagenomic analysis of mouse feces using sequencing revealed the relative proportions of these six phyla between the postbiotic and placebo groups. The results are shown in Table 1. Postbiotics altered the intestinal microbiota, increasing Firmicutes and decreasing Bacteroidetes and Verrucomicrobia. This study suggests that these unique changes in the mouse microbiome may promote intestinal barrier integrity.

[0066] Table 1

[0067]

[0068] 2.2 Ocular blood immune indicators

[0069] Blood samples were collected from the mice's eyes, and the test results, shown in Table 2, indicate that postbiotic treatment effectively reduced the concentrations of IL-2, IL-6, and IFN-γ, while increasing IL-10. This suggests that postbiotic treatment can effectively reduce the levels of pro-inflammatory factors in the mice's eyes and increase the levels of anti-inflammatory factors, significantly alleviating the mice's ocular condition and thus protecting their vision.

[0070] Table 2

[0071] index Epigenetic tuples control group IL-2 (pg / mL) 41.69±2.35 57.22±1.11 IL-6 (pg / mL) 63.56±1.52 79.28±2.16 IL-10 (pg / mL) 52.37±1.97 41.15±2.25 IFN-γ (pg / mL) 47.23±0.85 53.22±2.47

[0072] 3. Conclusion

[0073] Intervention with the postbiotic Lactobacillus rhamnosus ProSci-82 can effectively alleviate diabetic retinopathy and protect vision. Postbiotics protect vision by regulating intestinal flora and reducing the expression of related inflammatory cytokines.

[0074] Experimental Example 2: Human population study on the effects of postbiotics on diabetic retinopathy

[0075] 1. Experimental Methods

[0076] 1.1 Strain Source

[0077] The postbiotics of Lactobacillus rhamnosus ProSci-82 are the same as those in Experimental Example 1; the postbiotics of Lactobacillus rhamnosus LGG are the same as those in Comparative Example 1.

[0078] 1.2 Experimental subjects

[0079] Thirty subjects with diabetic retinopathy were selected from Hohhot, Inner Mongolia. Following the principles outlined in the Declaration of Helsinki and strictly adhering to the ethical and technical specifications of the International Human Microbiome Consortium, all subjects were informed of the detailed experimental procedures and signed informed consent before the study. Inclusion and exclusion criteria were determined according to the following:

[0080] ① Inclusion criteria: a. Subjects must be 18 years of age or older; b. No antibiotics or probiotics have been taken in the past two weeks; c. Participate in the experiment voluntarily and cooperate with the experiment as scheduled.

[0081] ② Exclusion criteria: a. Patients receiving drug treatment; b. Taking antibiotic preparations or probiotics in the past 2 weeks; c. Patients with other diseases; d. Pregnancy preparation, pregnancy or breastfeeding; e. Subjects who cannot cooperate with the experiment on time.

[0082] 1.3 Experimental Grouping

[0083] Postbiotic group: Taking the postbiotic Lactobacillus rhamnosus ProSci-82, 1×10 9 cells / 2g / d;

[0084] Control group: taking postbiotic Lactobacillus rhamnosus LGG, 1×10 9 cells / 2g / d;

[0085] Blank group: took the same dose of maltodextrin.

[0086] 1.4 Experimental methods

[0087] Thirty subjects were randomly divided into three groups, 10 in each, for a three-month intervention. Before and after the intervention, visual acuity and fundus examinations were performed, and blood samples were collected for serum platelet-derived growth factor-β (PDGF-B) levels. Data were processed using SPSS 24.0, and comparisons were made using t-tests. P < 0.05 was considered statistically significant.

[0088] 2. Experimental Results

[0089] 2.1 Visual acuity test results

[0090] Before and after the intervention, the same doctor performed visual acuity examination using a standard logarithmic visual acuity chart. The best corrected visual acuity was recorded using an optometrist combined with manual optometry and converted into the logarithm of the minimum angle of resolution, LogMAR = lg (1 / decimal visual acuity).

[0091] Compared with pre-intervention results, visual acuity in the postbiotic group improved significantly after intervention (P < 0.05). There was no significant change in visual acuity in the blank and control groups after intervention (P > 0.05). The results of visual acuity tests before and after intervention are shown in Table 3. This suggests that the postbiotic Lactobacillus rhamnosus ProSci-82 can improve visual acuity.

[0092] Table 3

[0093] Group Number of people Before intervention After intervention Blank group 10 0.387±0.113 0.389±0.110 Epigenetic tuples 10 0.391±0.120 0.227±0.053(*#) comparison group 10 0.390±0.118 0.354±0.044

[0094] Note: Compared with the group before treatment, *P<0.05; compared with the blank group, #P<0.05.

[0095] 2.2 Fundus examination results

[0096] Fundus color photography was used, and the same physician performed a dilated fundus examination before and after treatment to observe the number of retinal angiomas, hemorrhage, and exudation. The examination was performed with the center of the visual field as the center, and the adjacent visual field divided into nine sections. Five sections (the center section, upper left section, upper right section, lower left section, and lower right section) were selected as representatives, and the average value was calculated.

[0097] Markedly effective: The number of retinal microaneurysms decreases from (+++) to (++), or from (++) to (+), or from (+) to disappearance; the amount of fundus hemorrhage decreases from (+++) to (+), or from (++) to disappearance; the amount of exudate decreases from (+++) to (+), or from (++) to (+), or from (+) to disappearance. Two or more indicators of microaneurysms, hemorrhages, or exudates meet the requirements. Effective: The number of retinal microaneurysms decreases from (+++) to (++), or from (++) to (+), or from (+) to disappearance; the amount of fundus hemorrhage decreases from (+++) to (+), or from (++) to disappearance; the amount of exudate decreases from (+++) to (++), or from (++) to (+), or from (+) to disappearance. One or more indicators of microaneurysms, hemorrhages, or exudates meet the requirements. Ineffective: No change in the number of retinal microaneurysms, hemorrhages, or exudates. Worsening: Fundus photography reveals proliferative changes such as retinal neovascularization. (+) indicates a small number of retinal neovascularizations that are easy to count; (++) indicates a large number of retinal neovascularizations that are difficult to count; and (+++) indicates numerous, uncountable microaneurysms with extensive bleeding and exudation that are fused into a single sheet. Fundus examination results before and after intervention are shown in Table 4.

[0098] Compared with the blank group, both the postbiotic group and the control group improved the fundus condition of the subjects, with the postbiotic group showing a more significant improvement (P<0.05). This suggests that the postbiotic Lactobacillus rhamnosus ProSci-82 can significantly improve the fundus condition of subjects with diabetic retinopathy.

[0099] Table 4

[0100]

[0101] 2.3 Serum PDGF-B results

[0102] Studies have shown that anti-PDGF-B drugs may be used to treat diabetic retinopathy by reducing retinal neovascularization. PDGF-B levels were measured using a human PDGF-B ELISA kit (JSBOSSEN). Changes in serum PDGF-B levels before and after intervention are shown in Table 5.

[0103] After the intervention, there was no significant change in serum PDGF-B levels in the blank group, but PDGF-B levels in both the postbiotic group and the control group decreased significantly, with the decrease in the postbiotic group being more significant than that in the control group (P<0.05). This suggests that the postbiotic Lactobacillus rhamnosus ProSci-82 can significantly reduce serum PDGF-B levels in subjects with diabetic retinopathy.

[0104] Table 5

[0105] Group Number of people Before intervention After intervention Blank group 10 566.43±38.88 557.12±41.09 Epigenetic tuples 10 571.38±41.13 289.45±33.17(*#) comparison group 10 569.25±35.65 454.51±38.95(*#)

[0106] Note: Compared with the group before treatment, *P<0.05; compared with the blank group, #P<0.05.

[0107] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) ProSci-82, characterized in that The rhamnosus lactis ProSci-82 was deposited on April 24, 2024, with the deposit number CGMCC No.30412.

2. A postbiotic product, characterized in that: The postbiotic product includes a fermentation broth obtained by fermenting Lactobacillus rhamnosus ProSci-82.

3. The postbiotic product according to claim 2, characterized in that The postbiotic product is a solid preparation or a liquid preparation.

4. The postbiotic product according to claim 2, characterized in that The preparation steps of the postbiotic product are as follows: (1) inoculating the Lactobacillus rhamnosus ProSci-82 described in claim 1 into a fermentation medium to obtain a fermentation broth; (2) Sterilizing and inactivating the bacterial liquid by heat treatment to obtain the fermentation liquid; (3) The sterilized and inactivated fermentation broth was spray-dried to obtain the postbiotic Lactobacillus rhamnosus ProSci-82.

5. The postbiotic product according to claim 4, characterized in that In step (1), the culture is carried out at 30-37°C.

6. Use of the postbiotic product according to claim 2 in the preparation of a product for preventing, alleviating and / or treating diabetic retinopathy, wherein the product is a medicine.

7. The use according to claim 6, characterized in that The postbiotic product prevents, alleviates and / or treats diabetic retinopathy by reducing the levels of IL-2, IL-6 and IFN-γ in patients with diabetic retinopathy and increasing the anti-IL-10 level in patients with diabetic retinopathy.

8. The use according to claim 6, characterized in that The postbiotic product prevents, alleviates and / or treats diabetic retinopathy by reducing the PDGF-B level in the serum of patients with diabetic retinopathy.

9. The use according to claim 6, characterized in that The postbiotic product prevents, alleviates and / or treats diabetic retinopathy by protecting the vision of patients with diabetic retinopathy.

10. The use according to any one of claims 6 to 9, characterized in that: The medicine further includes pharmaceutical excipients, which include excipients, diluents, fillers and / or absorption enhancers.

11. The use according to any one of claims 6 to 9, characterized in that: The medicine is in the form of tablets, granules, powders, capsules, solutions, suspensions or freeze-dried preparations.

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