A kind of Pediococcus acidilactici and its application in preparing microcapsules for preventing and treating chronic heart failure

Abstract

The present invention provides Pediococcus acidilactici and its use in preparing microcapsules for the prevention and treatment of chronic heart failure. The Pediococcus acidilactici has a deposit number of CGMCC No. 30585. The material formula and process for preparing Pediococcus acidilactici microcapsules provided by the present invention can significantly improve the storage stability and environmental tolerance of Pediococcus acidilactici and can be used for future industrial-scale production. The Pediococcus acidilactici microcapsules provided by the present invention can improve myocardial cell function in experimental animals, improve ventricular pathological remodeling and / or heart failure, restore cardiac contractile function in mice with myocardial infarction, reduce cardiac fibrosis in mice with myocardial infarction, inhibit pathological myocardial hypertrophy in mice with myocardial infarction, and significantly increase the ejection fraction in mice with chronic heart failure.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to Pediococcus acidilactici and application thereof in preparing microcapsules for preventing and treating chronic heart failure. Background Art

[0002] Chronic heart failure (CHF) is a clinical syndrome characterized by decreased ventricular pumping and filling capacity due to structural and functional changes in the heart. Its primary manifestations include dyspnea, fatigue, and ankle edema. Chronic heart failure (CHF) is a serious, life-threatening clinical syndrome characterized by high morbidity, mortality, hospitalization rates, poor quality of life, and high medical costs. In developed countries, the prevalence of HF ranges from 1.1% to 5.5% of the general population, reaching as high as 6% to 10% in those aged 65 years and older. In my country, the prevalence of HF among adults aged 35-74 years is 0.9%. With the aging of my country's population, the significant increase in the incidence of chronic diseases such as metabolic syndrome, and continuous advancements in medical diagnosis and treatment, the prevalence of HF in my country is steadily increasing, with a mortality rate of 4.1% among hospitalized patients. HF has become the second leading cause of heart disease-related death after coronary artery disease. Due to the diverse pathophysiology and phenotypes of HF, effective therapeutic agents are currently lacking.

[0003] In recent years, the role of intestinal probiotics in the occurrence and development of major human diseases has received increasing attention. The number of intestinal microorganisms (intestinal flora) in the body is more than 10 times that of the body's own cells. Intestinal probiotics are very important for nutrient metabolism, human development, immunity and the occurrence of diseases. Dysbiosis of the intestinal microbiota can lead to the accumulation of toxic compounds such as trimethylamine oxide (TMAO) and endotoxins (LPS), as well as a reduction in beneficial products such as short-chain fatty acids (SCFA). Harmful microbial metabolites are transferred to the circulation, inducing immune and systemic inflammatory responses that directly or indirectly lead to tissue damage. Currently, several studies have confirmed that intestinal probiotics can change and affect host metabolism, and effectively alleviate the development of various metabolic and cardiovascular diseases such as hypertension, coronary heart disease and heart failure.

[0004] Pediococcus acidilactici is a Gram-positive bacterium with spherical cells. Colonies of Pediococcus acidilactici are approximately 1.5 to 2.5 mm in diameter, with a smooth, rounded, and off-white surface. They are commonly found in vegetables and meat products. Pediococcus acidilactici possesses unique physiological and biochemical properties. Research has shown that it possesses a variety of functions, including antibacterial, anti-inflammatory, antidepressant, and enteritis treatment. However, Pediococcus acidilactici has poor environmental tolerance. Currently, probiotic microcapsules utilize a suitable encapsulation material to encapsulate the bacteria, isolating them from the outside world and achieving their protective properties. Microencapsulation technology involves encapsulating a target core material (solid, liquid, or even gas) with an encapsulation material, creating microcapsules with a semipermeable or sealed membrane ranging in diameter from 1 to 5000 μm (typically 5 to 400 μm). This protects probiotics from external influences, improving their survival rate and facilitating storage. Summary of the Invention

[0005] The present invention provides Pediococcus acidilactici and application thereof in preparing microcapsules for preventing and treating chronic heart failure, thereby making up for the deficiency of current effective therapeutic drugs for chronic heart failure.

[0006] The present invention first provides a Pediococcus acidilactici HRQ-1 strain, which was deposited on May 10, 2024, at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 30585.

[0007] Another aspect of the present invention provides a use of the Pediococcus acidilactici HRQ-1 strain in the preparation of a product for preventing and / or treating chronic heart failure;

[0008] The product, as a specific description of the embodiment, is a microcapsule;

[0009] In another aspect, the present invention further provides a microcapsule, wherein the core material of the microcapsule comprises the Pediococcus acidilactici HRQ-1 strain.

[0010] The embedding agent of the microcapsule contains 3% sodium alginate, 1% chitosan and 2% calcium chloride;

[0011] The bacterial cells of the Pediococcus acidilactici HRQ-1 strain are mixed with a freeze-drying protective agent;

[0012] As a specific record of the embodiment, the freeze-dried protective agent has the following composition ratio: 13% sea skim milk powder, 12% glycerol, 14% sucrose, 3% mannitol and 0.8% tyrosine.

[0013] The bacterial cells of the Pediococcus acidilactici HRQ-1 strain are prepared by fermentation using MRS culture medium.

[0014] A specific preparation method of the microcapsules is as follows:

[0015] The bacterial slurry of Pediococcus acidilactici is diluted and fully stirred with a sodium alginate solution to make it uniform, and then further stirred with a freeze-drying protective agent to obtain a mixed bacterial solution. After the chitosan is completely dissolved, the mixed bacterial solution is uniformly dropped into a mixed solution of CaCl2 and chitosan to form wet microcapsules. The wet microcapsules are stirred and solidified so that the wet microcapsules are fully in contact with the solidifying liquid CaCl2, and then allowed to stand and solidify. After washing, the solution is freeze-dried in a freeze dryer to obtain Pediococcus acidilactici microcapsule particles.

[0016] The microcapsules provided by the present invention can be used to prepare orally administrable products for preventing and / or treating chronic heart failure.

[0017] The material formula and process for preparing Pediococcus acidilactici microcapsules provided by the present invention can significantly improve the storage stability and environmental tolerance of Pediococcus acidilactici, and can be used for future industrial-scale production. The Pediococcus acidilactici microcapsules provided by the present invention can improve myocardial cell function in experimental animals, ameliorate ventricular pathological remodeling and / or heart failure, restore cardiac contractile function in mice with myocardial infarction, reduce cardiac fibrosis in mice with myocardial infarction, inhibit pathological myocardial hypertrophy in mice with myocardial infarction, and significantly increase the ejection fraction (EF%) in mice with chronic heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0019] Figure 1 : Growth morphology of Pediococcus acidilactici in MRS plate culture medium.

[0020] Figure 2 :The cell morphology of Pediococcus acidilactici was observed under transmission electron microscope.

[0021] Figure 3 :The results of constructing the phylogenetic tree of Pediococcus acidilactici.

[0022] Figure 4 : Results of single factor optimization experiment of Pediococcus acidilactici microcapsule embedding agent.

[0023] Figure 5 : Single factor optimization test results of Pediococcus acidilactici microcapsule freeze-dried protective agent.

[0024] Figure 6 : The morphology of Pediococcus acidilactici microcapsules observed under transmission electron microscope.

[0025] Figure 7 :Results of in vitro simulated tolerance experiment of Pediococcus acidilactici microcapsules.

[0026] Figure 8 : Comparison of cardiac appearance in mice under different treatment groups.

[0027] Figure 9 : HE and Masson staining images of cardiac pathological sections of mice in different treatment groups.

[0028] Figure 10 : Histogram of cardiac ultrasound ejection fraction (EF%) results of mice in different treatment groups. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The culture medium used in the present invention comprises:

[0034] MRS medium (1000 mL) contains: 2.0 g dipotassium hydrogen phosphate, 2.0 g triammonium citrate, 5.0 g anhydrous sodium acetate, 0.25 g manganese sulfate heptahydrate, 0.58 g magnesium sulfate heptahydrate, 20.0 g glucose, 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, and 1 mL Tween-80. Adjust the pH to 6.2-6.4.

[0035] Add 20 g of agar to the solid culture medium and prepare calcium carbonate (add 5% (W / V) to make a solid plate, for example, prepare 25 g for 500 ml of culture medium), and then sterilize by high pressure at 115° C. for 30 min.

[0036] Luria-Bertani (LB) medium (g / L): 10.0% sodium chloride, 10.0% tryptone, 5.0% yeast extract, add distilled water to 1 L, and adjust the pH to 7.2.

[0037] In the present invention, culture media were sterilized by high-pressure steam at 120° C. for 20 min unless otherwise specified.

[0038] Example 1: Screening and identification of Pediococcus acidilactici

[0039] 1.1 Main materials:

[0040] Homemade kimchi samples, MRS culture, anaerobic box, anaerobic tube.

[0041] 1.2 The specific experimental methods are as follows:

[0042] Take 10g of kimchi sample in a 250ml triangular flask, add 90ml of sterile water to obtain a suspension, and dilute to 10 -1 ~10 -7 . Select 10 -4 ~10 -6 The dilutions were spread onto MRS screening medium plates, with three plates spread for each gradient. Each plate was coated with 200 μL of the diluted sample suspension. The plates were sealed and labeled after coating and stored. After 48 hours of anaerobic incubation at 37°C in an anaerobic box, individual colonies of different types were picked from different plates and isolated and purified using the three-zone streak method on the corresponding screening plates. The resulting single bacteria were stored at -80°C in 25% glycerol until further use. DNA was extracted from each strain obtained in the initial screening, and 16S rRNA was amplified by PCR using universal primers 27F and 1492R. The bacterial genus was identified by sequencing.

[0043] The single bacterium was initially isolated from a homemade kimchi sample by MRS screening culture medium plate. The growth on MRS plate and the morphology under transmission electron microscope were as follows. Figure 1 and Figure 2 As shown. According to the 16S rRNA sequencing results, the phylogenetic tree was constructed ( Figure 3 ), as well as a comprehensive analysis of the morphological characteristics of the strain, the genus of the screened strain was learned, and it was finally identified as Pediococcus acidilactici, named Pediococcus acidilactici HRQ-1.

[0044] Example 2: Single factor optimization test of Pediococcus acidilactici microcapsule embedding agent

[0045] 2.1 Effect of sodium alginate concentration on microcapsule embedding efficiency

[0046] Microcapsules were prepared under the conditions of chitosan concentration of 0.8%, CaCl2 concentration of 3%, curing time of 1.5h, and sodium alginate concentrations of 1%, 1.5%, 2%, 2.5%, and 3%, respectively. The embedding efficiency of the microcapsules was used as the evaluation index.

[0047] 2.2 Effect of CaCl2 concentration on microcapsule embedding efficiency

[0048] Microcapsules were prepared under the conditions of 2% sodium alginate concentration, 0.8% chitosan concentration, 1.5h curing time, and CaCl2 concentrations of 1%, 2%, 3%, 4%, and 5%, respectively. The embedding efficiency of the microcapsules was used as the evaluation index.

[0049] 2.3 Effect of chitosan concentration on microcapsule embedding efficiency

[0050] Microcapsules were prepared under the conditions of 2% sodium alginate concentration, 3% CaCl2 concentration, 1.5h curing time, and chitosan concentrations of 0.4%, 0.6%, 0.8%, 1.0%, and 1.2%, respectively. The embedding efficiency of the microcapsules was used as the evaluation index.

[0051] 2.4 Effect of fixation time on microcapsule embedding efficiency

[0052] Microcapsules were prepared under the conditions of 2% sodium alginate concentration, 0.8% chitosan concentration, 3% CaCl2 concentration and curing time of 0.5, 1, 1.5, 2 and 2.5h respectively. The embedding efficiency of microcapsules was used as the evaluation index.

[0053] 2.5 Orthogonal experiment

[0054] On the basis of single-factor experiments, the orthogonal experiment was used to optimize the preparation of microcapsules with four factors, namely, sodium alginate concentration, chitosan concentration, CaCl2 concentration, and curing time, as independent variables and embedding efficiency as the evaluation index. The factor levels are shown in Table 1.

[0055] Table 1: Orthogonal experiment optimization factor level table for microcapsule preparation

[0056]

[0057] 2.6 Determination of microcapsule embedding efficiency

[0058] Microcapsules with sodium alginate as the wall material can achieve rapid disintegration in sodium citrate solution. 1 mL of pre-freeze-dried microcapsules prepared from bacterial slurry were lysed in 9 mL of 3% sodium citrate solution. The solution was placed in a 37°C water bath for 60 minutes and then vortexed on a vortex oscillator for 5 minutes. This cycle was repeated 2-3 times until complete lysis. The lysate was diluted appropriately and then counted on a plate for viable bacterial counts. The calculation formula is as follows:

[0059]

[0060] Where N is the number of viable bacteria embedded in the microcapsules before freeze-drying, cfu / mL; M is the number of viable bacteria in the initial bacterial slurry, cfu / mL.

[0061] Depend on Figure 4 It can be seen from Figure 1 that when the concentration of sodium alginate is less than 3%, the embedding rate of Pediococcus acidilactici microcapsules increases with the increase of sodium alginate concentration. When the concentration of sodium alginate reaches 3%, the embedding rate of Pediococcus acidilactici reaches a maximum of 85.10%. Then, the embedding rate gradually decreases with the increase of sodium alginate concentration. Figure 4 b shows that when the concentration of chitosan is less than 0.8%, the embedding rate of Pediococcus acidilactici microcapsules increases with the increase of chitosan concentration. When the concentration of chitosan reaches 0.8%, the embedding rate of Pediococcus acidilactici reaches the highest of 86.74%, and then the embedding rate gradually decreases with the increase of chitosan concentration. Figure 4 c It can be seen that when the CaCl2 concentration is less than 3%, the embedding rate of the lactic acid bacteria microcapsules increases with the increase of the CaCl2 concentration. When the CaCl2 concentration reaches 3%, the embedding rate of lactic acid bacteria reaches a maximum of 90.28%. After that, the embedding rate gradually decreases with the increase of the CaCl2 concentration. When the cross-linking agent concentration is too low, the cross-linking degree is insufficient, the embedding is incomplete, and the contents are easy to lose. When the cross-linking agent concentration is too high, the pore size of the formed spatial network structure is low, the molecules are tightly connected, and the diffusion of molecules is affected. Figure 4As shown in the figure, the encapsulation efficiency of Pediococcus acidilactici reached a maximum of 87.43% when the curing time was 0.5 h, and then gradually decreased with increasing curing time. Based on the single-factor experiment affecting the encapsulation efficiency of Pediococcus acidilactici microcapsules, the optimal encapsulation conditions for Pediococcus acidilactici microcapsules were determined to be a sodium alginate concentration of 3%, a chitosan concentration of 0.8%, a CaCl2 concentration of 3%, and a curing time of 0.5 h, which were used as the conditions for subsequent orthogonal experiments.

[0062] An orthogonal experiment was conducted with the embedding rate obtained in the single-factor experiment as the indicator to determine the optimal conditions for preparing microcapsules. The four-factor three-level orthogonal analysis method was used to explore the effects of sodium alginate concentration, chitosan concentration, CaCl2 concentration, and curing time on the embedding rate of Pediococcus acidilactici microcapsules, so as to determine the optimal conditions for the final microcapsule preparation.

[0063] Table 2: Orthogonal test results

[0064]

[0065]

[0066] As shown in Table 2, the order of range is A > D > B > C. This indicates that sodium alginate concentration has the greatest impact on the microcapsule embedding efficiency, followed by curing time, chitosan concentration, and calcium chloride concentration. The highest values ​​among K1, K2, and K3 represent the optimal values ​​within the corresponding levels. Therefore, the combination A2B3C1D3 represents the optimal conditions for Pediococcus acidilactici microcapsule embedding: 3% sodium alginate concentration, 1% chitosan concentration, 2% calcium chloride concentration, and a curing time of 0.75 h. This condition demonstrated an embedding efficiency of 89.6% ± 0.02, significantly higher than the other combinations. Therefore, this condition was selected as the prerequisite for subsequent survival experiments.

[0067] Example 3: Single factor optimization test of Pediococcus acidilactici microcapsule freeze-dried protective agent

[0068] 3.1 Effect of skim milk concentration on microcapsule survival rate

[0069] Microcapsules were prepared under the conditions of glycerol concentration of 10%, sucrose concentration of 13%, mannitol concentration of 4%, tyrosine concentration of 0.8%, and skim milk concentration of 10%, 11%, 12%, 13%, and 14%, respectively. The survival rate of the microcapsules after freeze-drying was used as the evaluation index.

[0070] 3.2 Effect of glycerol concentration on microcapsule survival rate

[0071] Microcapsules were prepared under the conditions of skim milk concentration of 12%, sucrose concentration of 13%, mannitol concentration of 4%, tyrosine concentration of 0.8%, and glycerol concentrations of 8%, 9%, 10%, 11%, 12%, and 13%, respectively. The survival rate of the microcapsules after freeze-drying was used as the evaluation index.

[0072] 3.3 Effect of sucrose concentration on microcapsule survival rate

[0073] Microcapsules were prepared under the conditions of 12% skim milk concentration, 10% glycerol concentration, 4% mannitol concentration, 0.8% tyrosine concentration, and 11%, 12%, 13%, 14%, and 15% sucrose concentration respectively. The survival rate of the microcapsules after freeze-drying was used as the evaluation index.

[0074] 3.4 Effect of mannitol concentration on microcapsule survival rate

[0075] Microcapsules were prepared under the conditions of 12% skim milk concentration, 10% glycerol concentration, 13% sucrose concentration, 0.8% tyrosine concentration, and 2%, 3%, 4%, 5%, and 6% mannitol concentrations, and the survival rate of the microcapsules after freeze-drying was used as the evaluation index.

[0076] 3.5 Effect of tyrosine concentration on microcapsule survival rate

[0077] Microcapsules were prepared under the conditions of 12% skim milk concentration, 10% glycerol concentration, 13% sucrose concentration, 4% mannitol concentration, and 0.6%, 0.7%, 0.8%, 0.9% and 1% tyrosine concentration respectively, and the survival rate of the microcapsules after freeze-drying was used as the evaluation index.

[0078] 3.6 Orthogonal experiment

[0079] On the basis of single-factor experiments, orthogonal experiments were conducted to optimize the preparation of microcapsules with lyophilized protective agents using five factors, namely, skimmed milk powder concentration, glycerol concentration, sucrose concentration, mannitol concentration, and tyrosine concentration, as independent variables and survival rate as the evaluation index. The factor levels are shown in Table 3.

[0080] Table 3: Orthogonal experiment optimization of the preparation of microcapsule freeze-dried protective agent factor level table

[0081]

[0082] 3.7 Determination of microcapsule survival rate

[0083] Similarly, rapid disintegration was achieved in sodium citrate solution. 1 mL of freeze-dried microcapsules prepared from bacterial slurry were lysed in 9 mL of 3% sodium citrate solution. The solution was placed in a 37°C water bath for 60 minutes and then vortexed on a vortex oscillator for 5 minutes. This cycle was repeated 3-5 times until complete lysis. The lysate was appropriately diluted and then plated for viable bacterial count. The calculation formula is as follows:

[0084]

[0085] Where S is the number of viable bacteria in the microcapsules after freeze-drying, cfu / mL; M is the number of viable bacteria in the initial bacterial slurry, cfu / mL.

[0086] The positive effect of skim milk powder on freeze-dried microorganisms is that its loose and porous structure can store water for cells and provide a water environment. It has a positive effect on the survival rate of freeze-dried cells. The combination of skim milk and sucrose can achieve the best preservation effect of Candida sake cells. Figure 5 a It can be seen that with the increase of skim milk powder concentration, the freeze-drying survival rate of the microcapsules gradually increased. When the skim milk powder concentration was 13%, the freeze-drying survival rate reached a maximum of 75.91%. Subsequently, even if the skim milk powder concentration was higher, the freeze-drying survival rate did not continue to increase; sucrose, glycerol, sorbitol and skim milk were the most effective freeze-drying protectants for Lactobacillus bulgaricus LB14. The increase in skim milk concentration significantly improved the cell viability of freeze-dried L.bulgricus LB14 within the tested concentration range. At higher skim milk concentrations, increasing the glycerol concentration resulted in a significant increase in cell viability. Therefore, the interaction between skim milk and glycerol is very significant. Figure 5 b It can be seen that with the increase of glycerol concentration, the freeze-drying survival rate of microcapsules gradually increased. When the glycerol concentration was 11%, the freeze-drying survival rate reached a maximum of 71.03%. After that, with the increase of glycerol concentration, the freeze-drying survival rate of microcapsules decreased. Sucrose is the best freeze-drying protective agent. When corn flour is used as the supporting material, the drying speed is faster than glycerol and glucose during the freeze-drying process, and the survival and cell viability of yeast after drying are the highest. Figure 5 c It can be seen that with the increase of sucrose concentration, the freeze-drying survival rate of the microcapsules gradually increased. When the sucrose concentration was 14%, the freeze-drying survival rate reached a maximum of 75.25%. After that, with the increase of sucrose concentration, the freeze-drying survival rate of the microcapsules decreased instead. With the increase of sorbitol concentration, the cell viability of freeze-dried Lactobacillus LB14 decreased at low skim milk concentrations, but increased sharply at high skim milk concentrations. The interaction effect of sorbitol and skim milk (P<0.0001) is the reason for this behavior. Mannitol, as a filler, can better ensure the good appearance of the microcapsules during the freeze-drying process. Figure 5It can be seen from the results that with the increase of mannitol concentration, the freeze-dried survival rate of microcapsules gradually increased. When the mannitol concentration was 4%, the freeze-dried survival rate reached a maximum of 70.33%. After that, with the increase of mannitol concentration, lower concentrations of mannitol can promote the growth of bacteria, while higher concentrations of mannitol are harmful to bacteria. Figure 5 It can be seen from the data that with the increase of tyrosine concentration, the freeze-drying survival rate of the microcapsules gradually increased. When the tyrosine concentration was 0.9%, the freeze-drying survival rate reached a maximum of 61.55%. After that, with the increase of glycerol concentration, the freeze-drying survival rate of the microcapsules decreased.

[0087] Table 4: Orthogonal test results of freeze-drying protective agents

[0088]

[0089]

[0090] As can be seen from Table 4, the order of the range is B>A>C>D>E, that is, the glycerol concentration has the greatest impact on the freeze-dried survival rate of the microcapsules, followed by the skimmed milk powder concentration, sucrose concentration, mannitol concentration and tyrosine concentration. The largest value among k1, k2, and k3 is the optimal solution at the corresponding level. Therefore, the combination of A2B3C2D1E1 is the best freeze-dried protectant formula, that is, the freeze-dried survival rate of the microcapsules is the highest when the skimmed milk powder concentration is 13%, the glycerol concentration is 12%, the sucrose concentration is 14%, the mannitol concentration is 3% and the tyrosine concentration is 0.8%. It has been verified that under this experimental condition, the freeze-dried survival rate of the microcapsules reaches 76.42%.

[0091] Example 4: Preparation process and appearance characteristics determination of Pediococcus acidilactici microcapsules

[0092] 4.1 Preparation of Pediococcus acidilactici microcapsules

[0093] Second-generation activated Pediococcus acidilactici was centrifuged at 8000 rpm for 10 minutes. The collected sludge precipitate was diluted to an equal volume and thoroughly mixed with 2% sodium alginate. The mixture was then mixed with a lyoprotectant at a ratio of 2:2:1. To completely dissolve chitosan, 1% glacial acetic acid was added to the 0.8% chitosan solution. The sludge-sodium alginate-protectant mixture was uniformly added dropwise to the CaCl2-chitosan solution using a 1 mL tubular syringe (with a flat-tipped needle) to form wet microcapsules. The mixture was stirred to solidify, ensuring full contact between the wet microcapsules and the solidifying solution, and then allowed to solidify for 1.5 hours. The wet microcapsules were washed thoroughly with sterile distilled water 3-5 times to remove excess bacterial solution, solidifying solution, and chitosan coating from the surface. The mixture was then pre-frozen overnight at -80°C and freeze-dried in a freeze dryer for 24 hours to obtain Pediococcus acidilactici microcapsule particles.

[0094] 4.2 Determination of particle size of Pediococcus acidilactici microcapsules

[0095] Use a vernier caliper to randomly select 20 single-strain microcapsules and take the average value to obtain the particle size distribution of the probiotic microcapsules.

[0096] 4.3 Appearance characteristics of Pediococcus acidilactici microcapsules

[0097] In order to better observe the surface characteristics and internal structure of the microcapsules, a transmission electron microscope (TEM) was used to observe the microcapsules.

[0098] 4.1 Particle size of Pediococcus acidilactici microcapsules

[0099] Twenty probiotic microcapsules were randomly selected using a vernier caliper and their particle sizes were measured. The average particle size of the microcapsules was found to be 1.17±0.04 mm.

[0100] 4.2 Appearance characteristics of Pediococcus acidilactici microcapsules

[0101] Through transmission electron microscopy, we have a better understanding of the appearance and internal structure of microcapsules. Figure 6 It can be seen that the surface of the freeze-dried microcapsules is relatively smooth. The cross-section after cutting shows that the interior of the microcapsules is loose and porous due to freeze-drying and dehydration. After magnification, it can be seen that the raised parts in the pores are the encapsulated probiotics.

[0102] Example 5: In vitro simulated tolerance and slow release experiments of Pediococcus acidilactici microcapsules

[0103] 5.1 In vitro simulated continuous gastrointestinal fluid release experiment

[0104] The preparation of artificial gastric juice, artificial pancreatic juice and artificial colonic juice refers to the 2020 edition of the "Chinese Pharmacopoeia". Referring to Ren Guangyu's method, with slight modifications, 1mL of microcapsules made of bacterial mud were added to 9mL of gastric juice. After thorough shaking and mixing, they were placed in a 37°C shaker at 180r / min for 2h. Every 1h, the mixed solution was aspirated and appropriately diluted, then spread on MRS solid culture medium. After anaerobic culture in a constant temperature incubator at 37±1°C for 24h, live bacteria were counted. The microcapsules collected after gastric juice treatment were added to 9mL of intestinal fluid, shaken and mixed thoroughly, and placed in a 37°C shaker at 180r / min for 4h. The mixed solution was aspirated at 2h and 4h, respectively, and after appropriate dilution, it was spread on MRS solid culture medium. After anaerobic culture in a constant temperature incubator at 37±1°C for 24h, live bacteria were counted. The microcapsules collected after intestinal fluid treatment were added to 9 mL of colonic fluid, shaken thoroughly and placed in a shaker at 37°C and 180 r / min for 18 hours. The mixed solution was aspirated at 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, and 18 hours respectively. After appropriate dilution, it was spread on MRS solid culture medium and placed in a constant temperature incubator at 37±1°C for anaerobic culture for 24 hours, and then the viable bacteria were counted.

[0105] If probiotics are to reach the human body and play a beneficial role, it is necessary not only to ensure the activity and quantity of probiotics, but also to protect them so that they can withstand the harsh environment of the gastrointestinal tract, such as low pH and bile salts. Figure 7 It can be seen that the number of live bacteria encapsulated in the experiment simulating gastrointestinal digestion was 11.2 lg (cfu / mL). From the results, it can be seen that due to the high viscosity of sodium alginate under low pH conditions and the tight internal binding, the probiotics are not easily released, and only 2.45 lg (cfu / mL) is released, indicating that most of the probiotics in the probiotic microcapsules can pass through the stomach smoothly. Because sodium alginate has a low viscosity and a loose internal structure under higher conditions of pH 6-9, the probiotics are more easily released. After 4 hours in the intestinal fluid, the number of live bacteria released reached about 6 lg (cfu / mL). As the time under high pH conditions increases, the number of probiotics released increases. After 18 hours in the colonic fluid, about 10 lg (cfu / mL) has been released, which is about 4 orders of magnitude higher than that in pancreatic juice. At the same time, it can be seen that the probiotic microcapsules have a sustained release effect. Therefore, it can be concluded that the prepared probiotic microcapsules can well meet the requirements of targeted delivery of live bacterial drugs.

[0106] Example 6: Construction of ISO-induced chronic heart failure mouse model and gavage experiment

[0107] 6.1 Construction of a chronic heart failure mouse model

[0108] Twenty male C57BL / 6 mice were selected and bred for one week. After acclimation, each mouse received a daily subcutaneous injection of ISO 0.4 mg / 20 g (4 mg / ml, 0.1 ml / 20 g) for two consecutive weeks. The mice were then divided into a control group (HF group) and an intragastric administration group (HF-PA group). The HF-PA group received 0.1 g of microcapsules daily by intragastric administration; the HF group received 0.1 g of the embedding and protective material (i.e., a strain-free group containing only the embedding and protective material) daily by intragastric administration for four weeks. All mice were fed a normal diet and maintained at a temperature of (22 ± 3)°C, a humidity of (50 ± 15)°C, and a 12-hour light / 12-hour dark cycle. Mice were labeled with their ID number and date, and their condition was observed daily.

[0109] 6.2 Echocardiography and Pathological Section Examination

[0110] After 4 weeks of gavage, the mouse chest was dehaired and wiped clean, and the mouse was anesthetized with isoflurane, and its heart rate was stabilized at 450-500 beats per minute. A small animal cardiac ultrasound detection system (Visual Sonics, Vevo 2100) was used to detect the cardiac contractile function of the mouse, and the B-mode long axis image of the mouse left ventricle was collected and the M-mode image was collected at the maximum diameter of the left ventricle. Finally, the left ventricular ejection fraction (EF%) was calculated using the LVtrace tool. The mouse was euthanized, the heart was removed after dissection, a frontal photo of the mouse heart was taken, and the mouse heart was recorded. The heart tissue was collected, fixed and preserved, sliced ​​and prepared, and the cross-section was stained with HE and Masson.

[0111] By taking photos and comparing the characteristics of the heart samples, it was found that the heart volume of the mice in the HF-PA group was normal, while the heart of the mice in the HF group (HF group) was significantly enlarged and dilated ( Figure 8 ). After paraffin embedding and sectioning, HE and Masson staining were performed ( Figure 9 ) and detected changes in the cross-sectional area of ​​myocardial fibers. The staining results showed that the lactic acid Pediococcus group reduced the cross-sectional area of ​​myocardial fibers in mice with chronic heart failure, while the group that only received the supplementary material had no effect on reducing the area. The group that only received the supplementary material had thickening of the walls of the myocardial arterioles, narrowing of the lumen, increased collagen deposition, increased fibrosis area, and increased cross-sectional area of ​​myocardial cells. The results of small animal echocardiography showed that the group that only received the supplementary material had left or right ventricular hypertrophy, increased heart mass, and a significant decrease in ejection fraction (EF%). Figure 10 ); while the ventricles of mice in the oral gavage of Pediococcus acidilactici group were relatively normal, and the reduced ejection fraction (EF%) was improved, confirming that Pediococcus acidilactici has the effect of preventing and / or treating chronic heart failure.

[0112] In summary, the present invention provides Pediococcus acidilactici and microcapsules prepared therefrom. The preparation formula and process of the microcapsules can improve the number of live bacteria in the fermentation and pharmaceutical production process of Pediococcus acidilactici, facilitate the transportation of live bacteria, prolong the storage stability of the bacteria, enable targeted delivery in the intestine and slow release, and the microcapsules can treat chronic heart failure.

[0113] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Pediococcus acidilactici, characterized in that The Pediococcus acidilactici ( Pediococcus acidilactici ) is deposited in China with a number of CGMCC No. 30585.

2. Use of the Pediococcus acidilactici according to claim 1 in the preparation of a product for preventing and / or treating chronic heart failure.

3. The use according to claim 2, characterized in that The product is a microcapsule.

4. A microcapsule, characterized in that The core material of the microcapsule contains the Pediococcus acidilactici according to claim 1.

5. The microcapsule according to claim 4, wherein The embedding agent of the microcapsule contains 3% sodium alginate, 1% chitosan and 2% calcium chloride.

6. The microcapsule according to claim 4, wherein The Pediococcus acidilactici according to claim 1 is mixed with a freeze-drying protective agent and used as the core material.

7. The microcapsule according to claim 6, wherein The composition ratio of the freeze-dried protective agent is as follows: 13% sea skim milk powder, 12% glycerol, 14% sucrose, 3% mannitol and 0.8% tyrosine.

8. The method for preparing microcapsules according to claim 4, characterized in that: The preparation method comprises the following steps: diluting bacterial slurry of Pediococcus acidilactici and stirring the mixture with a sodium alginate solution to make it uniform, and then stirring the mixture with a freeze-drying protective agent to make it uniform to obtain a mixed bacterial solution; after completely dissolving chitosan, dripping the mixed bacterial solution into a mixed solution of calcium chloride and chitosan at a uniform speed to form wet microcapsules, stirring and solidifying the mixture so that the wet microcapsules are in full contact with the solidifying solution of calcium chloride, and then standing the mixture to solidify. After washing, the mixture is freeze-dried in a freeze dryer to obtain Pediococcus acidilactici microcapsule particles.

9. Use of the microcapsule according to claim 4 in the preparation of an orally administrable product capable of preventing and / or treating chronic heart failure.

10. An oral product capable of preventing and / or treating chronic heart failure, characterized in that: The product contains the microcapsules according to claim 4.

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