PH response type active bacterial raft and preparation method and application thereof

Abstract

The invention provides a pH response type active bacterium raft and a preparation method and application thereof, and belongs to the field of biomedical engineering.The preparation method of the pH response type active bacterium raft comprises the following steps that 1, calcium carbonate, sodium bicarbonate and a sodium alginate solution are mixed and stirred for the first time, and a mixed solution is obtained; (2) dropwise adding lactococcus lactis into the mixed solution obtained in the step (1), and carrying out secondary stirring to obtain a bacterium-sodium alginate mixed system; and 3) injecting the bacteria-sodium alginate mixed system into a cross-linking system through electrostatic spinning to obtain the pH response type active bacteria raft. The pH response type active bacteria raft and the lactococcus lactis are uniformly distributed in a hydrogel structure rich in bubbles, have a remarkable and acid-stable bubble structure, and can stably float in simulated gastric juice to regulate and control pH; the esophageal injury inhibition rate can reach 93.95% in the gastroesophageal reflux disease treatment process, and excellent clinical transformation prospects are shown.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, and in particular relates to a pH-responsive active bacterial raft, its preparation method, and its application. Background Technology

[0002] Gastroesophageal reflux disease (GERD) is a esophageal mucosal injury caused by the abnormal reflux of gastric contents. The pathogenesis of GERD involves the interaction of multiple factors, including hypersecretion of gastric acid (HCl) and dysfunction of the lower esophageal sphincter. Among these, lower esophageal sphincter failure and gastric reflux are the core factors triggering mucosal damage. Therefore, regulating gastric pH is of significant clinical importance in the management of GERD.

[0003] Currently, various strategies have been developed to regulate gastric pH, including neutralizing gastric acid and inhibiting gastric acid secretion. Weakly basic acid neutralizers, such as magnesium aluminum hydroxide and sodium bicarbonate, temporarily adjust pH through acid-base neutralization, but cannot achieve long-term control. Clinically, the gold standard for regulating gastric pH is proton pump inhibitors (PPIs), such as omeprazole. However, long-term inhibition of gastric acid can disrupt the overall acidic environment of the stomach, leading to adverse effects such as bacterial overgrowth and elevated gastrin levels. Therefore, there is an urgent need to develop local pH regulation strategies that do not interfere with the inherent acidic ecological balance of the stomach.

[0004] Urease possesses a unique enzymatic reaction that can continuously decompose urea into ammonia (N2). ) and carbon dioxide (C ), generated N It can effectively neutralize surrounding hydrogen protons ( This provides a potential means of regulating the local gastric pH. Currently, probiotics such as *Streptococcus thermophilus* and *Lactobacillus reuteri* have been developed that can produce acid-resistant urease and utilize a self-regulating feedback mechanism to stop the enzymatic reaction when the surrounding pH approaches neutral, demonstrating a dynamic response. The concept of conversion. Genetic engineering can optimize the codons and modify the molecular structure of acid urease genes, thereby achieving high levels of heterologous expression and significantly improving the enzyme's activity and stability. However, establishing a dynamic model based on acid urease... Significant challenges remain in the conversion system, ensuring its stable function in the complex gastric environment, and limiting its pH-regulating effects to localized areas. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a pH-responsive active bacterial raft, its preparation method, and its application; this invention establishes a dynamic pH-responsive raft by integrating engineered Lactococcus lactis with calcium alginate rafts. + Conversion Balance System (DH) + -CES), which allows for on-demand adjustment of the local pH at the esophagogastric junction to treat gastroesophageal reflux disease.

[0006] This invention provides a method for preparing pH-responsive active bacterial rafts, comprising the following steps: 1) Mix calcium carbonate, sodium bicarbonate and sodium alginate solution, stir for the first time to obtain a mixed solution; 2) Add Lactococcus lactis dropwise to the mixed solution obtained in step 1), and stir again to obtain a bacterial-sodium alginate mixed system; 3) The bacterial-sodium alginate mixture was injected into the cross-linking system via electrospinning to obtain pH-responsive active bacterial rafts; The crosslinking system includes a calcium chloride solution and a hydrochloric acid solution.

[0007] Preferably, the concentration of the sodium alginate solution in step 1) is 1~3% (w / v).

[0008] Preferably, the molar ratio of calcium carbonate, sodium bicarbonate and sodium alginate in the mixed solution in step 1) is 1:(2~3):1.

[0009] Preferably, the OD of the lactococcus lactis diluted 100 times in step 2) 600 The value is 0.9~1.1.

[0010] Preferably, the volume ratio of the lactococcus lactis to the mixed solution is 1:(8~12).

[0011] Preferably, in step 3), the injection is performed using a 25G needle, the injection rate is set to 20~30mL / h, and the injection voltage is 0kV.

[0012] Preferably, the concentration of the calcium chloride solution is 10~20 g / L, the pH of the hydrochloric acid solution is 0.9~1.1, and the volume ratio of the calcium chloride solution to the hydrochloric acid solution is 1:(3~5).

[0013] This invention provides a pH-responsive active bacterial raft prepared by the aforementioned preparation method.

[0014] Preferably, the pH-responsive active bacterial raft has a significant and acid-stable bubble structure.

[0015] This invention provides the application of the pH-responsive active bacterial rafts in the preparation of drugs for treating gastroesophageal reflux disease.

[0016] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a method for preparing a pH-responsive active bacterial raft. A mixed solution is obtained by mixing calcium carbonate, sodium bicarbonate, and sodium alginate solution. Lactococcus lactis is added dropwise to the mixed solution, and then injected into a cross-linking system via electrospinning to obtain a pH-responsive active bacterial raft. The pH-responsive active bacterial raft of the present invention has Lactococcus lactis uniformly distributed in a bubble-rich hydrogel structure, possessing a significant and acid-stable bubble structure, allowing it to float stably in simulated gastric fluid, achieving a 100% floating rate within 10 minutes. It can raise the pH of simulated gastric fluid from 2.0 to 6.0 within 0.5 hours, with the adjusted gastric fluid accounting for only 5.38% of the total volume. This effective pH adjustment with a specific site of action can continue for several hours.

[0017] The pH-responsive active bacterial rafts described in this invention can float on the surface of gastric juice, forming a natural physical barrier to prevent gastric acid reflux. Simultaneously, Lactococcus lactis expressing acid urease reacts with H+... + When activated, it converts protons into NH4. + This invention establishes a local chemical barrier at the esophagogastric junction. It will provide a local pH regulation platform for research on the treatment of gastroesophageal reflux, offering new insights into the complex process of gastric acid reflux and the development of methods to control it.

[0018] Compared with existing technologies, the pH-responsive active bacterial rafts described in this invention act as a physical-chemical barrier to regulate the local microenvironment at the gastroesophageal junction. They can inhibit esophageal damage by up to 93.95% during the treatment of gastroesophageal reflux disease, demonstrating excellent prospects for clinical translation. Attached Figure Description

[0019] Figure 1 To compare the bubble patterns inside alginate rafts with different sodium bicarbonate contents, a) shows the structure of the alginate rafts observed using an optical microscope, and b) shows the relative area of ​​bubbles in the raft structure. Figure 2 To compare the effect of crosslinking systems containing hydrochloric acid with different acidities (pH 1.0 and pH 2.0) on their structure, a) shows the structure of the alginate raft observed under an optical microscope, and b) shows the relative area of ​​the bubbles in the raft to the entire raft structure. Figure 3 To observe DH using a scanning microscope (MIRA4 LMH) and an optical microscope + -The structure of the CES system, where a is the prepared DH + - The CES system can float stably in simulated gastric fluid. b shows the significant presence of air bubbles in the structure. c shows, under scanning microscopy, Lactococcus lactis uniformly present in the porous DH structure. + -CES system; Figure 4 The buoyancy and physical barrier of pH-responsive active bacterial rafts are shown in the figure, where a is the buoyancy rate of stable floating in SGF for 48 h, b is the minimum volume of bubbles required for stable floating, c is the result of blocking liquid backflow, and d is the compression test result of the universal material instrument. Figure 5 To observe the spatial distribution of pH changes at different times in SGF containing the pH-sensitive dye bromocresol green, where a represents the spatial distribution at different times, b represents the proportion of the blue area to the total solution volume after 30 min, and c represents the pH changes of the solution after adding freshly prepared bromocresol green SGF solution at 20, 30, and 40 min. Figure 6 This study compares the performance of pH-responsive active bacterial rafts with the existing technology Gaviscon®. Specifically, (a) is a normalized comparison of pH-responsive active bacterial rafts and Gaviscon® in six aspects: acid neutralization range, floating stability, floating rate, floating percentage, storage stability, and physical barrier effect; (b) shows the floating behavior of the rafts after reaction at different initial pH values; (c) shows the average floating rate of the rafts formed by pH-responsive active bacterial rafts and Gaviscon®; (d) shows the floating rate of the rafts formed within 10 min; (e) shows the pH-adjusting effect of pH-responsive active bacterial rafts and Gaviscon® on SGF at 30 min; (f) shows the pH-time graphs of both technologies; and (g) explores the stability of the pH-adjusting performance of pH-responsive active bacterial rafts at different storage times under 4°C storage conditions. Figure 7 The study shows the lesion inhibition of a rat animal model after oral administration of a pH-responsive active bacterial raft. In the figure, a is the bright field image of the esophagus in the sham-operated group, control group, and treatment group; b is the quantitative map of the esophageal lesion extent; c is the serum level of interleukin-1β (IL-1β); d is the immunohistochemical image of TNF-α expression in the esophagus of the three groups; e is the expression level of positive cells in the immunohistochemical image; and f is the hematoxylin-eosin (H&E) staining image of the esophagus of the three groups. Detailed Implementation

[0020] This invention provides a method for preparing pH-responsive active bacterial rafts, comprising the following steps: 1) mixing calcium carbonate, sodium bicarbonate, and sodium alginate solution, followed by a first stirring to obtain a mixed solution; 2) adding Lactococcus lactis dropwise to the mixed solution obtained in step 1), followed by a second stirring to obtain a bacteria-sodium alginate mixed system; 3) injecting the bacteria-sodium alginate mixed system into a cross-linking system via electrospinning to obtain pH-responsive active bacterial rafts; wherein the cross-linking system comprises a calcium chloride solution and a hydrochloric acid solution.

[0021] In this invention, calcium carbonate, sodium bicarbonate, and sodium alginate solution are mixed and stirred to obtain a mixed solution. In this invention, the concentration of the sodium alginate solution is preferably 1-3% (w / v), more preferably 1.5-2.5% (w / v), and most preferably 2% (w / v). Preferably, the sodium alginate solution is prepared by dissolving sodium alginate in water, preferably deionized water. The sodium alginate is added to deionized water for dissolution, and during the dissolution process, stirring is preferably performed, preferably magnetic stirring. The dissolution time is preferably 10-14 hours. After obtaining the sodium alginate solution, calcium carbonate, sodium bicarbonate, and sodium alginate solution are mixed; the molar ratio of calcium carbonate, sodium bicarbonate, and sodium alginate is preferably 1:(2-3):1, more preferably 1:(2.8-2.8):1, and most preferably 1:2.5:1. In this invention, the first stirring is preferably magnetic stirring, the stirring speed is preferably 1000~2000 rpm, more preferably 1200~1800 rpm, most preferably 1500 rpm, and the stirring time is preferably 50~70 min, more preferably 55~65 min, most preferably 60 min.

[0022] In this invention, *Lactococcus lactis* is added dropwise to the obtained mixed solution, followed by a second stirring to obtain a bacterial-sodium alginate mixed system. In this invention, the OD of the *Lactococcus lactis* diluted 100 times... 600 The preferred ratio is 0.9 to 1.1, more preferably 1.0. In this invention, the *Lactococcus lactis* is preferably an engineered strain, and more preferably the *Lactococcus lactis* from the following literature (Document DOI: 10.1007 / s00253-014-5916-z). In this invention, the volume ratio of the *Lactococcus lactis* to the mixed solution is preferably 1:(8-12), more preferably 1:(9-11), and most preferably 1:10. In this invention, the second stirring is preferably magnetic stirring, the stirring speed is preferably 1000-2000 rpm, more preferably 1200-1800 rpm, and most preferably 1500 rpm, and the stirring time is preferably 50-70 min, more preferably 55-65 min, and most preferably 60 min.

[0023] In this invention, a pH-responsive active bacterial raft is obtained by injecting the bacterial-sodium alginate mixture into a cross-linking system via electrospinning. The bacterial-sodium alginate mixture is loaded into a microsyringe, installed in an injection pump, and connected to an electrospinning device; then injection is performed. The injection is preferably performed using a 25G needle, and the injection rate is preferably 20-30 mL / h, more preferably 22-28 mL / h, and most preferably 24 mL / h; the injection voltage is 0 kV. In this invention, the cross-linking system comprises a calcium chloride solution and a hydrochloric acid solution. The concentration of the calcium chloride solution is preferably 10-20 g / L, more preferably 13-17 g / L, and most preferably 15 g / L. The pH of the hydrochloric acid solution is preferably 0.9-1.1, more preferably 1.0; the volume ratio of the calcium chloride solution to the hydrochloric acid solution is preferably 1:(3-5), more preferably 1:4. In this invention, after observing the formation of bacterial-alginate rafts accompanied by the formation of a large number of bubbles, the rafts are left to stand for 1 minute and then quickly removed to obtain pH-responsive active bacterial rafts.

[0024] The electrospinning technology described in this invention is a common technique for preparing multifunctional microspheres. The instrument mainly consists of three parts: a high-voltage power supply, a microinjection pump, and a receiving device. It is inexpensive and easy to mass-produce multiple times. The *Lactococcus lactis* used in this invention has been widely used in oral microbial therapy due to its recognized biosafety, relatively simple metabolic pathway, and small genome size. Genetic engineering is used to promote high-level heterologous expression of *Lactococcus lactis*. Furthermore, this invention achieves 100% buoyancy and stable floating of active bacterial rafts through multiple optimizations, combining physical-chemical barrier protection with localized pH regulation of the microenvironment.

[0025] The present invention also provides a pH-responsive active bacterial raft prepared by the above preparation method; the pH-responsive active bacterial raft has a significant and acid-stable bubble structure.

[0026] The present invention also provides the application of the pH-responsive active bacterial rafts in the preparation of drugs for treating gastroesophageal reflux disease.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] Sodium alginate (80-120 degrees of polymerization) is from Fujifilm and Koichi Chemical Co., Ltd. Calcium carbonate, sodium bicarbonate, hydrochloric acid, and urea are from Sinopharm Chemical Reagent Company. Anhydrous calcium chloride is from Aladdin Biochemical Technology Co., Ltd.

[0030] D Preparation of the CES system: First, prepare a 2% (mass / volume) sodium alginate solution. Dissolve sodium alginate in deionized water overnight with magnetic stirring until completely dissolved. Accurately weigh 45.8 mg of calcium carbonate and 97.3 mg of sodium bicarbonate, and mix them with 5 mL of the 2% sodium alginate solution. Stir the mixture continuously with magnetic stirring for 1 h to obtain a homogeneous solution with a molar ratio of 1:2.5:1. Subsequently, add dropwise 500 μL of bacterial suspension (bacterial suspension diluted 100 times, OD...) 600 The concentration was set at 1.0 ± 0.05. The mixture was then stirred continuously for 1 hour to ensure uniform dispersion of the bacterial suspension, thus forming a stable bacterial-sodium alginate mixture. This system was then loaded into a 5 mL microsyringe, which was attached to an injection pump and connected to an electrospinning device. High-voltage electrostatic injection was performed using a 25G needle, injecting the mixture into the crosslinking system at an injection rate of 24.0 mL / h and a voltage of 0 kV. The crosslinking system consisted of a 15.0 g / L calcium chloride solution and a pH 1.0 hydrochloric acid solution in a volume ratio of 1:4. After observing the formation of bacterial-alginate rafts accompanied by a large number of bubbles, the mixture was allowed to stand for 1 minute, and then quickly removed to obtain D containing CaCO3. -CES system (i.e., pH-responsive active bacterial raft).

[0031] Comparative Example 1

[0032] ① Prepare a 2% sodium alginate solution by dissolving it in deionized water and stirring until completely dissolved. Accurately weigh 45.8 mg of calcium carbonate and add it to 5 mL of the 2% sodium alginate solution, stirring for 1 hour to form a homogeneous solution with a molar ratio of 1:0:1. To facilitate observation of bubble formation, no bacterial suspension was added. The system was then loaded into a 5 mL syringe, attached to a syringe pump, and connected to an electrospinning device. High-voltage electrostatic injection was performed using a 25G needle, injecting the solution into the crosslinking system at a rate of 24.0 mL / h and a voltage of 0 kV. The crosslinking system consisted of a 15.0 g / L calcium chloride solution and a pH 1.0 hydrochloric acid solution in a volume ratio of 1:4. Alginate raft formation was observed, but due to the lack of CO2-producing sodium bicarbonate, the gelation rate was faster than the bubble formation rate, resulting in no significant bubble generation.

[0033] ② Prepare a 2% sodium alginate solution and dissolve it in deionized water, stirring until completely dissolved. Accurately weigh 45.8 mg of calcium carbonate and 97.3 mg of sodium bicarbonate, place them in 5 mL of the 2% sodium alginate solution, and stir for 1 h to form a homogeneous solution with a molar ratio of 1:2.5:1. To facilitate observation of bubble formation, no bacterial suspension was added. The system was then loaded into a 5 mL syringe, attached to a syringe pump, and connected to an electrospinning device. High-voltage electrostatic injection was performed using a 25G needle, injecting the solution into the crosslinking system; the injection rate was set to 24.0 mL / h, and the voltage to 0 kV. The crosslinking system consisted of a 15.0 g / L calcium chloride solution and a pH 2.0 hydrochloric acid solution in a volume ratio of 1:4. Due to the acidity of the hydrochloric acid in the crosslinking system being 2.0, there were insufficient hydrogen protons to react with the sodium bicarbonate to produce CO2, resulting in a faster gelation rate than bubble production. Consequently, no bubble-rich alginate rafts were observed to form.

[0034] Experimental Example 1

[0035] D prepared in Example 1 Characterization of the -CES system, using scanning microscopy (MIRA4 LMH) and optical microscopy to observe D -The structure of the CES system; results are as follows Figure 3 As shown, where a represents the prepared D - The CES system can float stably in simulated gastric fluid. b shows the significant presence of air bubbles in the structure. c is a scanning microscope showing the engineered bacteria uniformly present in the porous structure. -CES system.

[0036] D - The buoyancy and physical barriers of the CES system, as shown in the following figures Figure 4 As shown, D, rich in bubble structure The CES system floated stably in SGF for up to 48 hours with a 100% buoyancy rate (a). Statistical analysis showed that the minimum bubble volume required for stable floating of the system was 0.47 mm. 3 (b). D Compared to the control group, the CES system effectively blocked the backflow of liquid with a pressure of 5 mmHg, demonstrating superior physical barrier properties (c). Compression experiments using a universal material analyzer showed that a D-type material with a diameter of 3-4 mm... -CES can withstand 56.5% deformation, approximately 0.2N of force, and 16KPa of compressive stress (d) before fracture. - The compressive strength of CES (16 kPa) far exceeds the maximum pressure in the stomach (about 10 kPa), indicating that it has sufficient strength to resist gastric peristalsis.

[0037] D -CES system Transformation process: D The CES system observed the spatial distribution of pH changes in SGF (initial pH 2.0) containing the pH-sensitive dye bromocresol green (0.1% w / v). Figure 5 (as shown in a), the system hydrolyzes urea chemical The conversion raises the pH of the solution, producing this change only in the vicinity, causing the bromocresol green in the surrounding area to change from yellow (pH < 3.8) to blue (pH > 5.4). After 30 minutes, the blue area comprised only 5.38% of the total solution volume. Figure 5 (b) indicates that the overall pH change of SGF is negligible, confirming its rapid and sustained local pH-regulating ability. Adding freshly prepared bromocresol green solution (pH 2.0) at 20, 30, and 40 min initially decreased the pH, but at D... - The rapid recovery around the CES system indicates that it is able to function under conditions of continuous gastric fluid turnover. Figure 5 (c in the text)

[0038] D - Comparison of the CES system with existing technology Gaviscon®: D -CES and Gaviscon®'s acid neutralization range is achieved through a combination of precision pH test strips and pH indicators to detect the pH level around the system.

[0039] Floating rate calculation: The bacterial-sodium alginate mixture was vertically injected from a syringe into a glass bottle containing a cross-linking agent. The floating rate was calculated by measuring the height of the cross-linking agent liquid level and the resulting D. -CES is the ratio of the time required for it to float from the bottom of the container to the highest point of the liquid surface; if it does not float, then it is 0. Floating stability is determined by randomly selecting 5 D particles. -CES was placed in pH 2.0 SGF to observe its buoyancy; D -CES buoyancy is measured in the bacterial-sodium alginate mixture system to D -CES calculations are based on observing the number of floaters after adding 20 drops; Gaviscon®'s flocculation rate is calculated by recording the weight of the floaters and the mass of SGF added to pH 1.0 (these two have different applications and calculation methods; commercial Gaviscon® is used for direct oral administration of the suspension, where it forms rafts in situ in the stomach, resulting in randomness, while D...) -CES is administered orally only after the rafts have stabilized.

[0040] Storage stability is compared to application stability, D - The presence of bacteria in CES slightly reduces its effectiveness compared to commercial Gaviscon®, which contains only inorganic materials and has a longer shelf life. This comparison highlights the differences between the two. Figure 6 In this context, 'a' represents the normalized result, such as acid neutralization capacity, D. -CES has better pH regulation capabilities, while Gaviscon® only uses internal calcium carbonate for temporary regulation, making it significantly inferior to D in this aspect. -CES, so Gaviscon® is 0, while D -CES is 1.

[0041] like Figure 6 As shown in a, DH + -CES exhibits superior performance in terms of acid neutralization range, floating rate, floating stability, and flocculation rate. Gaviscon® struggles to form rafts in HCl solutions at pH 2.0, and the raft formation process in the stomach requires a certain amount of time and is somewhat random, making it impossible to guarantee a 100% flocculation rate. Figure 6 (a~d) in the example. In contrast, D -CES does not depend on interaction with gastric contents, thus remaining effective over a wider pH range. Furthermore, Gaviscon®'s acid-neutralizing ability is entirely dependent on the response after oral administration; its pH-regulating function is lost once sodium bicarbonate is rapidly depleted. In contrast, D... - The CES system can maintain local pH regulation even under continuous acidic environmental challenges. Figure 6 (e, f in the text). Although after the incorporation of engineered bacteria, D -CES has slightly lower storage stability than Gaviscon®, but it can still be stored for more than 30 days at 4°C. Figure 6 g in (the middle part).

[0042] Experimental Example 2

[0043] Acute gastroesophageal reflux disease (GERD) was modeled in rats using forestomach ligation and complete pyloric ligation (post-modeling, macroscopic observation of congestion and erosion in the lower esophagus, and epithelial damage and inflammatory cell infiltration observed on HE staining confirmed successful model establishment). The rats were then randomly divided into three groups (n=5 per group): sham surgery group (laparotomy only, no ligation), control group (modeling, no treatment), and treatment group (modeling, DH). + -CES processing). Among them, DH + -CES treatment of pH-responsive bioactive bacterial rafts prepared in Example 1, administered orally at 150 mg; oral DH + - The CES system showed a significant reduction in lesion severity, with a lesion inhibition rate of 93.95%. Figure 7 (ab in the text). Gastric reflux can trigger a cytokine-mediated inflammatory response, increasing the level of pro-inflammatory cytokines in the esophagus. Serum analysis showed that DH... + -CES treatment significantly downregulated the expression of interleukin-1β (IL-1β). Figure 7 (c in the text)

[0044] Immunohistochemical analysis further confirmed this result, showing that DH + - The secretion of tumor necrosis factor-α (TNF-α) was significantly reduced in the CES group. Figure 7 The presence of hematoxylin and eosin (H&E) staining in the control group indicated a significantly reduced inflammatory response compared to the control group. Histological evaluation, using hematoxylin and eosin (H&E) staining, revealed that the control group exhibited discontinuous esophageal epithelial layers with defects and sloughed areas, enlarged intercellular spaces, tissue edema, and significant inflammatory cell infiltration. In contrast, the DH group showed significantly reduced inflammatory response. + -CES treatment preserves the integrity of the esophageal structure, with cells arranged in an orderly manner and no obvious signs of inflammation. Figure 7 f in the middle. These research results combined indicate that DH + -CES can effectively prevent the reflux of gastric contents, relieve mucosal inflammation, and maintain the integrity of the esophageal structure.

[0045] As can be seen from the above embodiments, the pH-responsive active bacterial raft provided by the present invention is convenient and simple to manufacture, can be mass-produced, and has an adaptive triggering feedback mechanism in a dynamic environment. Compared with the prior art, this system acts as a physical-chemical barrier to regulate the local microenvironment at the gastroesophageal junction. In the treatment of gastroesophageal reflux disease, the esophageal injury inhibition rate can reach 93.95%, showing excellent clinical translation prospects.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a pH-responsive active bacterial raft, characterized in that, Includes the following steps: 1) Mix calcium carbonate, sodium bicarbonate and sodium alginate solution, stir for the first time to obtain a mixed solution; 2) Add Lactococcus lactis dropwise to the mixed solution obtained in step 1), and stir again to obtain a bacterial-sodium alginate mixed system; 3) The bacterial-sodium alginate mixture was injected into the cross-linking system via electrospinning to obtain pH-responsive active bacterial rafts; The crosslinking system includes a calcium chloride solution and a hydrochloric acid solution.

2. The preparation method according to claim 1, characterized in that, The concentration of the sodium alginate solution in step 1) is 1~3% (w / v).

3. The preparation method according to claim 1 or 2, characterized in that, Step 1) The molar ratio of calcium carbonate, sodium bicarbonate and sodium alginate in the mixed solution is 1:(2~3):

1.

4. The preparation method according to claim 1, characterized in that, Step 2) The OD of the lactococcus lactis diluted 100 times 600 The value is 0.9 to 1.

1.

5. The preparation method according to claim 1 or 4, characterized in that, The volume ratio of the lactococcus lactis to the mixed solution is 1:(8~12).

6. The preparation method according to claim 1, characterized in that, Step 3) The injection is performed using a 25G needle, the injection rate is set to 20~30mL / h, and the injection voltage is 0kV.

7. The preparation method according to claim 1 or 6, characterized in that, The concentration of the calcium chloride solution is 10~20 g / L, and the pH of the hydrochloric acid solution is 0.9~1.1; the volume ratio of the calcium chloride solution to the hydrochloric acid solution is 1:(3~5).

8. The pH-responsive active bacterial raft prepared by the preparation method according to any one of claims 1 to 7.

9. The pH-responsive active bacterial raft according to claim 8, characterized in that, The pH-responsive active bacterial rafts have a significant and acid-stable bubble structure.

10. The use of the pH-responsive active bacterial rafts according to claim 8 or 9 in the preparation of a medicament for treating gastroesophageal reflux disease.

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