A Lactobacillus rhamnosus strain for alleviating age-related macular degeneration, its synbiotic preparation, process and application

Abstract

The present invention discloses a Lactobacillus rhamnosus strain for alleviating age-related macular degeneration, its synbiotic preparation, process and application, belonging to the field of microbial technology. The Lactobacillus rhamnosus is deposited in the China Center for Type Culture Collection, Wuhan, with the deposit date being May 10, 2024 and the deposit number being CCTCC NO: M 2024897. The synbiotic preparation encapsulates the Lactobacillus rhamnosus by layer-by-layer assembly with sodium alginate, carrageenan, hyaluronic acid and carrageenan to form a microcapsule preparation. The microcapsule preparation has good storage stability and antioxidant properties, improves the stability, cell viability and release rate of Lactobacillus rhamnosus under oral delivery conditions, and enhances the colonization of Lactobacillus rhamnosus in the intestine. The microcapsule preparation effectively reduces the contents of total cholesterol, phospholipids and triglycerides in the retinal pigment epithelium of rats, and inhibits the levels of pro-inflammatory cytokines and the production of reactive oxygen species, thereby alleviating age-related macular degeneration.

Description

Technical Field

[0001] The invention relates to a synbiotic preparation process of a Lactobacillus rhamnosus strain for alleviating age-related macular degeneration and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Age-related macular degeneration (AMD) is a spectrum of central retinal diseases ranging from early to late stages with varying degrees of severity, involving photoreceptors, retinal pigment epithelium, Bruch's membrane, and choriocapillaris (a neurovascular complex). According to the Global Vision Database, AMD caused blindness in 5% and 7% of the global population in 1990 and 2010, respectively. Currently, the global prevalence of the disease is about 196 million, and it is expected to increase to 288 million by 2040. The disease is an important public health issue with a huge impact on socioeconomic development. The pathological characteristics of AMD are that it is a chronic, progressive, degenerative and inflammatory disease. The genetic characteristics of AMD are polygenic and multifactorial inheritance. The strongest risk factor for AMD is advanced age, which is the leading cause of irreversible blindness in the elderly worldwide. Late AMD can be divided into two forms: non-exudative (or dry) and exudative (or wet). The non-exudative form is characterized by macular atrophy due to the accumulation of drusen beneath the retinal pigment epithelium (RPE) and Bruch's membrane, which damages the RPE and may lead to indirect photoreceptor cell death; the exudative form is characterized by choroidal neovascularization (CNV), which leads to detachment of the RPE and photoreceptor cell death.

[0003] Multiple risk factors are involved in the pathogenesis of AMD, including smoking, age, genetics, and diet. However, the exact mechanisms behind these associations, especially lifestyle and environmental factors, remain unclear. Interestingly, some environmental risk factors associated with AMD, such as diet, can affect the composition and function of the gut microbiota. In addition, dysbiosis of the gut microbiota can lead to the formation of an inflammatory environment and the development of metabolic disorders, both of which are pathological conditions associated with AMD. Gut microbiota has become a promising area of ​​research with important implications for mechanistic and therapeutic studies. As a mediator of metabolic and immune regulation, disturbances in the gut microbiota may have significant effects on the distal neural retina and its adjacent tissues, the so-called gut-retinal axis.

[0004] Probiotics are live microorganisms that have been shown to have many health benefits for humans, including improving gastrointestinal disorders, inflammation and oxidative stress, and helping to maintain a healthy intestinal microbial balance. Lactic acid bacteria are a group of safe Gram-positive microorganisms that are widely used in various fermented foods. Lactic acid bacteria are also an important component of the human intestinal flora and have multiple physiological functions, including immune cell balance, metabolite regulation, and intestinal flora. Related studies have found that probiotics have a mitigating effect on a variety of diseases, such as: probiotic preparations can shorten the duration of diarrhea and reduce the incidence of diarrhea. They have a certain effect on various types of diarrhea, including infectious diarrhea and antibiotic-related diarrhea; probiotic preparations can improve the function of the digestive system and reduce gastrointestinal discomfort and symptoms of indigestion; probiotic preparations have a certain auxiliary therapeutic effect on intestinal inflammatory diseases, such as inflammatory bowel disease (such as Crohn's disease and ulcerative colitis). Therefore, it is of great practical significance to develop probiotic preparations for alleviating age-related macular degeneration. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Lactobacillus rhamnosus YBT20 for alleviating / preventing age-related macular degeneration.

[0006] At the same time, the purpose of the present invention is to provide a synbiotic preparation containing a strain of Lactobacillus rhamnosus for alleviating age-related macular degeneration, which can resist the harsh environment of the gastrointestinal tract, help the strain to better colonize in the intestine, and exert its probiotic effect.

[0007] At the same time, the purpose of the present invention is to provide a process for preparing a synbiotic preparation.

[0008] At the same time, the purpose of the present invention is to provide a synbiotic preparation for use in preparing a drug for alleviating / preventing age-related macular degeneration.

[0009] At the same time, the purpose of the present invention is to provide a strain of Lactobacillus rhamnosus for alleviating age-related macular degeneration and its use in preparing a drug for alleviating / preventing age-related macular degeneration.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0011] In view of the above problems, the purpose of the present invention is to provide a Lactobacillus rhamnosus, a composition, an oral agent and a use thereof for protecting the retina. The Lactobacillus rhamnosus and the oral agent are administered through the gastrointestinal tract and have a good protective effect on the retina.

[0012] To achieve the above objectives, the present invention first provides a strain of Lactobacillus rhamnosus YBT20 for alleviating age-related macular degeneration. The Lactobacillus rhamnosus was deposited in the China Center for Type Culture Collection, Wuhan, on May 10, 2024, with a deposit number of CCTCC NO: M 2024897.

[0013] The invention discloses a synbiotic preparation comprising a strain of Lactobacillus rhamnosus for alleviating age-related macular degeneration. The synbiotic preparation is a four-layer microcapsule synbiotic preparation in which Lactobacillus rhamnosus is embedded in sodium alginate / carrageenan / hyaluronic acid / carrageenan.

[0014] The process of the synbiotic preparation of the present invention comprises the following steps:

[0015] The first step is to add 1 mL of 35% w / v whey protein solution to 10 mL of rhamnosus lactobacillus suspension to obtain a mixture of rhamnosus lactobacillus and whey protein, add 10 mL of rhamnosus lactobacillus and whey protein mixture to 2.5 mL of 1.2 M sodium alginate solution, stir for 30 min at 400 rpm using a magnetic stirrer, add 1 mL of 0.6 M sterile calcium chloride solution during the stirring process; then add 1.5 mL of 2 mol / L Tween 80, stir for 10 min at 3000 rpm using a magnetic stirrer to obtain a mixture I, recorded as microcapsule I;

[0016] Step 2: The mixed solution 1 obtained in the first step was filtered and washed three times, and then 5.0 mL of 0.5 mol / L carrageenan was added and stirred at 200 rpm for 20 min to obtain a mixed solution 2, which was recorded as microcapsule II;

[0017] Step 3: The mixed solution II obtained in step 2 was filtered and washed three times, and then 7.5 mL of 0.5 mol / L hyaluronic acid was added and stirred at 200 rpm for 20 min to obtain mixed solution III, which was recorded as microcapsule III;

[0018] Step 4: The mixed solution III obtained in the third step was filtered and washed three times, and then 10.0 mL of 0.5 mol / L carrageenan was added and stirred at 200 rpm for 20 min to obtain a mixed solution IV, and a four-layer microcapsule synbiotic preparation of sodium alginate / carrageenan / hyaluronic acid / carrageenan encapsulating Lactobacillus rhamnosus was obtained, which was recorded as microcapsule IV;

[0019] Then, the obtained microcapsule synbiotic preparations encapsulating Lactobacillus rhamnosus in each layer are freeze-dried and stored in a -20°C refrigerator for later use.

[0020] Preferably, the preparation of the Lactobacillus rhamnosus suspension is as follows: Lactobacillus rhamnosus is inoculated into a sterile MRS medium at room temperature and incubated in a constant temperature incubator at 37°C for 24 hours; then the Lactobacillus rhamnosus is centrifuged at 3000 rpm for 10 minutes, the supernatant is removed and the precipitate is harvested, the precipitate is washed twice with sterile phosphate buffered saline (PBS), and the washed precipitate is diluted with PBS to a cell concentration of 10 7 CFU / mL, and obtain Lactobacillus rhamnosus suspension.

[0021] Preferably, the preparation of 35% w / v whey protein solution: dissolve an appropriate amount of whey protein solution in a sterile PBS solution, and stir with a magnetic stirrer at 500 rpm for 90 min to prepare a 35% w / v whey protein solution, and sterilize the 35% w / v whey protein solution through a 0.22 μm filter membrane before use.

[0022] Preferably, the preparation of 1.2 mol / L sodium alginate solution: dissolve an appropriate amount of sodium alginate in 50°C distilled water to prepare a 1.2 mol / L sodium alginate solution, stir at 500 rpm for 30 min in a constant temperature water bath magnetic stirrer, sterilize at 121°C for 15 min after the sodium alginate is fully dissolved, and cool to room temperature for later use;

[0023] Preparation of 0.5 mol / L carrageenan: dissolve an appropriate amount of carrageenan in 50°C distilled water to prepare a 0.5 mol / L carrageenan solution, stir at 500 rpm for 30 min in a constant temperature water bath magnetic stirrer, sterilize at 121°C for 15 min after the carrageenan is fully dissolved, and cool to room temperature for later use;

[0024] Preparation of 0.5 mol / L hyaluronic acid solution: Dissolve an appropriate amount of hyaluronic acid in 50°C distilled water to prepare a 0.5 mol / L hyaluronic acid solution, stir at 500 rpm for 30 min in a constant temperature water bath with a magnetic stirrer, sterilize at 121°C for 15 min after the hyaluronic acid is fully dissolved, and cool to room temperature for use.

[0025] The synbiotic preparation obtained by the invention has an embedding rate of 68.51% to 93.73% for lactobacillus rhamnosus and a particle size distribution of 100 to 1000 nm.

[0026] Application of the synbiotic preparation of the present invention in the preparation of a drug for alleviating / preventing age-related macular degeneration.

[0027] Preferably, the synbiotic preparation improves the damage to the retinal pigment epithelium of rats induced by a high-fat diet and blue light, effectively reduces the total cholesterol, phospholipid and triglyceride contents in the retinal pigment epithelium of rats, inhibits the levels of pro-inflammatory cytokines and the production of reactive oxygen species, thereby alleviating / preventing age-related macular degeneration.

[0028] Use of a strain of Lactobacillus rhamnosus for alleviating age-related macular degeneration in the preparation of a drug for alleviating / preventing age-related macular degeneration.

[0029] Preferably, after oral administration, the Lactobacillus rhamnosus with the deposit number of CCTCC NO: M 2024897 exerts a retinal protective effect by reducing the levels of total cholesterol, phospholipids and triglycerides, the levels of pro-inflammatory cytokines and reactive oxygen species in the retinal pigment epithelium.

[0030] The retinal protectant can be used as a drug to prevent and alleviate retinal damage induced by inflammation, oxidative stress, etc., including but not limited to retinal damage induced by blue light radiation and / or hyperlipidemia, age-related macular degeneration; for example, retinal damage induced by blue light, retinal damage induced by hyperlipidemia, etc.

[0031] The Lactobacillus rhamnosus synbiotic preparation obtained by the process of the present invention is a multi-layer microcapsule preparation.

[0032] Synbiotic preparations promote intestinal colonization by Lactobacillus rhamnosus strains.

[0033] The present invention has the following beneficial effects:

[0034] The microcapsule preparation of the present invention has four layers, and sodium alginate, carrageenan, hyaluronic acid, and carrageenan are assembled layer by layer to encapsulate Lactobacillus rhamnosus. The microcapsule preparation of the present invention has an embedding rate of Lactobacillus rhamnosus of 68.51% to 93.73%, and as the number of microcapsule wall material layers increases, the embedding rate of Lactobacillus rhamnosus in the microcapsule gradually increases. The overall particle size distribution of the microcapsule preparation is between 100 and 1000 nm, and as the number of microcapsule layers increases, the maximum intensity of the particle size distribution becomes larger and larger.

[0035] The microcapsule preparation of the present invention is mainly assembled through electrostatic interaction and hydrogen bonding.

[0036] The microcapsule preparation of the present invention has good storage stability, and as the number of microcapsule layers increases, the survival rate of Lactobacillus rhamnosus also increases.

[0037] The microcapsule preparation of the present invention can well protect Lactobacillus rhamnosus from the influence of the external environment and prolong its storage time.

[0038] The DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of the microcapsule preparation are significantly increased, and the microcapsule can effectively promote the health benefits of Lactobacillus rhamnosus.

[0039] Microcapsule preparations can improve the cell viability of Lactobacillus rhamnosus in saliva, gastric juice and intestinal juice.

[0040] The efficiency of releasing Lactobacillus rhamnosus from the microcapsule preparation showed an obvious upward trend in the intestinal fluid stage. The release rates of microcapsules Ⅰ and Ⅱ in simulated saliva and gastric juice were greater than those of microcapsules Ⅲ and Ⅳ. After entering the intestinal fluid, the release rates of microcapsules Ⅲ and Ⅳ increased significantly and were higher than those of microcapsules Ⅰ and Ⅱ. Finally, microcapsule Ⅲ showed the greatest release performance.

[0041] The microcapsule preparation of the present invention can improve the antioxidant activity of Lactobacillus rhamnosus and promote Lactobacillus rhamnosus to exert its health effects.

[0042] With the increase of the number of encapsulation layers of the microcapsule preparation of the present invention, the embedding stability of Lactobacillus rhamnosus is significantly improved, and the ability of Lactobacillus rhamnosus to resist oral and gastric acid is increased.

[0043] The invention provides a multi-layer microcapsule preparation containing Lactobacillus rhamnosus, which can greatly improve the stability, cell viability and release rate of Lactobacillus rhamnosus under oral delivery conditions and improve the colonization of Lactobacillus rhamnosus in the intestine.

[0044] The microcapsule preparation of the present invention can effectively reduce the content of total cholesterol, phospholipids and triglycerides in the retinal pigment epithelium of rats.

[0045] The microcapsule preparation of the present invention can inhibit the level of pro-inflammatory cytokines and the generation of reactive oxygen species, thereby alleviating age-related macular degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 .The encapsulation efficiency of Lactobacillus rhamnosus in microcapsules;

[0047] Figure 2 . Potential (A) and particle size distribution (B) of microcapsule preparations;

[0048] Figure 3 . Storage stability of microcapsule preparations;

[0049] Figure 4 DPPH (A) and hydroxyl radical scavenging ability (B) of microcapsule preparations;

[0050] Figure 5 . Cell viability of Lactobacillus rhamnosus in microcapsule formulations after simulated digestion, where SSF stands for simulated saliva, SGF stands for simulated gastric fluid, and SIF stands for simulated intestinal fluid;

[0051] Figure 6 . Release rate of Lactobacillus rhamnosus in microcapsule formulations during simulated digestion, where SSF stands for simulated saliva, SGF stands for simulated gastric fluid, and SIF stands for simulated intestinal fluid;

[0052] Figure 7.Changes in the contents of total cholesterol (A), phospholipids (C) and triglycerides (B) in rat retinal pigment epithelium;

[0053] Figure 8 . Pathological changes of small intestine in rats, including blank group (A), model group (B), Lactobacillus rhamnosus powder group (C), microcapsule Ⅰ preparation group (D), microcapsule Ⅱ preparation group (E), microcapsule Ⅲ preparation group (F), microcapsule Ⅳ preparation group (G), positive control group (H);

[0054] Fig. 9 .The inhibitory effect of microcapsule preparation on reactive oxygen species in rat retinal pigment epithelium;

[0055] Fig.10 Inflammatory cytokines in rat retinal pigment epithelium, including IL-6 (A), IL-8 (B), IL-1β (C), and TNF-α (D). DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0057] Example 1

[0058] Preparation and characterization of physicochemical properties of microcapsules

[0059] 1. Materials and Methods

[0060] 1.1. Experimental Materials

[0061] 1.1.1. Experimental strains

[0062] Lactobacillus rhamnosus was isolated from traditional yogurt.

[0063] 1.1.2 Experimental strain culture medium

[0064] MRS medium: 12 g peptone, 6 g yeast extract, 6 g beef extract, 18 g glucose, 5 g sodium acetate, 2.15 g ammonium citrate, 1 g Tween 80, 0.58 g magnesium sulfate, 0.05 g manganese sulfate, 2 g dipotassium hydrogen phosphate, make up to 1 L with pure water, adjust the pH to 6.5, sterilize in a high-pressure steam autoclave at 121°C for 15 min for later use.

[0065] 1.1.3. Experimental instruments and equipment

[0066] Ultra Turrax IKAI25 Agitator

[0067] 1.2 Experimental methods

[0068] 1.2.1. Microbial activation and preparation of bacterial suspension

[0069] Lactobacillus rhamnosus was stored at -80°C in 20% glycerol (v / v). Before the experiment, Lactobacillus rhamnosus was inoculated in sterile MRS medium at room temperature and incubated in a constant temperature incubator at 37°C for 24 h for three generations; then Lactobacillus rhamnosus was centrifuged at 3000 rpm for 10 min, the supernatant was removed and the precipitate was harvested, and the precipitate was washed twice with sterile phosphate buffered saline (PBS). The washed precipitate was diluted with PBS to a cell concentration of 10 7 CFU / mL, and obtain Lactobacillus rhamnosus suspension.

[0070] 1.2.2. Preparation of synbiotic preparations

[0071] Preparation of 35% (w / v) whey protein solution: Dissolve an appropriate amount of whey protein solution in sterile PBS solution and stir with a magnetic stirrer at 500 rpm for 90 min to prepare a 35% (w / v) whey protein solution. Before use, the 35% (w / v) whey protein solution was sterilized by passing through a 0.22 μm filter membrane.

[0072] Preparation of 1.2 mol / L sodium alginate solution: dissolve an appropriate amount of sodium alginate in 50°C distilled water to prepare a 1.2 mol / L sodium alginate solution, stir at 500 rpm for 30 min in a constant temperature water bath with a magnetic stirrer, sterilize at 121°C for 15 min after the sodium alginate is fully dissolved, and cool to room temperature for use.

[0073] The preparation method of 0.5 mol / L carrageenan and 0.5 mol / L hyaluronic acid solution is the same as that of 1.2 mol / L sodium alginate solution.

[0074] Preparation of synbiotic preparations:

[0075] The first step is to add 1 mL of 35% w / v whey protein solution to 10 mL of rhamnosus lactobacillus suspension to obtain a mixture of rhamnosus lactobacillus and whey protein, add 10 mL of rhamnosus lactobacillus and whey protein mixture to 2.5 mL of 1.2 M sodium alginate solution, stir for 30 min at 400 rpm using a magnetic stirrer, add 1 mL of 0.6 M sterile calcium chloride solution during the stirring process; then add 1.5 mL of 2 M Tween 80, stir for 10 min at 3000 rpm using a magnetic stirrer to obtain a mixture I, recorded as microcapsule I;

[0076] Step 2: The mixed solution 1 obtained in the first step was filtered and washed three times, and then 5 mL of 0.5 M carrageenan was added and stirred at 200 rpm for 20 min to obtain mixed solution 2, which was recorded as microcapsule II;

[0077] Step 3: The mixed solution II obtained in step 2 was filtered and washed three times, and then 7.5 mL of 0.5 M hyaluronic acid was added and stirred at 200 rpm for 20 min to obtain mixed solution III, which was recorded as microcapsule III;

[0078] Step 4: The mixed solution III obtained in step 3 was filtered and washed three times, and then 10 mL of 0.5 M carrageenan was added and stirred at 200 rpm for 20 min to obtain a mixed solution IV, and a four-layer microcapsule synbiotic preparation of sodium alginate / carrageenan / hyaluronic acid / carrageenan encapsulating Lactobacillus rhamnosus was obtained, which was recorded as microcapsule IV;

[0079] Then, the obtained microcapsule synbiotic preparations encapsulating Lactobacillus rhamnosus were freeze-dried and stored in a -20°C refrigerator for later use (due to the needs of subsequent experiments, the stored materials here are microcapsules I, II, III and IV).

[0080] 1.2.3. Determination of the encapsulation efficiency of Lactobacillus rhamnosus

[0081] The microcapsules were added to 15 mL of preheated 0.1 M, pH 7.4 phosphate buffer. Then centrifuged at 4000 r / min for 20 min to completely release the embedded Lactobacillus rhamnosus. The sample was diluted in sterile saline solution and then inoculated on MRS agar. The number of live bacteria was calculated after culturing at 37°C under anaerobic conditions for 48 h. The formula for calculating the embedding rate is:

[0082]

[0083] Where: C is the number of live cells embedded in the microcapsule (log CFU / mL); C 0 is the total number of viable cells in the bacterial suspension (log CFU / mL).

[0084] 1.2.4. Zeta potential and particle size determination of microcapsule preparations

[0085] 0.5 g of each microcapsule was weighed, dispersed in 10 mL of ultrapure water, and stirred at 300 r / min for 10 min to prepare a microcapsule re-dissolved suspension. The particle size and Zeta potential of the microcapsules were measured using a Malvern Zetasizer (ZS90, Malvern Instruments Ltd., UK).

[0086] 1.2.5. Storage stability of microcapsule preparations

[0087] To simulate the long-term storage properties of multilayer microcapsules encapsulating Lactobacillus rhamnosus, multilayer microcapsules loaded with Lactobacillus rhamnosus and free cells suspended in 0.1 M, pH 7.4 phosphate buffer were stored at 4°C for 31 days under aerobic conditions. The experimental process was carried out in a fume hood with loosely sealed samples to keep the oxygen level close to that of air. Cell viability was tested after 2, 4, 7, 11, 16, 22 and 31 days.

[0088] 1.2.6. Antioxidant properties of microcapsule preparations

[0089] The freeze-dried powder of each layer of microcapsules encapsulating Lactobacillus rhamnosus was mixed into a solution (0.5 mg / mL) and a 0.1 mM DPPH ethanol solution, and the mixture was kept in a dark place for 30 min. The wavelengths of sample group A and sample group B were read at a wavelength of 528 nm. S , control group A C and blank group A 0 In addition, in order to determine the hydroxyl radical scavenging ability of the microcapsule preparation, the above-mentioned microcapsule solutions encapsulating Lactobacillus rhamnosus were mixed with 9mM ferrous sulfate solution, 9mM salicylic acid-ethanol solution, and 8.8mM hydrogen peroxide solution, and kept in the dark for 30min. The absorbance values ​​of sample group A and sample group B were read at a wavelength of 510nm. S , control group A C and blank group A 0 The ratio of each group is shown in Table 1. The DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of each layer of microcapsules encapsulating Lactobacillus rhamnosus were calculated by the following formula.

[0090]

[0091] Table 1 DPPH and hydroxyl radical scavenging ability ratio of each group of reagents

[0092]

[0093] 1.2.7. Data processing

[0094] The experimental data are expressed as mean ± standard deviation. The data were repeated at least 3 times. SPSS 27.0 software was used to perform one-way analysis of variance on the data between different groups. The Spearman correlation coefficient was used to evaluate the correlation between two statistical variables, and GraphPad Prism 8.0 and Origin 2022 software were used for drawing. p < 0.05 indicated a significant difference.

[0095] 2. Results and Analysis

[0096] 2.1. Encapsulation efficiency of Lactobacillus rhamnosus

[0097] Depend on Figure 1 It can be seen that the encapsulation rate of microcapsules for Lactobacillus rhamnosus is between 68.51% and 93.73%, and with the increase in the number of microcapsule wall material layers, the encapsulation rate of microcapsules for Lactobacillus rhamnosus gradually increases, which indicates that more layers of microcapsules can provide more adsorption surface area for Lactobacillus rhamnosus; at the same time, this also indirectly indicates the formation of multi-layer microcapsules.

[0098] Zeta potential and particle size of microcapsule preparations

[0099] The size and particle size distribution of microcapsule particles have a great influence on the bioavailability of synbiotic preparations. Smaller particles are more easily adhered to biological tissues and have a larger specific surface area, which increases the contact area between the preparation and the surrounding environment. The more uniform the particle size of the microcapsule particles, the more evenly the probiotics can be wrapped in the microcapsule particles, thereby providing a stable environment for the probiotics and playing a protective role, preventing the probiotics from being damaged by the harsh environment of the gastrointestinal tract and reducing the occurrence of adverse reactions. Figure 2 As shown in A, the zeta potential of free Lactobacillus rhamnosus is -17.81 mV. This is mainly because Lactobacillus rhamnosus belongs to Gram-positive bacteria. As we all know, the cell wall of Gram-positive bacteria is mainly composed of peptidoglycan and acidic polysaccharides containing teichoic acid. Teichoic acid is negatively charged, resulting in a negative charge on the cell surface as a whole. 2+ Crosslinked with sodium alginate to form microcapsule I, which is negatively charged as a whole. With the addition of carrageenan, the zeta potential of microcapsule II is 6.85mV, and microcapsules III and IV also show positive and negative potential fluctuations, which indicates that the aggregation force between microcapsules I, microcapsules II, microcapsules III and IV is electrostatic interaction. Figure 2 B shows the particle size distribution of microcapsules with different numbers of layers. The overall particle size distribution of the microcapsules is between 100 and 1000 nm. As the number of microcapsule layers increases, the maximum intensity of the particle size distribution becomes larger and larger, which indicates that the particle size of the microcapsules increases with the number of layers.

[0100] 2.3. Storage stability of microcapsule preparations

[0101] The effect of the probiotic delivery system on the cell viability of the drug during storage is crucial to the shelf life of the product. Figure 3As shown in the figure, the survival rate of free Lactobacillus rhamnosus on the 31st day was only 40.13% of the initial survival rate. After Lactobacillus rhamnosus was embedded in microcapsules, its survival rate increased significantly, with a maximum increase of 38.09%. This shows that microcapsules can well protect Lactobacillus rhamnosus from the influence of the external environment and prolong its storage time. Moreover, with the increase of the number of microcapsule layers, the survival rate of Lactobacillus rhamnosus also increased, indicating that with the increase of wall material, the protective effect of microcapsules on Lactobacillus rhamnosus is more obvious.

[0102] 2.4. Antioxidant properties of microcapsule preparations

[0103] like Figure 4 As shown in the figure, compared with free Lactobacillus rhamnosus, the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of the microcapsule preparation increased significantly, with the maximum increase of 37.01% and 23.23% respectively, indicating that microcapsules can effectively promote Lactobacillus rhamnosus to play its health effect. In addition, with the increase of the number of layers of the embedding wall material, the DPPH free radical scavenging ability of the microcapsule preparation gradually increased, from 63.47% of microcapsule I to 81.54% of microcapsule IV; the hydroxyl free radical scavenging ability of microcapsule III was the largest, which increased by 16.88% compared with microcapsule I, indicating that the increase of microcapsule wall material can improve the antioxidant activity of Lactobacillus rhamnosus.

[0104] In vitro digestion simulation of microcapsules

[0105] 1. Materials and Methods

[0106] 1.1. Experimental Materials

[0107] Potassium chloride (KCl, purity 98%), mucin, α-amylase, pepsin, trypsin.

[0108] 1.2 Experimental methods

[0109] 1.2.1. Preparation of simulated digestive fluid and digestion simulation steps

[0110] Preparation of simulated saliva (SSF): 12.0 mg NaCl, 15.0 mg KCl, 100.0 mg mucin and 7.5 mg α-amylase were dissolved in 50.0 mL distilled water to prepare artificial saliva.

[0111] Preparation of simulated gastric fluid (SGF): 12.0 mg NaCl, 15.0 mg KCl and 100.0 mg mucin were mixed with 7.5 mg α-amylase in human saliva, and then the mixture was dissolved in 50.0 mL of 2.0 mg / mL pepsin solution. After suspension, 0.1 M HCl was added to adjust the pH to 2.0 to obtain artificial gastric fluid.

[0112] Simulated intestinal fluid (SIF) was prepared by placing 4.0 mg / mL bile salts, 2.0 mg / mL trypsin and 1.0 mg / mL lipase in 8.0 M PBS adjusted to pH 1, containing 0.1 M HCl.

[0113] Simulation steps:

[0114] The control group was composed of freeze-dried powder of Lactobacillus rhamnosus, and the experimental group was composed of microcapsule preparations of various layers encapsulated with Lactobacillus rhamnosus.

[0115] 2g of each layer of microcapsule synbiotic preparation encapsulated with Lactobacillus rhamnosus was inoculated into a shake flask containing 20mL SSF, incubated in a 37°C incubator for 6min, and then centrifuged (1200rpm, 5min) to obtain the microcapsule synbiotic preparation after saliva digestion, and the synbiotic preparation after saliva digestion was added to a shake flask containing 20mL SGF and placed in a constant temperature shaker (37°C, 3rpm, 2h). Centrifugation (1200rpm, 5min) was used to obtain the microcapsule synbiotic preparation digested by gastric juice, and it was added to a shake flask containing 20mL SIF, placed in a constant temperature shaker (37°C, 2rpm, 4h), and then the microcapsule synbiotic preparation digested by intestinal juice was harvested. Samples were taken once every 1 hour during gastrointestinal digestion, and cell viability was determined by viable count, unit log CFU / mL.

[0116] 1.2.2. Determination of the release rate of Lactobacillus rhamnosus

[0117] At each stage of digestion, only the corresponding digestion fluid was collected, and the cells were counted using the above method. Then, the release rate of Lactobacillus rhamnosus during the digestion process was calculated according to the following formula.

[0118]

[0119] Data processing

[0120] The experimental data are expressed as mean ± standard deviation. Each sample was repeated at least 3 times. SPSS27.0 software was used to perform one-way analysis of variance on the data between different groups. The Spearman correlation coefficient was used to evaluate the correlation between two statistical variables, and GraphPad Prism 8.0 and Origin 2022 software were used for drawing graphs. p<0.05 indicated a significant difference.

[0121] 2. Results and Analysis

[0122] 2.1. Cell viability of Lactobacillus rhamnosus

[0123] The cell survival rate is an important indicator for evaluating whether microencapsulated probiotics have probiotic effects in vivo, such as Figure 5As shown in the figure, after simulated gastrointestinal digestion, the cell viability of free Lactobacillus rhamnosus is close to 0, while the cell viability of Lactobacillus rhamnosus encapsulated in microcapsules reaches a maximum of 5.53 CFU / mL, indicating that microcapsules can effectively improve the cell viability of Lactobacillus rhamnosus in saliva, gastric juice and intestinal juice, and the cell viability of Lactobacillus rhamnosus is positively correlated with the number of layers of microcapsule wall material. It is worth mentioning that the microcapsule preparation exhibits a very excellent protective effect during gastric juice digestion. During the gastric juice digestion stage, the cell viability of free Lactobacillus rhamnosus decreased from the initial 6.51% to 1.06%, while the Lactobacillus rhamnosus in the microcapsule preparation decreased by a maximum of 2.48% during this stage.

[0124] 2.2. Release rate of Lactobacillus rhamnosus

[0125] Probiotics must eventually colonize in the intestines to exert their health benefits. Therefore, the efficiency of probiotic delivery carriers in releasing probiotics after reaching the intestines is an important indicator for evaluating the performance of the carrier. Figure 6 As shown in the figure, the efficiency of releasing Lactobacillus rhamnosus from the microcapsule preparation showed a significant upward trend in the intestinal fluid stage. The release rates of microcapsules Ⅰ and Ⅱ in simulated saliva and gastric fluid were greater than those of microcapsules Ⅲ and Ⅳ. After entering the intestinal fluid, the release rates of microcapsules Ⅲ and Ⅳ increased significantly and were higher than those of microcapsules Ⅰ and Ⅱ. Interestingly, microcapsule Ⅲ finally showed the greatest release performance. The above phenomenon may indicate that too few or too many layers of microcapsule wall material cannot achieve the ideal release effect of Lactobacillus rhamnosus. Too few layers may lead to premature release in the oral cavity or gastric fluid, while too many layers may not lead to Lactobacillus rhamnosus being released from the microcapsule until the digestion and absorption in the small intestine is completed.

[0126] Animal experiments

[0127] 1. Materials and Methods

[0128] 1.1. Experimental Materials

[0129] Hexane (purity 99.0%, Shanghai Yuanye Biotechnology Co., Ltd., China), isopropanol (purity ≥99.9%, Shanghai MacLean Biochemical Technology Co., Ltd., China), 2,7-dichlorofluorescein diacetate (purity ≥97%, Sigma-Aldrich, UK), Conbercept ophthalmic injection (Chengdu Kanghong Biotechnology Co., Ltd., national medicine standard S20130012), Amplex Red cholesterol test kit (Thermo Fisher, UK), phospholipid test kit (Sigma-Aldrich, UK) and EnzyChrom triglyceride test kit (BioAssay system, UK).

[0130] 1.2 Experimental methods

[0131] 1.2.1. Animal experiment design

[0132] 20-month-old female SD rats (weight 300-400 g) were purchased from the Second Affiliated Hospital of Harbin Medical University and housed in a specific pathogen-free standard laboratory: 25±2℃, 50±5% humidity, 12h / 12h light-dark cycle. After 1 week of adaptive feeding, they were randomly divided into blank group (A), model group (B), Lactobacillus rhamnosus powder group (C), microcapsule I preparation group (D), microcapsule II preparation group (E), microcapsule III preparation group (F), microcapsule IV preparation group (G), and positive control group (H), with 6 rats in each group. The feeding methods and dosages of each group are shown in Table 2. After 9 weeks, all SD female rats were humanely euthanized after fasting for 24 hours, and blood samples were collected and serum was separated; mouse eyeballs were collected, the anterior segment and neural retina were removed, and the retinal pigment epithelium was biochemically analyzed. The small intestine and its contents were collected and stored at -80℃.

[0133] Table 2 Rat feeding method

[0134]

[0135] 1.2.2. Determination of rat organ index

[0136] After the rats were dissected, the heart, spleen, liver, and kidneys were separated, washed with physiological saline, and the surface moisture was absorbed with filter paper. Then, they were quickly weighed and the organ index was calculated according to the following formula:

[0137]

[0138] 1.2.3. Pathological observation of rat small intestine

[0139] The changes of intestinal pathological structure were observed by hematoxylin-eosin (HE) staining.

[0140] 1.2.4. Measurement of total cholesterol, phospholipids and triglycerides in rat retinal pigment epithelium

[0141] Total cholesterol, phospholipids, and triglycerides were determined by Amplex Red cholesterol assay kit, phospholipid assay kit, and EnzyChrom triglyceride assay kit, respectively. Lipids were extracted from rat retinal pigment epithelium / choroid using n-hexane:isopropanol (3:2, v / v), the extracts were centrifuged at 1500×g for 10 min, the organic phase of the extracts was transferred to a new tube, dried under nitrogen to remove the organic solvent, the dried lipids were resuspended in the corresponding assay buffer, and then total cholesterol, phospholipids, and triglycerides were determined according to the instructions.

[0142] 1.2.5. Measurement of reactive oxygen species and inflammatory cytokines in rat retinal pigment epithelium

[0143] According to the manufacturer's instructions, the total reactive oxygen species in rat retinal pigment epithelium were quantified using the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA). Briefly, rat retinal pigment epithelial cells were collected and prepared into a cell suspension; 200 μL of 10 μM DCFH-DA was added to the cell suspension and incubated at 37°C for 30 min. After incubation, the cells were rinsed three times with 0.01 M PBS, and then the fluorescence signal was measured using a FluoStar Optima MBG-Labtech fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Inflammatory factors IL-6, IL-1β, IL-8, and TNF-α were determined using the corresponding kits.

[0144] Data processing

[0145] The experimental data are expressed as mean ± standard deviation. The data were repeated at least 3 times. SPSS 27.0 software was used to perform one-way analysis of variance on the data between different groups. The Spearman correlation coefficient was used to evaluate the correlation between two statistical variables, and GraphPad Prism 8.0 and Origin 2022 software were used for drawing. p < 0.05 indicated a significant difference.

[0146] 2. Results and Analysis

[0147] 2.1. Organ index

[0148] In order to explore whether the high-fat diet and daily blue light induction have negative effects on rat organs, the organ indexes of rats were measured after the experiment. The results are shown in Table 3. There was no significant difference in the organ indexes of all groups, indicating that the experimental modeling method has no toxic effect on rats, and the microcapsule wall material has no significant toxic effect on rats.

[0149] Table 3 Effects of different interventions on organ indexes in rats

[0150]

[0151] Note: All data in the table are mean ± standard deviation; different letters in the same column indicate significant differences (p<0.05).

[0152] 2.2. Total cholesterol, phospholipids and triglycerides in rat retinal pigment epithelium

[0153] Cholesterol is the major sterol in the retina and is widely distributed in drusen, a hallmark of early age-related macular degeneration. Abnormal lipid metabolism and dyslipidemia are recognized risk factors for the development of cardiovascular disease, obesity, and age-related macular degeneration. Figure 7 As shown in A, starting from a high-fat diet and daily blue light induction for 4 hours, by the end of the 8th week of the experiment, the total cholesterol content in the retinal pigment epithelium of the model group rats was 29.04μg / mg higher than that of the blank group, with a significant difference (p < 0.05); compared with the blank group, the total cholesterol content of the Lactobacillus rhamnosus powder group was 16.13μg / mg higher, with a significant difference (p < 0.05); while the total cholesterol content in the retinal pigment epithelium of the microcapsule group and the positive control group rats was significantly lower than that of the model group and the Lactobacillus rhamnosus powder group, and there was no significant difference compared with the blank group. In addition, triglycerides ( Figure 7 .B) and phospholipids ( Figure 7 .C) content and total cholesterol ( Figure 7 .A) The changes in the content were similar. This indicates that the microcapsule preparation can effectively improve the damage of the retinal pigment epithelium of rats induced by high-fat diet and blue light.

[0154] 2.3. Rat small intestinal histopathology

[0155] The morphological and structural integrity of the animal intestine is closely related to its ability to resist pathogens and absorb nutrients. Maintaining the integrity of the basic structure of the intestine plays a vital role in maintaining normal intestinal function. The results of HE staining reflect the histopathological changes of the small intestine of rats in each experimental group, and are used to evaluate the effects of microcapsule preparations on the intestinal mucosal tissue structure and villi. Figure 8 Compared with the blank group ( Figure 8 .A), Lactobacillus rhamnosus powder group ( Figure 8 .C), microcapsule preparation group ( Figure 8 .DG) and positive control group ( Figure 8 .H) The intestinal mucosal tissue structure of rats was intact, without necrosis or shedding, the glands were neatly arranged, without inflammation or necrosis, the villi were neatly arranged and tight, with uniform thickness, and no significant difference; while the model group ( Figure 8 .B) The villi and glands of the small intestine of rats are abnormal. As we all know, the intestine and the retina are closely related and affect each other, so this may be caused by high-fat diet and blue light induction. The above HE staining results show that the microcapsule preparation does not affect the normal physiological structure of the small intestine of rats.

[0156] 2.4. Inhibition of Reactive Oxygen Species in Rat Retinal Pigment Epithelium by Microcapsule Preparation

[0157] The retina is prone to oxidative stress, which may increase with age. This is due to the large amount of light reaching the photoreceptor membranes, which contain a large amount of polyunsaturated fatty acids, which are the main substrates for oxidation. The photoreceptor membrane is unique in that 50% is composed of the phospholipid docosahexaenoic acid, which is the fatty acid with the highest content of polyunsaturated fatty acids naturally occurring. Because the phospholipid docosahexaenoic acid has a large number of double bonds, it is particularly susceptible to lipid peroxidation, which can disrupt its barrier function and ultimately lead to photoreceptor degeneration. Photosensitizers are chemicals that absorb light to promote chemical reactions, and they cause photochemical damage to the retina and retinal pigment epithelium by releasing reactive oxygen species when they absorb visible or UV light. As Fig. 9 As shown, the fluorescence intensity of the model group was significantly greater than that of the blank group, indicating that high-fat diet and blue light induced oxidative damage to the retinal pigment epithelium of rats; compared with the blank group, there was no significant difference in the fluorescence intensity between the microcapsule preparation group and the positive control group, which shows that the microcapsule preparation can effectively inhibit the oxidative damage caused by high-fat diet and blue light, and has an excellent protective effect on the retinal pigment epithelium of rats.

[0158] 2.5. Microcapsule preparation inhibits inflammatory cytokines in rat retinal pigment epithelium

[0159] Fig.10 The effect of microcapsule preparation on retinal pigment epithelial inflammation in rats was shown. Compared with the blank group, the inflammatory factor IL-6 ( Fig.10 .A) and IL-8( Fig.10 .B) IL-1β( Fig.10 .C) and TNF-α( Fig.10 .D) increased by 77.78%, 131.25%, 74.36% and 29.17% respectively, with significant differences (p < 0.05); the inflammatory factor IL-6 ( Fig.10 .A) and IL-8( Fig.10 .B) IL-1β( Fig.10 .C) and TNF-α( Fig.10 .D) was higher than that of the blank group, with significant difference (p<0.05); IL-6 ( Fig.10 .A) and IL-8( Fig.10 .B) and TNF-α( Fig.10 .D) compared with the blank group, interestingly, the microcapsule preparation group IL-1β ( Fig.10.C) content compared with the blank group and the model group, there are significant differences (p < 0.05), and no significant difference with the positive control group. Based on the above experimental results, it can be found that high-fat diet and blue light induction can cause inflammation of the retinal pigment epithelium of rats, and the microcapsule preparation can effectively prevent the inflammatory reaction and play a protective role on the retinal pigment epithelium of rats.

[0160] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus that relieves age-related macular degeneration ( Lactobacillus rhamnosus ) YBT20, characterized in that, Lactobacillus rhamnosus was deposited in China Center for Type Culture Collection, Wuhan, with a deposit date of May 10, 2024 and a deposit number of CCTCC NO: M 2024897.

2. A synbiotic preparation, characterized in that: The synbiotic preparation is a four-layer microcapsule synbiotic preparation of sodium alginate / carrageenan / hyaluronic acid / carrageenan encapsulating Lactobacillus rhamnosus, and the Lactobacillus rhamnosus is the Lactobacillus rhamnosus for relieving age-related macular degeneration according to claim 1 ( Lactobacillus rhamnosus )YBT20.

3. A method for preparing a synbiotic preparation, characterized in that: The following steps are involved: Step 1: Add 1 mL of 35% w / v whey protein solution to 10 mL of rhamnosus lactobacillus suspension to obtain a mixture of rhamnosus lactobacillus and whey protein, add 10 mL of rhamnosus lactobacillus and whey protein mixture to 2.5 mL of 1.2 M sodium alginate solution, stir for 30 min at 400 rpm using a magnetic stirrer, add 1 mL of 0.6 M sterile calcium chloride solution during stirring; then add 1.5 mL of 2 mol / L Tween 80, stir for 10 min at 3000 rpm using a magnetic stirrer to obtain a mixture I, recorded as microcapsule I; Step 2: The mixed solution 1 obtained in the first step was filtered and washed three times, and then 5.0 mL of 0.5 mol / L carrageenan was added and stirred at 200 rpm for 20 min to obtain mixed solution 2, which was recorded as microcapsule II; Step 3: The mixed solution II obtained in step 2 was filtered and washed three times, and then 7.5 mL of 0.5 mol / L hyaluronic acid was added and stirred at 200 rpm for 20 min to obtain mixed solution III, which was recorded as microcapsule III; Step 4: The mixed solution III obtained in step 3 was filtered and washed three times, and then 10.0 mL of 0.5 mol / L carrageenan was added and stirred at 200 rpm for 20 min to obtain a mixed solution IV, and a four-layer microcapsule synbiotic preparation of sodium alginate / carrageenan / hyaluronic acid / carrageenan encapsulating Lactobacillus rhamnosus was obtained, which was recorded as microcapsule IV; Then, the obtained microcapsule synbiotic preparations encapsulating Lactobacillus rhamnosus are freeze-dried and stored in a -20°C refrigerator for later use; The synbiotic preparation is the synbiotic preparation according to claim 2.

4. The method of synbiotic preparation according to claim 3, characterized in that: Preparation of Lactobacillus rhamnosus suspension: Lactobacillus rhamnosus was inoculated into sterile MRS medium at room temperature and incubated in a constant temperature incubator at 37 °C for 24 h; then Lactobacillus rhamnosus was centrifuged at 3000 rpm for 10 min, the supernatant was removed and the precipitate was harvested, and the precipitate was washed twice with sterile phosphate buffered saline (PBS), and the washed precipitate was diluted with PBS to a cell concentration of 10 7 CFU / mL, and obtain Lactobacillus rhamnosus suspension.

5. The method of synbiotic preparation according to claim 3, characterized in that: Preparation of 35% w / v whey protein solution: Dissolve an appropriate amount of whey protein solution in sterile PBS solution and stir with a magnetic stirrer at 500 rpm for 90 min to prepare a 35% w / v whey protein solution. Before use, the 35% w / v whey protein solution was sterilized by passing through a 0.22 μm filter membrane.

6. The method of synbiotic preparation according to claim 3, characterized in that: Preparation of 1.2 mol / L sodium alginate solution: Dissolve an appropriate amount of sodium alginate in 50 ℃ distilled water to prepare a 1.2 mol / L sodium alginate solution, stir at 500 rpm for 30 min in a constant temperature water bath magnetic stirrer, sterilize at 121 ℃ for 15 min after the sodium alginate is fully dissolved, and cool to room temperature for use; Preparation of 0.5 mol / L carrageenan: Dissolve an appropriate amount of carrageenan in 50 °C distilled water to prepare a 0.5 mol / L carrageenan solution, stir at 500 rpm for 30 min in a constant temperature water bath magnetic stirrer, sterilize at 121 °C for 15 min after the carrageenan is fully dissolved, and cool to room temperature for later use; Preparation of 0.5 mol / L hyaluronic acid solution: Dissolve an appropriate amount of hyaluronic acid in 50 ℃ distilled water to prepare a 0.5 mol / L hyaluronic acid solution, stir at 500 rpm for 30 min in a constant temperature water bath with a magnetic stirrer, sterilize at 121 ℃ for 15 min after the hyaluronic acid is fully dissolved, and cool to room temperature for use.

7. The synbiotic preparation obtained by the method for preparing a synbiotic preparation according to any one of claims 3 to 6, characterized in that: The encapsulation rate of synbiotic preparation for Lactobacillus rhamnosus was between 68.51% and 93.73%, and the particle size distribution of synbiotic preparation was between 100 and 1000 nm.

8. Use of the synbiotic preparation according to claim 2 in the preparation of a drug for alleviating / preventing age-related macular degeneration.

9. The use according to claim 8, characterized in that: Synbiotic preparations improve the damage to the retinal pigment epithelium of rats induced by a high-fat diet and blue light, effectively reduce the levels of total cholesterol, phospholipids and triglycerides in the retinal pigment epithelium of rats, inhibit the levels of proinflammatory cytokines and the production of reactive oxygen species, thereby alleviating / preventing age-related macular degeneration.

10. The strain of Lactobacillus rhamnosus for alleviating age-related macular degeneration according to claim 1 ( Lactobacillus rhamnosus ) Application of YBT20 in the preparation of drugs for alleviating / preventing age-related macular degeneration.

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