A strain of Lactobacillus plantarum with uric acid-lowering effect, microcapsule, preparation method and application thereof

Abstract

The present invention belongs to the technical field of microorganisms and hyperuricemia disease prevention and treatment, and discloses a plant lactobacillus with uric acid lowering effect, a microcapsule, a preparation method and an application thereof, which is named SHS01, and the classification name is: plant lactobacillus (Lactobacillus plantarum), the deposit number is: CGMCC No.31000, the deposit date is: June 19, 2024, and the deposit unit is: General Microbiological Center of China Microbiological Culture Collection Administration Committee, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. In the in vitro degradation, the plant lactobacillus SHS01 of the present invention has a degradation rate of 89.44% for inosine and a degradation rate of 100% for guanosine. Therefore, as a plant lactobacillus that can efficiently degrade nucleosides in vitro, it has broad application prospects in lowering uric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and hyperuricemia disease prevention and treatment, in particular to a plant lactobacillus strain with uric acid-lowering effect, a microcapsule, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a metabolic disorder caused by abnormal uric acid metabolism. It not only causes joint damage, but also increases the risk of metabolic diseases, cardiovascular diseases, kidney diseases and diabetes. These diseases are all related to excessive purine intake and can be life-threatening in severe cases. Uric acid is the final metabolite produced by purine metabolism. The causes of hyperuricemia can usually be divided into two aspects: increased uric acid production and reduced uric acid excretion. This process is affected by many factors, including genetics and lifestyle habits. Generally speaking, about two-thirds of the body's uric acid is excreted through the kidneys, and the other one-third is excreted through the intestines. Today, the mechanism of action of uric acid-lowering drugs mainly covers reducing the synthesis of uric acid, increasing the decomposition of uric acid, and promoting the excretion of uric acid in the kidneys. Common uric acid-lowering drugs include allopurinol, febuxostat and benzbromarone.

[0003] Commonly used drugs for the treatment of hyperuricemia not only cause side effects and enhance human drug resistance, but also cause harm to the human body in severe cases. Allopurinol reduces the synthesis of uric acid by inhibiting the activity of xanthine oxidase, but surveys show that allopurinol has a fatal risk, especially for Asians. Febuxostat, like allopurinol, is an effective inhibitor of xanthine oxidase and is better than allopurinol in inhibiting uric acid synthesis. However, some reports indicate that febuxostat may increase the mortality rate of cardiovascular disease and also cause adverse reactions such as muscle pain. Benzbromarone can inhibit the repeated absorption of uric acid by the renal tubules, thereby reducing uric acid, but studies have shown that benzbromarone can cause diseases such as kidney stones and liver poisoning. Existing drugs that promote uric acid excretion mainly act on the kidneys, which will increase the renal burden of patients with chronic kidney disease. Therefore, the intestine has great potential for lowering uric acid. As an important physiological flora in the human intestine, probiotics have been shown in a large number of studies to improve uric acid levels. Therefore, regulating uric acid levels through probiotics is a safer and more effective way.

[0004] Lactobacillus plantarum is a type of lactic acid bacteria. Its health functions that have been experimentally proven include: immune regulation, antagonism against pathogenic bacteria, and prevention of cardiovascular diseases by lowering serum cholesterol. In addition, Lactobacillus plantarum has many functions such as maintaining the balance of intestinal flora, promoting nutrient absorption, alleviating lactose intolerance, and inhibiting the formation of tumor cells, and has broad application prospects. However, there are still few strains and studies on Lactobacillus plantarum as a probiotic for hyperuricemia. Therefore, it is of great significance to find Lactobacillus plantarum that can relieve hyperuricemia and further explore its conditioning effect on hyperuricemia and such metabolic diseases.

[0005] Through searching, the following patent disclosure document related to the patent application of the present invention was found: A strain of Lactobacillus plantarum capable of alleviating hyperuricemia and its use (CN112795500A). The Lactobacillus plantarum is Lactobacillus plantarum grx03, which was isolated from the intestines of people in the longevity village of Bama, Guangxi. The strain of the present invention fermented the traditional Chinese herbal medicine chicory and showed good uric acid-lowering ability, and can be combined with other probiotic lactic acid bacteria to develop uric acid-lowering fermented milk products or lactic acid bacteria beverages.

[0006] The uric acid-lowering ability of Lactobacillus plantarum shown in the above patent disclosure is reflected in the uric acid-lowering ability of the strain in vitro in synergy with fermented chicory. The present invention further tests the uric acid-lowering ability of Lactobacillus plantarum itself at the cellular level, and the present invention provides a safe and effective system to achieve the delivery of Lactobacillus plantarum, so that it can smoothly reach the intestine and colonize in large quantities, providing a new way to enhance the activity of probiotics and improve uric acid levels. In addition, the above patent disclosure only focuses on the treatment and relief of Lactobacillus plantarum as a probiotic in hyperuricemia.

[0007] Therefore, by comparison, the patent application of the present invention is essentially different from the above-mentioned patent publications. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a strain of Lactobacillus plantarum with uric acid-lowering effect, a microcapsule and a preparation method and application thereof.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] A strain of Lactobacillus plantarum with uric acid-lowering effect, named SHS01, classified as Lactobacillus plantarum, with a preservation number of CGMCC No.31000, a preservation date of June 19, 2024, and a preservation unit of China National Culture Collection Center for General Microbiology (CGMCC for short), No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0011] Furthermore, the plant lactobacillus has the ability to degrade inosine and guanosine in vitro.

[0012] Furthermore, the morphological characteristics of the plant lactobacillus are:

[0013] Lactobacillus plantarum is a type of lactic acid bacteria, which is straight or curved rod-shaped, either alone, in pairs or in chains;

[0014] It forms small white colonies with slightly raised round outlines on MRS broth agar medium;

[0015] Lactobacillus plantarum is a facultative anaerobic bacteria with an optimum growth temperature of 37°C and an optimum pH of 7.0.

[0016] Furthermore, the formula of the MRS broth agar medium is: 10.0 g of peptone, 8.0 g of beef powder, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 1.0 g of Tween 80, 20 g of agar, 1000 mL of distilled water is fully dissolved and the pH value is adjusted to 7.0, and sterilized at 121°C and 0.1 MPa for 20 min.

[0017] Use of the Lactobacillus plantarum as described above in the preparation of drugs for degrading inosine and / or guanosine.

[0018] A microcapsule for improving the activity and adverse environmental resistance of Lactobacillus plantarum as described above.

[0019] Furthermore, the embedding rate of Lactobacillus plantarum in the microcapsule is 84.49%.

[0020] The method for preparing the microcapsules as described above comprises the following steps:

[0021] The Lactobacillus plantarum SHS01 bacterial suspension was centrifuged at 8000 r / min for 10 minutes at 4°C, the bacterial cells were collected, and resuspended with physiological saline. This process was repeated twice, and the obtained bacterial suspension was placed in a 4°C refrigerator for standby use; the bacterial suspension was diluted to a suitable concentration and the colony count was performed using the plate counting method;

[0022] After sterilizing a 2% sodium alginate solution and a 2% wolfberry polysaccharide solution, the two were mixed in a volume ratio of 1:1 to prepare a sodium alginate-wolfberry polysaccharide solution; the bacterial suspension was added to the sodium alginate-wolfberry polysaccharide solution, and the volume ratio of the bacterial suspension to the sodium alginate-wolfberry polysaccharide solution was 1:10. After mixing evenly, the mixed solution was aspirated and slowly dripped into a sterile CaCl2 solution with a concentration of 0.2 mol / L. After standing for 30 minutes, the CaCl2 solution remaining on the surface of the microcapsules was washed with sterile water, and the microcapsules were collected after filtering with gauze and named SLG gel; the SLG gel was allowed to stand for another 30 minutes and then added to a 1% chitosan solution, 50 mL of SLG gel was added to every 100 mL of the 1% chitosan solution, and magnetic stirring was performed at 25°C for 1 hour; the microcapsules were washed with sterile water, filtered with gauze to remove excess water, and then placed at 4°C for standby use. The capsules were named SLCG capsules;

[0023] The 1% chitosan solution is prepared by dissolving 1 g of chitosan in 1 mL of 1% glacial acetic acid solution.

[0024] Furthermore, the optimal preparation conditions of the SLCG capsules are: the mass concentration of sodium alginate is 2%, the mass concentration of wolfberry polysaccharides is 2%, the mass concentration of chitosan is 1%, the concentration of the cross-linking agent CaCl2 is 0.2 mol / L, and the static fixation time is 30 min. At this time, the encapsulation rate of Lactobacillus plantarum is 84.49%.

[0025] The advantages and positive effects achieved by the present invention are:

[0026] 1. The Lactobacillus plantarum of the present invention is derived from the shrimp paste of Beitang, Binhai New Area, Tianjin, and is a recognized safe edible fungus.

[0027] 2. In the in vitro degradation, the degradation rate of inosine by the Lactobacillus plantarum SHS01 of the present invention is 89.44%, and the degradation rate of guanosine is 100%. Therefore, as a plantarum Lactobacillus that can efficiently degrade nucleosides in vitro, it has broad application prospects in lowering uric acid.

[0028] 3. The plant lactobacillus of the present invention has excellent ability to degrade inosine and guanosine in vitro, and can alleviate hyperuricemia by lowering uric acid levels.

[0029] 4. The Lactobacillus plantarum of the present invention has the effect of lowering uric acid.

[0030] 5. The present invention provides a safe and effective system to achieve the delivery of Lactobacillus plantarum, allowing it to reach the intestine smoothly and colonize in large quantities, providing a new way to enhance the activity of probiotics and improve uric acid levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 2 is the colony morphology and cell morphology of the strain in the present invention;

[0032] Figure 2 The purified colony morphology of the strain of the present invention is shown in FIG.

[0033] Figure 3 is the Gram staining diagram of the strain of the present invention;

[0034] Figure 4 is a liquid phase diagram of the inosine standard product in the present invention;

[0035] Figure 5 is a liquid phase diagram of the guanosine standard in the present invention;

[0036] Figure 6 is a standard curve diagram of inosine in the present invention;

[0037] Figure 7 is a standard curve diagram of guanosine in the present invention;

[0038] Figure 8 This is a liquid phase diagram of the inosine guanosine mixed standard product of the present invention after being treated with Lactobacillus plantarum SHS01;

[0039] Fig. 9 The morphology of the SLCG freeze-dried microcapsules used in the present invention for improving the activity of the strain and the resistance to adverse environments;

[0040] Fig.10 This is a graph showing the effect of the amount of sodium alginate added on the microcapsule embedding efficiency in the present invention;

[0041] Fig.11 This is a graph showing the effect of the amount of Lycium barbarum polysaccharide added on the microcapsule embedding efficiency in the present invention;

[0042] Fig.12 This is a graph showing the effect of chitosan addition on microcapsule embedding efficiency in the present invention;

[0043] Fig.13 Graph showing the effect of cross-linking agent concentration on microcapsule embedding efficiency in the present invention;

[0044] Fig.14 This is a graph showing the effect of the fixation time on the microcapsule embedding efficiency in the present invention;

[0045] Fig.15 Graph showing the effects of different microcapsule systems on the encapsulation rate and loading amount of Lactobacillus plantarum in the present invention;

[0046] Fig.16 It is a graph showing the acid resistance of different samples in the present invention in simulated gastric juice digestion;

[0047] Fig.17 Graph showing the release characteristics of different samples in simulated intestinal fluid in the present invention;

[0048] Fig.18 It is the release characteristic diagram of the gastrointestinal continuous experiment of different samples in the present invention.

[0049] A strain of Lactobacillus plantarum with uric acid-lowering effect, named SHS01, classified as Lactobacillus plantarum, with a preservation number of CGMCC No.31000, a preservation date of June 19, 2024, and a preservation unit of China National Culture Collection Center for General Microbiology (CGMCC for short), No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0051] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0052] A strain of Lactobacillus plantarum with uric acid-lowering effect, named SHS01, classified as Lactobacillus plantarum, with a preservation number of CGMCC No.31000, a preservation date of June 19, 2024, and a preservation unit of China National Culture Collection Center for General Microbiology (CGMCC for short), No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0053] Preferably, the Lactobacillus plantarum has the ability to degrade inosine and guanosine in vitro.

[0054] Preferably, the morphological characteristics of the plant lactobacillus are:

[0055] Lactobacillus plantarum is a type of lactic acid bacteria, which is straight or curved rod-shaped, either alone, in pairs or in chains;

[0056] It forms small white colonies with slightly raised round outlines on MRS broth agar medium;

[0057] Lactobacillus plantarum is a facultative anaerobic bacteria with an optimum growth temperature of 37°C and an optimum pH of 7.0.

[0058] Preferably, the formula of the MRS broth agar medium is: 10.0 g of peptone, 8.0 g of beef powder, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 1.0 g of Tween 80, 20 g of agar, 1000 mL of distilled water is fully dissolved and the pH value is adjusted to 7.0, and sterilized at 121°C and 0.1 MPa for 20 min.

[0059] Use of the Lactobacillus plantarum as described above in the preparation of drugs for degrading inosine and / or guanosine.

[0060] A microcapsule for improving the activity and adverse environmental resistance of Lactobacillus plantarum as described above.

[0061] Preferably, the encapsulation rate of Lactobacillus plantarum in the microcapsules is 84.49%.

[0062] The method for preparing the microcapsules as described above comprises the following steps:

[0063] The Lactobacillus plantarum SHS01 bacterial suspension was centrifuged at 8000 r / min for 10 minutes at 4°C, the bacterial cells were collected, and resuspended with physiological saline. This process was repeated twice, and the obtained bacterial suspension was placed in a 4°C refrigerator for standby use; the bacterial suspension was diluted to a suitable concentration and the colony count was performed using the plate count method;

[0064] After sterilizing a 2% sodium alginate solution and a 2% wolfberry polysaccharide solution, the two were mixed in a volume ratio of 1:1 to prepare a sodium alginate-wolfberry polysaccharide solution; the bacterial suspension was added to the sodium alginate-wolfberry polysaccharide solution, and the volume ratio of the bacterial suspension to the sodium alginate-wolfberry polysaccharide solution was 1:10. After mixing evenly, the mixed solution was aspirated and slowly dripped into a sterile CaCl2 solution with a concentration of 0.2 mol / L. After standing for 30 minutes, the CaCl2 solution remaining on the surface of the microcapsules was washed with sterile water, and the microcapsules were collected after filtering with gauze and named SLG gel; the SLG gel was allowed to stand for another 30 minutes and then added to a 1% chitosan solution, 50 mL of SLG gel was added to every 100 mL of the 1% chitosan solution, and magnetic stirring was performed at 25°C for 1 hour; the microcapsules were washed with sterile water, filtered with gauze to remove excess water, and then placed at 4°C for standby use. The capsules were named SLCG capsules;

[0065] The 1% chitosan solution is prepared by dissolving 1 g of chitosan in 1 mL of 1% glacial acetic acid solution.

[0066] Preferably, the optimal preparation conditions of the SLCG capsules are: the mass concentration of sodium alginate is 2%, the mass concentration of wolfberry polysaccharides is 2%, the mass concentration of chitosan is 1%, the concentration of the cross-linking agent CaCl2 is 0.2 mol / L, and the static fixation time is 30 min, at which time the encapsulation rate of Lactobacillus plantarum is 84.49%.

[0067] Specifically, the relevant preparation and detection are as follows:

[0068] The screening steps of plant lactobacillus in the present invention are as follows:

[0069] Beitang shrimp paste was purchased from the local market in Tanggu, Binhai New Area, Tianjin. 1.0 g of shrimp paste sample was taken with a pipette, mixed with a sodium chloride solution with a mass concentration of 0.9%, and diluted with a sodium chloride solution with a mass concentration of 0.9%, and the dilution was evenly spread on an MRS solid plate medium containing 2% agar. After the culture dish was placed in a constant temperature anaerobic incubator at 37°C and cultured for 48 hours, white single colonies were observed on the plate. Then, a single colony was selected using a clean inoculation loop, streaked onto the MRS solid culture medium, and evenly spread with a sterile spreader; after it was absorbed, it was placed in a 37°C constant temperature incubator for anaerobic culture for 2 days; a single colony on the plate after culture was streaked and separated, and this separation and purification step was repeated 2-3 times to obtain a purified single colony with consistent morphology, and obtain plant lactobacillus that can efficiently degrade uric acid.

[0070] Example 1 Isolation, purification and identification of bacterial strains

[0071] 1. Isolation of strains:

[0072] Weigh 1.0 g shrimp paste sample, mix it with 0.9% sodium chloride solution, and dilute it with 0.9% sodium chloride solution in a gradient manner (such as 1:100, 1:1000, 1:10000, etc.). Then, take 0.1 mL of different fermented yogurt dilutions and spread them on MRS solid culture medium. After constant temperature cultivation at 37°C for 48 h, observe the colony morphology. The results are as follows: Figure 1 As shown, the strain has a translucent bulge in the middle, smooth dot-shaped colonies on the surface, and regular edges without wrinkles.

[0073] 2. Purification of strains:

[0074] Pick a typical single colony (round or irregularly round, milky white, opaque, tight texture, smooth edges, rough surface, dry and dull, thin colonies and small colonies) on the plate, streak it on the solid plate for 2 to 3 generations to isolate the pure colony. The result is as follows: Figure 2 As shown, Lactobacillus plantarum is a type of lactic acid bacteria, which is straight or curved rod-shaped, single, paired or chain-shaped; it forms small white colonies on MRS broth agar medium with a slightly raised circular outline.

[0075] 3. Morphological observation and preservation of strains:

[0076] The suspected lactic acid bacteria were determined to be Gram-positive and catalase-negative. Gram staining was first performed, and the purple colonies were selected and H2O2 was added. If no bubbles were produced, it was the target strain. The bacterial morphology was observed under a microscope, and the strains were frozen and stored in glycerol at -80°C. The results were as follows Figure 3 As shown, the strain turns blue-purple under the action of Gram staining reagent, indicating that it is a Gram-positive bacterium. The colony morphology in the microscopic field of view is uniform, and the cell morphology is rod-shaped (some are long and some are short), and the arrangement morphology is single or chain-like.

[0077] 4. Identification of bacterial species

[0078] SHS01 was sequenced for 16S rDNA, and the sequencing results were:

[0079]

[0080] Example 2 In vitro degradation of inosine and guanosine by bacterial strains

[0081] Since the precursors of uric acid in the production process are xanthine, hypoxanthine, inosine, guanosine, etc., their contents are related to the final uric acid content in the blood. Therefore, the ability of the above strains to degrade inosine and guanosine was measured using a phosphate buffer system. The strain was allowed to stand at 37°C for 18-24 h to cultivate the seed solution, and 1 mL was added to 9 mL of blank MRS medium and incubated for 10 h. Take 2 ml of the above activated second-generation plant lactobacillus bacterial solution, centrifuge at 4°C and 5000 r / min for 10 minutes, and then collect the bacteria. Resuspend and wash with 1 mL of physiological saline, and then centrifuge at 4°C and 5000 r / min for 10 minutes, repeat twice and collect the bacteria. Weigh 33.7 mg of inosine and 35.8 mg of guanosine, dissolve them in potassium phosphate buffer respectively. If guanosine is not dissolved, heat it in a water bath at 80℃, and dilute to 50 mL respectively. Prepare the prepared inosine-neutral potassium phosphate solution and guanosine-neutral potassium phosphate solution. The molar concentration of the solution is 2.5128 mM and 2.52789 mM. Add 375 μL of guanosine-neutral potassium phosphate solution and inosine-neutral potassium phosphate solution to the washed cells to resuspend the cells, and then shake at 37℃ and 120 r / min for 1 hour. After the reaction is completed, centrifuge at 4℃ and 5000 r / min for 10 minutes. Take 720 μL of the supernatant and add 80 μL of perchloric acid solution (the volume ratio of supernatant to terminator is 9:1) to terminate the reaction. Filter the supernatant with a filter membrane and determine the content of inosine and guanosine by high performance liquid chromatography.

[0082] The conditions of high performance liquid chromatography are as follows:

[0083] Chromatographic conditions: Shimadzu high performance liquid chromatograph

[0084] Reverse phase chromatography column: C18 (250mmx4.6mm)

[0085] Mobile phase: 20 mmol / L KH2PO4

[0086] Flow rate: 1 mL / min

[0087] Column temperature: 30°C

[0088] Retention time: 10 min

[0089] Measurement wavelength: 254 nm

[0090] The chromatographic results are as follows Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, Figure 4 is the liquid phase diagram of inosine standard. Figure 5 This is the liquid phase diagram of guanosine standard. Figure 6 , Figure 7 For the standard curve of inosine and guanosine, Figure 8 This is the liquid phase diagram of the mixed standard of inosine and guanosine after being treated with Lactobacillus plantarum SHS01. Figure 8 It can be seen that after the reaction, the degradation rate of inosine was 89.44%, and the degradation rate of guanosine was 100%, indicating that this strain has a good ability to degrade uric acid precursors.

[0091] Example 3 Microencapsulation method for improving strain activity and resistance to adverse environments.

[0092] Add the bacterial suspension of Lactobacillus plantarum SHS01 to a centrifuge tube, centrifuge at 8000 r / min for 10 minutes at 4°C, collect the bacteria, and resuspend them with physiological saline. Repeat this process twice, and put the obtained bacterial suspension into a 4°C refrigerator for later use. Take 1 mL of the bacterial suspension for gradient dilution to an appropriate concentration and use the plate count method to count the colonies.

[0093] After sterilizing a 2% sodium alginate solution and a 2% Lycium barbarum polysaccharide solution, the two were mixed in a volume ratio of 1:1 to prepare a sodium alginate-lycium barbarum polysaccharide solution. The bacterial suspension prepared above was added to the sodium alginate solution and the sodium alginate-lycium barbarum polysaccharide solution (the volume ratio of the bacterial suspension to the sodium alginate-lycium barbarum polysaccharide solution was 1:10), mixed evenly, and the mixed solution was sucked with a 10 mL sterile syringe and slowly dripped into a 0.2 mol / L sterile CaCl2 solution. After standing for 30 minutes, the CaCl2 solution remaining on the surface of the microcapsules was washed with sterile water, and the microcapsules were collected after gauze filtration, and named SAG gel and SLG gel, respectively. After standing for another 30 minutes, 50 mL of SLG gel was added to 100 mL of 1% chitosan solution (1 g chitosan was dissolved in 1 mL of 1% glacial acetic acid solution by volume), and magnetically stirred at 25°C for 1 hour. The microcapsules were washed with sterile water, filtered through gauze to remove excess water, and then placed at 4°C for later use. The capsules were named SLCG capsules. Fig. 9 As shown, the freeze-dried microcapsules are light brown in appearance, spherical in shape, uniform in size, and have a loose and porous surface.

[0094] Example 4 Verification of the relationship between the embedding rate and loading amount of microcapsules and the amount of sodium alginate added

[0095] The specific conditions of the relevant implementation method and verification method are the same as those of Example 3, except that the mass concentration of sodium alginate is set to 1%, 1.5%, 2%, 2.5% and 3%.

[0096] The embedding rate refers to the ratio of the actual number of live Lactobacillus plantarum bacteria contained in the microcapsule to the theoretical maximum number of Lactobacillus plantarum bacteria contained in the microcapsule. The maximum loading capacity refers to the maximum number of live bacteria contained per unit mass of the microcapsule product. Both the embedding rate and loading capacity reflect the binding effect and encapsulation ability of the microcapsule material on Lactobacillus plantarum.

[0097] The embedding rate and loading capacity of microcapsules are closely related to the amount of sodium alginate added, that is, its mass concentration. Alginate is widely used in the preparation of microcapsules for bacterial preparations due to its non-toxicity, good biocompatibility with microorganisms, and the high surface porosity of the prepared microcapsules. Fig.10 It can be seen that in the range of 1%-3% mass concentration of sodium alginate, the embedding rate and loading capacity of Lactobacillus plantarum first increase with the increase of the added amount, and then decrease with the addition. When the mass concentration of sodium alginate is 1%, the embedding rate is 39.87%; when the mass concentration of sodium alginate is 1.5%, the embedding rate is 62.50%; when the mass concentration of sodium alginate reaches 2%, the embedding rate is 78.52%; when the mass concentration of sodium alginate is 2.5%, the embedding rate is 74.49%, and when the mass concentration of sodium alginate is 3%, the embedding rate is 62.83%. In summary, when the mass concentration of sodium alginate is 2%, the embedding rate and loading rate of Lactobacillus plantarum reach the maximum value, and the embedding rate at this time is 78.52%, which is significantly different from other concentrations, so the mass concentration of sodium alginate is finally selected as 2%.

[0098] Example 5 Verification of the relationship between the embedding rate and loading amount of microcapsules and the amount of Lycium barbarum polysaccharide added

[0099] The specific conditions of the relevant implementation method and verification method are the same as those of Example 3, except that the mass concentration of Lycium barbarum polysaccharides is set to 1%, 1.5%, 2%, 2.5% and 3%.

[0100] Lycium barbarum polysaccharides are non-toxic, biocompatible, and can be used as a prebiotic and have the potential to form a suitable microcapsule structure to sustain cells. Fig.11It can be seen that within the range of 1%-3% of the addition amount of Lycium barbarum polysaccharide, the embedding rate and loading capacity of Lactobacillus plantarum first increase with the increase of the addition amount, and then decrease with the addition. When the mass concentration of Lycium barbarum polysaccharide is 1%, the embedding rate is 57.42%; when the mass concentration of Lycium barbarum polysaccharide is 1.5%, the embedding rate is 74.1%; when the mass concentration of Lycium barbarum polysaccharide is 2%, the embedding rate is 79.13%; when the mass concentration of Lycium barbarum polysaccharide is 2.5%, the embedding rate is 78.08%; when the mass concentration of Lycium barbarum polysaccharide is 3%, the embedding rate is 70.45%. In summary, when the mass concentration of Lycium barbarum polysaccharide is 2%, the embedding rate and loading capacity reach the highest value, the embedding rate is 79.13%, and there is a significant difference with other concentrations, so the mass concentration of Lycium barbarum polysaccharide is finally selected as 2%.

[0101] Example 6 Verification of the relationship between the embedding efficiency and loading amount of microcapsules and the amount of chitosan added

[0102] The specific conditions of the relevant implementation method and verification method are the same as those of Example 3, except that the mass concentration of chitosan is set to 0.5%, 1%, 1.5%, 2% and 2.5%.

[0103] Chitosan is a biodegradable and biotolerant polymer that enables controlled release of compounds and is a suitable polymer for cell encapsulation. Its mechanical and physicochemical properties are indeed easily adaptable, thus offering the possibility to bypass, correct and adjust these properties to suit the encapsulation of different cells. Fig.12 It can be seen that when the amount of chitosan added is within the range of 0.5%-2.5%, the embedding rate and load of Lactobacillus plantarum increase with the increase of the amount added, and then decrease with the addition. When the mass concentration of chitosan is 0.5%, the embedding rate is 61.43%; when the mass concentration of chitosan is 1%, the embedding rate is 80.58%; when the mass concentration of chitosan is 1.5%, the embedding rate is 78.5%; when the mass concentration of chitosan is 2%, the embedding rate is 70.45%; when the mass concentration of chitosan is 2.5%, the embedding rate is 64.58%. In summary, when the mass concentration of chitosan is 1%, the embedding rate and load reach the highest value of 80.58%, and there is a significant difference with other concentrations, so the mass concentration of chitosan is finally selected as 1%.

[0104] Example 7 Verification of the relationship between the embedding rate and loading amount of microcapsules and the concentration of CaCl2

[0105] The specific conditions of the relevant implementation method and verification method are the same as those of Example 3, except that the concentration of the CaCl2 solution is set to 0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L and 0.5mol / L.

[0106] The interaction of salts in the gel structure also has a positive effect on the entrapment efficiency. 2+ Crosslinking in the presence of ions occurs due to the interchain interactions of polysaccharides, resulting in alginate-Ca 2+ Bead formation. Fig.13 It can be seen that in the range of 0.1-0.5 mol / L of the cross-linking agent concentration, i.e., the CaCl2 solution concentration, the embedding rate and load of Lactobacillus plantarum first increase with the increase of the cross-linking agent concentration, and then decrease with the increase of the concentration. When the cross-linking agent concentration is 0.1 mol / L, the embedding rate is 64.48%; when the cross-linking agent concentration is 0.2 mol / L, the embedding rate is 81.79%; when the cross-linking agent concentration is 0.3 mol / L, the embedding rate is 76.88%; when the cross-linking agent concentration is 0.4 mol / L, the embedding rate is 67.36%; when the cross-linking agent concentration is 0.5 mol / L, the embedding rate is 48.72%; in summary, when the cross-linking agent concentration is 0.2 mol / L, the embedding rate and load reach the highest value, the embedding rate is 81.79%, and there is a significant difference with other concentrations, so the cross-linking agent CaCl2 concentration of 0.2 mol / L was finally selected.

[0107] Example 8 Verification of the relationship between the embedding efficiency and loading amount of microcapsules and the fixation time of the crosslinking agent

[0108] The specific conditions of the relevant implementation method and verification method are the same as those of Example 3, except that: the mixture is dripped into a sterile CaCl2 solution with a concentration of 0.2 mol / L, and the fixed standing time is set to 10 minutes, 20 minutes, 30 minutes, 40 minutes and 50 minutes.

[0109] The static fixation time refers to the time it takes for the raw material solution to remain static after being dropped into the crosslinking agent, and for the ions in the raw material to react with the calcium ions in the crosslinking agent. The length of the static fixation time determines whether the bonding reaction is complete. Fig.14 It can be seen that within the range of 10-50 minutes of static fixation time, the embedding rate and load of Lactobacillus plantarum first increased significantly with the increase of fixation time, and then fluctuated up and down with the increase of static fixation time. When the static fixation time was 10 minutes, the embedding rate was 10.41%; when the static fixation time was 20 minutes, the embedding rate was 55.37%; when the static fixation time was 30 minutes, the embedding rate was 82.78%; when the static fixation time was 40 minutes, the embedding rate was 81.2%; when the static fixation time was 50 minutes, the embedding rate was 81.91%. In summary, when the static fixation time was 30 minutes, the embedding rate and load reached the highest value, the embedding rate was 82.78%, and there was a significant difference with other times, so the best static fixation time of 30 min was finally selected.

[0110] In summary, the optimal preparation conditions of the SLCG capsules are: the mass concentration of sodium alginate is 2%, the mass concentration of wolfberry polysaccharides is 2%, the mass concentration of chitosan is 1%, the concentration of the cross-linking agent CaCl2 is 0.2 mol / L, and the static fixation time is 30 min. Fig.15 As shown in the figure, the embedding rate of SAG capsule is 33.87%, the maximum load is 9.279 log CFU / g, the embedding rate of SLG capsule is 56.50%, the maximum load is 9.501log CFU / g, and the embedding rate of SLCG capsule is 84.49%, the maximum load is 9.676 log CFU / g. Compared with other embedding methods, SLCG has a higher embedding rate and load, and there are obvious differences.

[0111] At the same time, it can be seen from Examples 3 to 8 that in the method for preparing microcapsules using plant lactobacillus SHS01 of the present invention, the mass concentration of sodium alginate, the mass concentration of lycium barbarum polysaccharides, the mass concentration of chitosan, the concentration of CaCl2 solution, and the fixing time of the cross-linking agent have a synergistic effect between the two conditions, and can synergistically improve the relevant properties of the prepared microcapsules. In particular, when the mass concentration of sodium alginate is 2%, the mass concentration of lycium barbarum polysaccharides is 2%, the mass concentration of chitosan is 1%, the concentration of the cross-linking agent CaCl2 is 0.2 mol / L, and the static fixing time is 30 min, the relevant properties of the prepared microcapsules can be significantly synergistically improved.

[0112] Example 9 The microcapsules prepared in Example 3 were used to conduct gastric acid resistance test and intestinal release test

[0113] Microcapsule acid resistance experiment: Add 2g sodium chloride and 3.2g pepsin to 1L distilled water, and then adjust the pH to 2.0 with hydrochloric acid to prepare simulated gastric fluid. Weigh 1g of microcapsules and put them into sterile centrifuge tubes respectively. At 0 hours, 0.5 hours, 1 hour, 1.5 hours, and 2 hours, add artificial gastric juice (9 mL) with a pH of 2.0 to the centrifuge tubes, and then place the centrifuge tubes in a constant temperature water bath oscillator at 37°C for oscillation reaction at a speed of 100r / min. At 2 hours, take out all centrifuge tubes and centrifuge at a low speed of 4000g for 5 minutes to collect microcapsules. Then add PBS buffer to the centrifuge tube, stir for 30 minutes and homogenize for 30 seconds to ensure that the microcapsules are completely broken. The mixed solution is then diluted in a gradient manner and counted using the plate count method. At the same time, a control experiment was carried out on the obtained bacterial suspension (i.e., the bacterial suspension prepared in Example 3 and stored in a 4°C refrigerator) under the same reaction conditions. The experimental results are as follows Fig.16As shown, the acid resistance curves of Lactobacillus plantarum in different matrices under simulated gastric fluid conditions were compared, with the bacterial solution as the blank group and SAG and SLG as the control groups. The original concentration of the bacterial suspension was 9.749 1og CFU / mL. In the gastric acid environment of pH=2, the number of bacteria suddenly decreased significantly after one hour of reaction to 8.415 log CFU / mL, and after two hours of reaction to 7.699 1og CFU / mL, a decrease of 2.05log CFU / mL compared with the original concentration; the original concentration of SAG was 9.279 1og CFU / mL. In the gastric acid environment of pH=2, the concentration after two hours of reaction was 9.086 1og CFU / mL, a decrease of 0.193 log CFU / mL compared with the original concentration; the original concentration of SLG was 9.501 1og CFU / mL. In the gastric acid environment of pH=2, the concentration after two hours of reaction was 9.3561og CFU / mL, a decrease of 0.1449 log CFU / mL compared with the original concentration; the original concentration of SLCG was 9.676 1og CFU / mL. CFU / mL, in the gastric acid environment of pH=2, the concentration after two hours of reaction was 9.615 log CFU / mL, which was 0.061 log CFU / mL lower than the original concentration. It can be concluded that the microcapsule SLCG has good acid resistance.

[0114] Microcapsule intestinal release experiment: Add potassium dihydrogen phosphate with a final concentration of 0.65%, 1g of trypsin and 18g of ox bile salt to 1L of distilled water, adjust the pH to 7.4 with sodium hydroxide to prepare simulated intestinal fluid. Subsequently, filter and sterilize it with a 0.22 μm filter membrane, and store it in a refrigerator at 4°C for use. In order to explore the intestinal release ability of the microcapsules, first take out 1g of microcapsules and put it in a sterile centrifuge tube. Next, add simulated intestinal fluid (9 mL) with a pH value of 7.4 to the centrifuge tube. The centrifuge tube was then placed in a constant temperature water bath oscillator at 37°C and oscillated at 100 r / min for reaction. Samples were taken at 1 hour, 2 hours, 3 hours, 4 hours and 5 hours of reaction. The intestinal fluid after the reaction was taken out and appropriately diluted, and then the microcapsules were counted using the spread plate counting method. The experimental results are as follows Fig.17 As shown in the figure, after the reaction, the cell release of SAG capsules was 5.357 log CFU / mL, and the release of SLG capsules was 8.362 log CFU / mL. After 5 hours of reaction, the release rate of SLCG microcapsules reached 92.36%, and the release of Lactobacillus plantarum was 9.574 log CFU / mL, which was much higher than that of SAG and SLG capsules, indicating that SLCG microcapsules had good intestinal release performance.

[0115] Microcapsule gastrointestinal continuous release experiment: 1g of microcapsules were placed in artificial gastric juice (9 mL) and shaken at 100 r / min at 37°C for 2 hours, and then the gastric juice after reaction was sampled and counted by plate counting method. The gastric juice was filtered, and the microcapsules were collected by centrifugation and transferred to artificial intestinal juice (9 mL), and the reaction was shaken at 100 r / min at 37°C to fully release Lactobacillus plantarum. The intestinal juice was sampled at 1h, 2h, 3h, 4h, and 5h of the reaction, and the intestinal juice was diluted and counted by plate counting method, and a control experiment was carried out with Lactobacillus plantarum suspension (i.e., the bacterial suspension prepared in Example 3 and kept in a 4°C refrigerator) under the same reaction conditions.

[0116] Conducting gastric acid resistance test and intestinal release test separately can minimize the influence of other factors and more directly evaluate the cell survival rate in gastric juice and cell release rate in intestinal juice under different encapsulation methods. On the other hand, continuous gastrointestinal simulation experiment can obtain more accurate data and obtain the number of probiotic cells released when they reach the large intestine after gastrointestinal digestion.

[0117] pass Fig.18 It can be seen that in the continuous gastrointestinal simulation experiment, the release rate of Lactobacillus plantarum was 88.16% after 5 hours of reaction of SLCG, and the cumulative release reached 9.657 log CFU / mL. The number of live bacteria in free bacterial cells was 0 after 5 hours of reaction; the cumulative release of SAG capsules was 5.284 log CFU / mL after 5 hours of reaction; the cumulative release of SLG capsules was 8.23 ​​log CFU / mL after 5 hours of reaction. Compared with free bacterial cells and SAG and SLG capsules, SLCG capsules have a higher content of Lactobacillus plantarum cells in the intestinal fluid. The continuous gastrointestinal digestion simulation experiment confirmed that SLCG capsules can achieve sustained release at a high release count of 9.657 log CFU / mL, and the release rate at this time is as high as 88.16%.

[0118] Compared with the prior art:

[0119] Qian Luan et al. prepared a nanocellulose-based macrosphere by alginate / chitosan multi-layer coatings for delivery of probiotics (Electrostatically reinforced and sealed nanocellulose-based macrosphere by alginate / chitosan multi-layer coatings for delivery of probiotics, Food Hydrocolloids, Volume 142, September 2023, 108804). When Lactobacillus plantarum cells were encapsulated in this macrosphere, the survival rate of the cells was 80.74% under gastric fluid simulation conditions, and the release amount was 7.64 log CFU / mL. The present invention uses a composite method of sodium alginate, wolfberry polysaccharide and chitosan to encapsulate plant lactobacillus. The survival rate in the continuous gastrointestinal simulation experiment was 88.16%, and the release amount was 9.657 log CFU / mL. Compared with this macrosphere, the present invention has a higher survival rate and release amount, and can better achieve the delivery of plant lactobacillus.

[0120] Huiyi Song et al. studied the use of alginate-chitosan microencapsulation technology to microencapsulate probiotics (Saccharomyces cerevisiae Y235) in the article "Improved probiotic viability in stress environments with post-culture ofalginate-chitosan microencapsulated low density cells" to improve its survival rate in stress environments such as freeze-drying, storage and simulated gastrointestinal fluid. (Improved probiotic viability in stress environments with post-culture ofalginate-chitosan microencapsulated low density cells, Carbohydr Polym, 2014, Aug 8, 108:10-6) The release amount of the capsule in gastric fluid is 6.29 log CFU / g, and the present invention has a higher release amount than this capsule.

[0121] Therefore, this study developed a safe and effective system to achieve the delivery of Lactobacillus plantarum, allowing it to reach the intestine smoothly and colonize in large numbers, providing a new way to enhance the activity of probiotics and improve uric acid levels.

[0122] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A plant lactobacillus having uric acid lowering effect, characterized in that: Its name is SHS01 and its classification name is: Lactobacillus plantarum Lactobacillus plantarum , the deposit number is: CGMCC No.31000, the deposit date is: June 19, 2024, the deposit unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of plant lactobacillus as claimed in claim 1 in preparing medicines for degrading inosine and / or guanosine.

3. A microcapsule for improving the acid resistance of Lactobacillus plantarum as claimed in claim 1, characterized in that: The preparation method of the microcapsule comprises the following steps: The Lactobacillus plantarum SHS01 bacterial solution of claim 1 was centrifuged at 8000 r / min for 10 minutes at 4°C, the bacterial cells were collected, and the cells were resuspended with physiological saline. This process was repeated twice, and the obtained bacterial suspension was placed in a 4°C refrigerator for standby use; the bacterial suspension was diluted to a suitable concentration and the colony count was performed using the plate count method; After sterilizing a 2% sodium alginate solution and a 2% wolfberry polysaccharide solution, the two were mixed in a volume ratio of 1:1 to prepare a sodium alginate-wolfberry polysaccharide solution; the bacterial suspension was added to the sodium alginate-wolfberry polysaccharide solution, and the volume ratio of the bacterial suspension to the sodium alginate-wolfberry polysaccharide solution was 1:

10. After mixing evenly, the mixed solution was aspirated and slowly dripped into a sterile CaCl2 solution with a concentration of 0.2 mol / L. After standing for 30 minutes, the CaCl2 solution remaining on the surface of the microcapsules was washed with sterile water, and the microcapsules were collected after filtering with gauze and named SLG gel; the SLG gel was allowed to stand for another 30 minutes and then added to a 1% chitosan solution, 50 mL of SLG gel was added to every 100 mL of the 1% chitosan solution, and magnetic stirring was performed at 25°C for 1 hour; the microcapsules were washed with sterile water, filtered with gauze to remove excess water, and then placed at 4°C for standby use. The capsules were named SLCG capsules; The 1% chitosan solution is prepared by dissolving 1 g of chitosan in 1 mL of 1% glacial acetic acid solution.

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