A method for cultivating bovine gallstones in vivo based on temperature-sensitive gel

Abstract

The application discloses a method for cultivating bovine bezoar in vivo based on temperature-sensitive gel, which comprises the following steps: implanting a bovine bezoar bed into a bovine gallbladder; then, mixing prepared temperature-sensitive polyacrylamide hydrogel with cultured bovine bezoar conversion bacteria, and injecting the mixture into the bovine bezoar bed; or, first, injecting the prepared temperature-sensitive polyacrylamide hydrogel into the bovine bezoar bed, and finally, injecting the cultured bovine bezoar conversion bacteria into the bovine bezoar bed. The gel solution is injected into the bovine bezoar bed in the form of injection, so that the generated wound is small, the health of the bovine is not adversely affected, and the operation is convenient and fast. The gel is injected into the bovine bezoar bed, the bovine bezoar bed protects the gel, so that the gel is not rapidly degraded, the viscosity of the gel is maintained for a long time, the stable and continuous enrichment of bovine bezoar small particles is realized, the initial core of the bovine bezoar is formed, and the efficiency and quality of the in-vivo cultivation of the bovine bezoar are improved, so that the method has good practicability.

Description

Technical Field

[0001] This invention belongs to the technical field of in vivo culture of bezoar, specifically relating to a method for in vivo culture of bezoar based on thermosensitive gel. Background Technology

[0002] Bezoar is a gallstone found in the liver of bovine animals (Bovidae), belonging to the class Mammalia, phylum Chordata. When it forms in the gallbladder, it's called "gallbladder yolk" or "egg yolk"; when it forms in the bile ducts, it's called "duct yolk"; and when it forms in the hepatic ducts, it's called "liver yolk." Traditional artificial bezoar production suffers from poor formation and low quality, primarily due to several factors: firstly, the lack of additional E. coli infection prevents bacterial or parasitic formation of the gallbladder or bile ducts, hindering the formation of consistently effective bezoar particles; secondly, the absence of a suitable structure to enrich these particles during formation results in extremely slow core formation, leading to either very little or no bezoar in the final product. Therefore, a bezoar bed is necessary. The bezoar bed (core) is the substrate for bezoar formation and is crucial for bezoar cultivation.

[0003] Existing patent CN202210870068.X discloses a method for in vivo cultivation of three-dimensional composite bezoar, including the following steps: S1: implantation into a bezoar bed; S2: implantation of bezoar fungi; S3: feeding; S4: feeding with additives. This method of in vivo cultivation of three-dimensional composite bezoar has the characteristic of better adhesion and is more likely to aggregate free bile salt particles in bile. Existing patent CN202010124954.9 discloses a detachable bezoar bed and a bezoar cultivation method. The detachable bezoar bed includes a mesh-like skeleton and an adsorption film. The adsorption film is disposed on the inner surface of the skeleton. The skeleton includes an upper cover, a bed body, and a lower cover from top to bottom. The upper cover, bed body, and lower cover are movably connected to each other. Matching pores are provided in the upper cover, bed body, and lower cover. A buffer layer is provided on the side of the skeleton. However, the bezoar beds disclosed above have the following defects:

[0004] ① The skeletal structure of the bezoar bed is fixed, and its overall shape cannot be changed. The large incision required for implantation into the cow hinders its recovery. In gallbladder cannulation for bezoar bed implantation, the incision is typically 10cm. The three-dimensional composite bezoar in vivo cultivation method requires an 8cm incision via open surgery, with an additional 1.5cm opening in the gallbladder. The open surgery used in detachable bezoar beds and bezoar cultivation methods requires a 4-6cm incision. All these methods require complex suturing and at least a week of meticulous post-operative care before normal feeding can resume. The entire process is complex, requires specialized personnel and equipment, and may adversely affect the cow's health and fattening.

[0005] ②The overall stability of the bezoar bed with the main body combined connection structure is poor. When the bile inside the bezoar bed hardens and clumps, the resulting expansion may cause the connectors to loosen and fail, which may further cause the bezoar bed components to detach and affect the health of the implanted cattle.

[0006] ③ The multi-layered structure in the bezoar bed can easily obstruct bile flow, making it difficult for bile to pass through the interior of the bed. Furthermore, the bezoar is collected in a unidirectional manner, which leads to a large loss of small bezoar particles and poor quality of cultivated bezoar.

[0007] Existing patent CN118020715 A discloses a method for in vivo cultivation of bezoar, comprising: puncturing the bovine gallbladder with a syringe to extract bile; step two, after extracting bile, first injecting a calcium salt solution into the gallbladder, and then immediately injecting a polysaccharide solution into the gallbladder, causing the two solutions to undergo a cross-linking reaction within the gallbladder to form a network-like gel-like bezoar bed. This invention uses an injection method to produce an organic bezoar bed within the bovine gallbladder, exhibiting excellent adsorption within the gallbladder, which is beneficial for the adsorption of bezoar crystals, shortening the bezoar production cycle, and effectively solving various problems associated with previous surgical implantation of bezoar beds, such as stimulation of the cattle, poor bezoar adsorption, and surgical infections causing various diseases. However, because the fluid within the bovine gallbladder is dynamic, this method cannot guarantee the stable formation of a network-like gel-like bezoar bed within the bovine gallbladder. Furthermore, the calcium salt and polysaccharide solutions form a weak gel, and the degree of cross-linking and network structure of the resulting network-like gel-like bezoar bed are uncontrollable, failing to ensure non-degradation during the initial formation of the bezoar core. This method also cannot guarantee the formation of regularly shaped bezoar beds, making it difficult to achieve the enrichment effect of ordinary bezoar beds. Summary of the Invention

[0008] The purpose of this invention is to provide a method for in vivo culture of bovine gallstones based on a thermosensitive gel, aiming to solve the aforementioned problems. This invention is applicable to the enrichment of stones or crystals in animals such as cattle and sheep.

[0009] This invention is mainly achieved through the following technical solutions:

[0010] A method for in vivo culture of bovine gallstones based on thermosensitive gel includes the following steps:

[0011] Step S1: Prepare a room-temperature injectable thermosensitive polyacrylamide hydrogel;

[0012] Step S2: Insert the bezoar bed into the bovine gallbladder;

[0013] Step S3: Mix the thermosensitive polyacrylamide hydrogel prepared in step S1 with bezoar transforming bacteria and inject it into the bezoar bed;

[0014] Alternatively, the thermosensitive polyacrylamide hydrogel prepared in step S1 can be injected into the bezoar bed first, and then the bezoar transforming bacteria can be injected into the bezoar bed.

[0015] Step S4: Proceed with normal feeding.

[0016] To better realize the present invention, further, in step S1, N-isopropylacrylamide, acrylamide and N,N′-methylenebisacrylamide are polymerized to obtain a room-temperature injectable thermosensitive polyacrylamide hydrogel; the molar ratio of N-isopropylacrylamide and acrylamide is 10-3:1; the content of N,N′-methylenebisacrylamide is 1%-2% of the total molar number of monomers.

[0017] To better implement the present invention, step S1 further includes the following steps:

[0018] Step S11: Add N-isopropylacrylamide, acrylamide, N,N′-methylenebisacrylamide and surfactant to the reactor and stir to dissolve;

[0019] Step S12: Heat to 40-80℃ in an inert gas environment, then add an initiator to the reactor to react and obtain a gel dispersion aqueous solution.

[0020] To better realize the present invention, further, in a 50 ml solution, the amounts of N-isopropylacrylamide, acrylamide, and N,N′-methylenebisacrylamide are 42 mmol, 7.4 mmol, and 0.53 mmol, respectively; the initiator is potassium persulfate, the surfactant is sodium dodecyl sulfate, and the amounts of sodium dodecyl sulfate and potassium persulfate are 0.08 g and 0.22 g, respectively.

[0021] To better realize the present invention, step S1 further includes step S13: sodium alginate and compatible monomers and / or compatibilizers are added to the gel dispersion aqueous solution prepared in step S12 and mixed and stirred evenly; then, mucin and nutrients of bezoar transforming bacteria are added and mixed and stirred evenly to obtain an injectable gel aqueous solution; in step S3, the injectable gel aqueous solution prepared in step S13 is first introduced into the bezoar bed, and then the bezoar transforming bacteria and calcium salt solution are injected into the bezoar bed.

[0022] To better realize the present invention, step S1 further includes step S13: sodium alginate and compatible monomers and / or compatibilizers are added to the gel dispersion aqueous solution prepared in step S12 and mixed and stirred evenly; then, bezoar transforming bacteria and mucin and nutrients of bezoar transforming bacteria are added and mixed and stirred evenly to obtain an injectable gel aqueous solution; in step S3, the injectable gel aqueous solution prepared in step S13 is first introduced into the bezoar bed, and then the calcium salt solution is injected into the bezoar bed.

[0023] To better realize the present invention, further, in step S13, the molar ratio of the compatible monomer, compatibilizer, sodium alginate and calcium salt is 0.01-0.1:0.015-0.2:1-20:0.5-5; the compatible monomer is any one or more of dopamine, tannic acid, anthocyanin, catechin and lignin, and the compatibilizer is any one or more of polyvinyl alcohol, polyethylene glycol, starch and cellulose.

[0024] To better realize the present invention, the compatible monomer is tannic acid, the compatibility agent is polyvinyl alcohol, and the polyvinyl alcohol is of type 1788, and the calcium salt is calcium lactate; in a 50 ml solution, the amounts of tannic acid, polyvinyl alcohol, sodium alginate and calcium lactate are 0.044 mmol, 0.067 mmol, 8.7 mmol and 2.3 mmol, respectively.

[0025] To better realize the present invention, in step S2, the bezoar bed is a compressible structure, and the compressed bezoar bed is implanted into the bovine gallbladder, where the bezoar bed is in an expanded state.

[0026] To better realize this invention, a minimally invasive method is employed to implant a compressed bezoar bed into the bovine gallbladder. This invention allows for the minimally invasive implantation of a compressed bezoar bed into the bovine gallbladder. The minimally invasive implantation method is existing technology, and can be achieved using an endoscope or a puncture needle, etc., and will not be elaborated further. This invention utilizes a compressible bezoar bed and injectable thermosensitive polyacrylamide hydrogel, achieving the introduction of bezoar into the bovine gallbladder through a tiny incision. This solves the problem of excessively large incisions in existing bezoar in vivo culture methods, improving the yield and quality of cultured bezoar, and demonstrating good practicality.

[0027] To better realize the present invention, the bezoar bed further includes a bezoar bed body, a cover body, and an elastic frame. The two ends of the bezoar bed body are respectively provided with wave-shaped elastic frames along the circumference. The bezoar bed body has a hollow structure, and the two ends of the bezoar bed body are respectively provided with flexible covers. The middle of the cover body is provided with a flow hole.

[0028] To better realize the present invention, the main body of the yellow bed is made of a mesh, the cover is made of PET mesh, and the elastic skeleton is made of shape memory alloy.

[0029] like Figure 1 As shown, the nucleation mechanism of bezoar is as follows:

[0030] A cow's liver secretes cholesterol and glucuronide. Cholesterol is further synthesized into bile acids, which can be broken down into free bile acids.

[0031] Glucuronate is converted into glucuronide bilirubin, and β-glucuronidase secreted by bovine transforming bacteria (Escherichia coli) promotes the breakdown of glucuronide bilirubin into free bilirubin.

[0032] Free bilirubin and free bile acids react with calcium ions to form bilirubin calcium. Bilirubin calcium further reacts with mucin to form bilirubin calcium protein polymers, namely bezoar granules.

[0033] Furthermore, *Escherichia coli*, a bacterium that transforms bezoar, can secrete β-glucuronidase, which hydrolyzes soluble conjugated bilirubin (CB) into unconjugated bilirubin (UCB). The unconjugated bilirubin then combines with metal ions such as calcium and magnesium to form calcium bilirubin salt precipitates, which further polymerize with protein-polysaccharide complexes to form small bezoar particles. During its activity, *E. coli* produces acid, lowering the pH of bile, and this acidic environment causes the precipitation of proteins in the bile. Simultaneously, it utilizes large molecules such as proteins and carbohydrates in the bile, resulting in a decrease in bile viscosity. Newly formed small bezoar particles are easily suspended in high-viscosity bile, making them less prone to sedimentation and adhesion; however, in low-viscosity bile, they easily precipitate, adhere, and aggregate, forming large bezoar clumps. Once these small bezoar particles rapidly form large clumps, they are less likely to be lost due to bile flow.

[0034] Secondly, *Escherichia coli*, a bacterium that transforms bezoar, can degrade bile acids in bile, leading to a decrease in bile acid content. Bile acids in bile have a solubilizing effect on free bilirubin (UCB) and inhibit the binding of UCB with metal ions such as calcium and magnesium; bile acids themselves can also bind with calcium ions, inhibiting the formation of bilirubin calcium salts. The reduced content of bile and bile acids in bile facilitates the binding of UCB with metal ions such as calcium and magnesium, thereby promoting the formation of bezoar.

[0035] Bezoar transformant bacteria (Escherichia coli) can produce mucus, which on the one hand increases the viscosity of bile, which is conducive to the aggregation of bezoar particles; on the other hand, it may participate in the formation of bezoar.

[0036] The formation of bezoar mainly involves three stages: a chemical stage, including changes in bile composition, cholesterol crystallization, and the precipitation of bile pigment particles; a physical stage, where the precipitated components solidify into a cohesive form through a core and framework; and a growth stage, where, under conditions where the nascent gallstone is not washed away by bile flow, the precipitated components in bile gradually accumulate and precipitate on the stone. The precipitation of bile pigment particles is a crucial step in this process. Figure 1 As shown, the aggregation mechanism of bezoar particles in the bezoar bed inside the gallbladder is as follows:

[0037] Bile carries these substances into the luteal bed, where small bezoar particles form inside the bed and are directly deposited. However, because bile is constantly flowing, this process is relatively rare. More often, bile carries the bezoar particles into the bed, where they are adsorbed by the gel, and the bile then flows out through the pores of the bed.

[0038] In the early stages of bezoar core formation, the gelation effect is significant. Firstly, the gel inside the bezoar bed carries mucin and nutrients. In the initial stages, bezoar-transforming bacteria (E. coli) and nutrients are released in a burst. E. coli enters the bile ducts, gallbladder, and other tissues, infecting and multiplying the bovine tissues, providing the basic conditions for the formation of small bezoar particles. Secondly, the gel inside the bezoar bed also enriches the small bezoar particles. Without the gel to adsorb these particles, bile would carry them out through the flow pores of the bezoar bed. Even if some adhere to the wall, they are scattered and cannot form a core. Without the protection of the bezoar bed, the gel degrades quickly, and its viscosity is short-lived, making it impossible to stably collect small bezoar particles for 2-3 months.

[0039] The formation time of the bezoar core is approximately 2-4 months. The degradation time of the gel prepared in this invention meets this requirement, and the gel formed with the bezoar core forms a core-shell structure. Generally, the degradation rate of gels in vivo is a gradually accelerating process because the initial degradation structure is well-crosslinked, resulting in a slower degradation rate. As the structure and crosslinking network are disrupted, the degradation rate gradually increases. However, due to this core-shell structure, the gel is encapsulated by bezoar, which slows down its degradation time. As the gel structure is disrupted, the specific surface area of ​​the gel also increases, providing more vacancies for the bezoar particles. In the later stages of bezoar formation, a dynamic equilibrium is reached between the gel degradation rate and the growth of the bezoar.

[0040] This invention prepares a cross-linked polyacrylamide hydrogel, i.e., a gel dispersion aqueous solution, by free radical polymerization of N-isopropylacrylamide (NIPAAm), acrylamide (AAm), and N,N′-methylenebisacrylamide (MBAAm) monomers. Potassium persulfate (KPS) acts as an initiator for the polymerization reaction, accelerating the generation of free radicals and the polymerization of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) monomers; N,N′-methylenebisacrylamide (MBAAm) acts as a cross-linking agent, covalently linking the molecular chains formed by the polymerization of acrylamide (AAm) to form a three-dimensional polymer network structure in the polyacrylamide hydrogel.

[0041] During polymerization, the carbon-carbon double bond of N-isopropylacrylamide and acrylamide is opened by the initiator (ammonium persulfate), resulting in a carbocation. The carbocation attacks the other double bond, similar to the chain growth part of the photoreaction of alkane. However, due to the addition of N,N′-methylenebisacrylamide, it can also be attacked by the carbocation and participate in its polymerization reaction. Therefore, it appears between the single chains of polyacrylamide, forming a three-dimensional spatial network structure, which prolongs the degradation time of the gel preparation and meets the cycle of the initial core formation of bezoar.

[0042] The NIPAAm contains hydrophobic isopropyl groups and hydrophilic amide groups, thus exhibiting thermosensitive properties. Increasing the content of hydrophilic groups can raise the LCST temperature; therefore, introducing acrylamide (AAm), which also contains amide bonds, can increase the proportion of hydrophilic groups. Thus, this invention also increases the LCST temperature of the gel, allowing the entire hydrogel to undergo a phase transition at conditions closer to bovine body temperature (37-40°C). This facilitates injection into the bovine gallbladder while rapidly transforming into a viscous state that readily enriches bezoar particles, thereby improving the efficiency of bezoar cultivation.

[0043] Based on the polyacrylamide hydrogel prepared above, this invention utilizes the characteristic that calcium ions can instantly form a cross-linked gel network by forming coordination bonds with the carboxyl groups on the sodium alginate (SA) molecular chain. Ultimately, this results in a thermosensitive gel with an interpenetrating network structure of two interconnected networks. The addition of tannic acid (TA) and polyvinyl alcohol (PVA) enhances the compatibility of the two gel networks. PVA is rich in hydroxyl groups, which can form hydrogen bonds with tannic acid (TA) and polyacrylamide hydrogel, enhancing the connectivity and cross-linking strength of the thermosensitive gel with the interpenetrating network structure. Furthermore, tannic acid (TA) is rich in polyphenol groups, which not only oxidize to produce quinone groups, forming covalent bonds with the amino and thiol groups of proteins, but also form hydrogen bonds with hydroxyl and carbonyl groups. Therefore, it can effectively adhere to mucoproteins, promoting the enrichment and formation of bezoar particles on the gel surface, and possessing excellent adsorption function for small bezoar particles.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) This invention uses minimally invasive instruments to introduce a compressible bezoar bed into the bovine gallbladder, and injects a gel solution into the bezoar bed, resulting in an incision of only 1 cm. It does not require complex surgical instruments or open abdominal suturing, and does not require complicated postoperative care. The cattle can be fed normally one day later, without any adverse effects on the health or fattening of the cattle.

[0046] (2) The phase transition temperature of the thermosensitive polyacrylamide hydrogel prepared in this invention is 37-40℃, which meets the requirements for injection at room temperature. Furthermore, it exhibits a viscous state within the bovine gallbladder, increasing the adhesion area and stickiness of the bezoar particles. In this invention, the gel is injected into a bezoar bed, where the thermosensitive gel accumulates to form the initial bezoar core. The bezoar bed protects the gel, preventing rapid degradation. Simultaneously, the protection of the bezoar bed prolongs the gel's viscosity maintenance time, achieving stable and continuous accumulation of bezoar particles to form the initial bezoar core. This is beneficial for the efficiency and quality of bezoar in vivo cultivation and has good practicality.

[0047] (3) In this invention, bezoar-transforming bacteria and their nutrients are directly mixed into the gel. In the bovine gallbladder, the gel simultaneously enriches bezoar particles and releases bezoar-transforming bacteria. The continuously released bezoar-transforming bacteria can cause bacterial infection in the gallbladder or bile duct, forming continuously effective bezoar particles. This invention, by mixing bezoar-transforming bacteria with the gel and then implanting it into the bezoar bed, can ensure the bacterial concentration, ensure that the bezoar bed complex is suspended in the bile, and increase the collection efficiency of bezoar particles, thus having good practicality.

[0048] (4) In order to rapidly infect the gallbladder or bile duct of cattle, the present invention first injects a thermosensitive polyacrylamide hydrogel into a bezoar bed, and then immediately injects bezoar transforming bacteria into the bezoar bed. Part of the bezoar transforming bacteria will rapidly infect the cattle gallbladder, and part will be loaded in the gel. The high concentration of released bezoar transforming bacteria will rapidly infect the cattle gallbladder or bile duct, initially promoting the formation of a large number of small bezoar particles. The bezoar transforming bacteria loaded on the gel surface will continue to be released, ensuring the stable formation of the bezoar core in the later stage, which has good practicality.

[0049] (5) Bezoar transforming bacteria are an important part of the bezoar formation process, and they can create conditions for bezoar formation. On the one hand, the gel can load bezoar transforming bacteria to ensure that they are not washed away by the flowing bile. At the same time, it can also load growth factors and nutritional proteins needed to promote the growth of bezoar transforming bacteria. On the other hand, the gel itself is a nutrient substance and can serve as a nutrient source for the growth of bezoar transforming bacteria. Furthermore, the gel can adsorb bezoar particles free in the bile, ensuring the formation of the bezoar core. The bezoar bed can ensure that the gel complex has a regular shape when it solidifies in situ in the bile.

[0050] (6) The phase transition temperature (LCST) of the existing traditional uncrosslinked N-isopropylacrylamide (NIPAAm) polymer is 30-32℃. Adding acrylamide (AAm) can improve the thermosensitive properties of polyacrylamide hydrogel, making its phase transition temperature closer to the bovine body temperature. This allows the entire hydrogel to undergo a phase transition at a temperature closer to the bovine body temperature (37-40℃), making it easier to inject the polyacrylamide hydrogel into the bovine gallbladder. It can also quickly transform the polyacrylamide hydrogel into a viscous state that is easy to enrich bezoar particles, thus improving the efficiency of bezoar cultivation.

[0051] (7) This invention first prepares an injectable gel-water solution that can undergo phase transition in bovine tissue, and then prepares a thermosensitive gel with an interpenetrating network structure based on the injected calcium salt solution. The degradation time of the gel prepared by this invention meets the time requirements for the initial core formation of bezoar and also meets the requirements for convenient operation, making it easy to enrich bezoar particles. The thermosensitive gel with an interpenetrating network structure prepared by this invention not only prolongs the gel degradation time but also increases the area that can be attached in the gel and improves the adhesion strength of the gel to bezoar particles, effectively improving the quality of bezoar cultivation and having good practicality.

[0052] (8) The combination of bezoar bed and gel complex can make the density of the entire bezoar bed complex similar to or slightly higher than that of bile, allowing it to be immersed in bile. The bezoar bed itself is small in volume and light in weight. Before the bezoar core is formed, the entire complex can be well suspended in the gallbladder because the center is filled with a biodegradable gel complex and the degree of gel degradation is low. After the bezoar core is formed, the degree of gel degradation increases, the weight of the bezoar core increases, and the mass of the entire bezoar bed does not change. Therefore, the formation of the bezoar core and the degradation of the gel can be in a balanced state throughout the entire cultivation process, ensuring that the entire bezoar bed is suspended in bile from the implantation of the bezoar bed, the formation of the bezoar core, the complete degradation of the gel, until the final harvesting of the bezoar. Only in the final stage, when a large amount of bezoar accumulates to cover the entire bezoar bed, it is semi-suspended or sinks to the bottom of the gallbladder. Since the bezoar bed is covered by bezoar at this time, it will hardly come into direct contact with the inner wall of the gallbladder. Only the formed bezoar can contact the gallbladder wall. Its surface is smooth and has little irritation to the gallbladder epithelial cells and tissues, ensuring that the health of the implanted cattle is not affected. Attached Figure Description

[0053] Figure 1 A schematic diagram illustrating the formation mechanism of bezoar;

[0054] Figure 2 This is a flowchart of Example 3;

[0055] Figure 3 This is a schematic diagram of the structure of the bezoar bed in Example 5;

[0056] Figure 4 A schematic diagram of the connection structure between the cover and the main body of the yellow bed;

[0057] Figure 5 This is a schematic diagram of the bezoar enriched in Example 4.

[0058] Among them: 1-Yellow bed body, 2-Cover body, 3-Elastic skeleton, 4-Flow hole. Detailed Implementation

[0059] Example 1:

[0060] A method for in vivo culture of bovine gallstones based on thermosensitive gel includes the following steps:

[0061] Step 1: Isolate bile-resistant bezoar-transforming bacteria from fresh bezoar-producing bile and culture the bezoar-transforming bacteria;

[0062] Step 2: Prepare a room-temperature injectable thermosensitive polyacrylamide hydrogel;

[0063] Step 21: As shown in Table 1, first add 50 mL of water to a three-necked flask equipped with a reflux condenser and a gas delivery device, then add 4.76 g of N-isopropylacrylamide (NIPAAm), 0.53 g of acrylamide (AAm), 0.081 g of N,N′-methylenebisacrylamide (MBAAm) monomer and 0.08 g of sodium dodecyl sulfate (SDS), and dissolve them under magnetic stirring;

[0064] Step 22: After purging nitrogen gas into a three-necked flask for 30 minutes, heat the flask to 70°C. Then, add 0.22 g of potassium persulfate (KPS) and react in an N2 atmosphere for a period of time to obtain a gel dispersion aqueous solution.

[0065] Step 3: The compressed bezoar bed is implanted into the bovine gallbladder in a minimally invasive manner, and the bezoar bed inside the bovine gallbladder is in an expanded state;

[0066] Then, the thermosensitive polyacrylamide hydrogel prepared in step 2 is mixed with the bezoar transforming bacteria cultured in step 1, and then injected into the bezoar bed.

[0067] Step 4: Closely observe the diet, defecation and urination of the transplanted cattle within 1 day after surgery to help them recover to a normal diet and growth state as soon as possible and to carry out normal feeding.

[0068] Bezoar testing method: After feeding the cattle with cultured bezoar for one year, the cultured bezoar in the gallbladder was obtained through surgical laparotomy. The obtained bezoar was dried and weighed, and the yield of cultured bezoar for each cow was recorded. Then, the content of bilirubin and other major components of the cultured bezoar was determined according to the Chinese Pharmacopoeia (2020 edition).

[0069] The results showed that the experimental cattle produced an average of 18.35±0.31g / head / year of bezoar. The cultivated bezoar was light in weight and had a bilirubin content of more than 35%, which met the standards for high-quality bezoar.

[0070] Example 2:

[0071] A method for in vivo culture of bovine gallstones based on thermosensitive gel includes the following steps:

[0072] Step 1: Isolate bile-resistant bezoar-transforming bacteria from fresh bezoar-producing bile and culture the bezoar-transforming bacteria;

[0073] Step 2: Prepare a room-temperature injectable thermosensitive polyacrylamide hydrogel;

[0074] Step 21: As shown in Table 1, first add 50 mL of water to a three-necked flask equipped with a reflux condenser and a gas delivery device, then add 4.76 g of N-isopropylacrylamide (NIPAAm), 0.53 g of acrylamide (AAm), 0.081 g of N,N′-methylenebisacrylamide (MBAAm) monomer and 0.08 g of sodium dodecyl sulfate (SDS), and dissolve them under magnetic stirring.

[0075] Step 22: After purging nitrogen gas into a three-necked flask for 30 minutes, heat the flask to 70°C. Then, add 0.22 g of potassium persulfate (KPS) and react in an N2 atmosphere for a period of time to obtain a gel dispersion aqueous solution.

[0076] Step 3: The compressed bezoar bed is implanted into the bovine gallbladder in a minimally invasive manner, and the bezoar bed inside the bovine gallbladder is in an expanded state;

[0077] Then, the thermosensitive polyacrylamide hydrogel prepared in step 2 is injected into the bezoar bed, and then the bezoar transforming bacteria cultured in step 1 is injected into the bezoar bed.

[0078] Step 4: Closely observe the diet, defecation and urination of the transplanted cattle within 1 day after surgery to help them recover to a normal diet and growth state as soon as possible and to carry out normal feeding.

[0079] The bezoar testing method was the same as in Example 1. The results showed that the average bezoar production of the experimental cattle was 21.46±0.12g / head / year. The cultivated bezoar was light in weight and had a bilirubin content of more than 35%, which met the standards for high-quality bezoar.

[0080] Example 3:

[0081] A method for in vivo culture of bovine gallstones based on thermosensitive gel, such as Figure 2 As shown, it includes the following steps:

[0082] Step 1: Isolate bile-resistant bezoar-transforming bacteria from fresh bezoar-producing bile and culture the bezoar-transforming bacteria;

[0083] Step 2: Prepare a room-temperature injectable thermosensitive polyacrylamide hydrogel;

[0084] Step 21: As shown in Table 1, first add 50 mL of water to a three-necked flask equipped with a reflux condenser and a gas delivery device, then add 4.76 g of N-isopropylacrylamide (NIPAAm), 0.53 g of acrylamide (AAm), 0.081 g of N,N′-methylenebisacrylamide (MBAAm) monomer and 0.08 g of sodium dodecyl sulfate (SDS), and dissolve them under magnetic stirring.

[0085] Step 22: After purging nitrogen gas into a three-necked flask for 30 minutes, heat the flask to 70°C. Then, add 0.22 g of potassium persulfate (KPS) and react in an N2 atmosphere for a period of time to obtain a gel dispersion aqueous solution.

[0086] Step 23: Add 0.075g tannic acid, 5g polyvinyl alcohol and 2g sodium alginate to the gel dispersion aqueous solution prepared in step 22. The polyvinyl alcohol is type 1788. Mix and stir evenly. Then, add mucin and nutrients of bezoar transformant bacteria. Mix and stir evenly to obtain an injectable gel aqueous solution.

[0087] Step 3: The compressed bezoar bed is implanted into the bovine gallbladder in a minimally invasive manner, and the bezoar bed inside the bovine gallbladder is in an expanded state;

[0088] Then, the injectable gel aqueous solution prepared in step 23 is first introduced into the bezoar bed, and then the bezoar transforming bacteria and calcium salt solution are injected into the bezoar bed. The calcium salt is calcium lactate, and the calcium lactate content is 0.4g.

[0089] Step 4: Closely observe the diet, defecation and urination of the transplanted cattle within 1 day after surgery to help them recover to a normal diet and growth state as soon as possible and to carry out normal feeding.

[0090] The bezoar testing method was the same as in Example 1. The results showed that the average bezoar production of the experimental cattle was 31.78±0.11g / head / year. The cultivated bezoar was light in weight and had a bilirubin content of more than 35%, which met the standards for high-quality bezoar.

[0091] Example 4:

[0092] A method for in vivo culture of bovine gallstones based on thermosensitive gel includes the following steps:

[0093] Step 1: Isolate bile-resistant bezoar-transforming bacteria from fresh bezoar-producing bile and culture the bezoar-transforming bacteria;

[0094] Step 2: Prepare a room-temperature injectable thermosensitive polyacrylamide hydrogel;

[0095] Step 21: As shown in Table 1, first add 50 mL of water to a three-necked flask equipped with a reflux condenser and a gas delivery device, then add 4.76 g of N-isopropylacrylamide (NIPAAm), 0.53 g of acrylamide (AAm), 0.081 g of N,N′-methylenebisacrylamide (MBAAm) monomer and 0.08 g of sodium dodecyl sulfate (SDS), and dissolve them under magnetic stirring.

[0096] Step 22: After purging nitrogen gas into a three-necked flask for 30 minutes, heat the flask to 70°C. Then, add 0.22 g of potassium persulfate (KPS) and react in an N2 atmosphere for a period of time to obtain a gel dispersion aqueous solution.

[0097] Step 23: Add 0.075g tannic acid, 5g polyvinyl alcohol and 2g sodium alginate to the gel dispersion aqueous solution prepared in step 22. The polyvinyl alcohol is type 1788. Mix and stir evenly. Then, add the bezoar transforming bacteria cultured in step S1 as well as mucin and nutrients of bezoar transforming bacteria. Mix and stir evenly to obtain an injectable gel aqueous solution.

[0098] Step 3: The compressed bezoar bed is implanted into the bovine gallbladder in a minimally invasive manner, and the bezoar bed inside the bovine gallbladder is in an expanded state;

[0099] Then, the injectable gel aqueous solution prepared in step 23 is first introduced into the bezoar bed, and then the calcium salt solution is injected into the bezoar bed. The calcium salt is calcium lactate, wherein the calcium lactate content is 0.4g.

[0100] Step 4: Closely observe the diet, defecation and urination of the transplanted cattle within 1 day after surgery to help them recover to a normal diet and growth state as soon as possible and to carry out normal feeding.

[0101] The method for testing bezoar was the same as in Example 1. The results showed that the average bezoar production of the experimental cattle was 26.78 ± 0.11 g / head / year. Figure 5 As shown, the cultivated bezoar is light in weight and has a bilirubin content of over 35%, meeting the standards for high-quality bezoar.

[0102] Example 5:

[0103] This embodiment is an optimization based on any one of embodiments 1-4, such as... Figure 3 and Figure 4As shown, the bezoar bed includes a main body 1, a cover 2, and an elastic frame 3. The main body 1 has wavy elastic frames 3 arranged circumferentially at both ends. The main body 1 has a hollow structure, and flexible covers 2 are provided at both ends of the main body 1. A flow hole 4 is provided in the middle of each cover 2. Preferably, the main body 1 is made of mesh, the cover 2 is made of PET mesh, and the elastic frame 3 is made of shape memory nickel-titanium alloy.

[0104] Specifically, this invention adds a layer of flexible mesh to the outside of a corrugated nickel-titanium alloy metal skeleton. The mesh size of the flexible mesh is controlled between 20 and 60 mesh, preferably 40 mesh. Both sides of the mesh are provided with a funnel-shaped cover 2. The inner portion of the cover 2 is recessed into the body of the bezoar bed 1, and the end of the cover 2 reaches 1 / 2 to 1 / 10 of the height of the entire bezoar bed 1, preferably at 1 / 3. The opening size of the flow hole 4 is 0.1-5 cm, preferably 1.5 cm. The volume of the gel complex added inside the bezoar bed is between 1 and 30 g, preferably 10-15 g.

[0105] The bezoar bed designed in this invention increases the interception area of ​​bezoar particles, ensuring bile flow regardless of the orientation of the bezoar bed body 1 within the gallbladder, thus increasing the collection rate of bezoar particles. Secondly, during gallbladder contraction, the inwardly concave structure of the cap 2 forms a chamfer, preventing the accumulated bezoar particles or the formed bezoar core from being washed away by the moving bile. Furthermore, the wave-shaped memory metal skeleton increases the elasticity and stability of the bezoar bed body 1, preventing deformation or even destruction of the bezoar bed during gallbladder contraction.

[0106] This invention utilizes a flexible mesh to rub against the gallbladder wall and collect bezoar particles. The flow holes 4 are used to increase the flow rate of bile within the gallbladder bed body 1. Figure 4 As shown, the cone-shaped cover 2 on both sides serves as a bidirectional collection net, effectively guiding bile into the collection net and trapping bezoar particles in both directions. This encourages more bezoar particles to be collected in the bezoar bed body 1, effectively improving the quality of bezoar cultivation in the bezoar bed and demonstrating good practicality.

[0107] This invention enables simultaneous lateral and longitudinal compression, effectively adjusting the size of the bezoar bed. When implanting the bezoar bed into the cow, the compressed bezoar bed is pressed into the loader. Under the action of the elastic skeleton 3, the bezoar bed will not detach from the loader, improving implantation stability, reducing the size of the incision required for implantation, and lowering the operational difficulty of bezoar bed implantation, thus demonstrating good practicality.

[0108] Comparative Example 1:

[0109] A method for in vivo culture of bezoar includes the following steps:

[0110] 1) Preparation of room-temperature injectable thermosensitive polyacrylamide hydrogel;

[0111] a. As shown in Table 1, first add 50 mL of water to a three-necked flask equipped with a reflux condenser and a gas delivery device, then add 4.76 g of N-isopropylacrylamide (NIPAAm), 0.53 g of acrylamide (AAm), 0.081 g of N,N′-methylenebisacrylamide (MBAAm) monomer and 0.08 g of sodium dodecyl sulfate (SDS), and dissolve them under magnetic stirring.

[0112] b. After purging nitrogen gas into a three-necked flask for 30 minutes, heat to 70°C, then add 0.22 g of potassium persulfate (KPS), and react in a N2 atmosphere for a period of time to obtain a gel dispersion aqueous solution.

[0113] c. Add 0.075g tannic acid, 5g polyvinyl alcohol, and 2g sodium alginate to the prepared gel dispersion aqueous solution, and mix thoroughly. Then, add the bezoar-transforming bacteria, mucin, and nutrients for the bezoar-transforming bacteria, and mix thoroughly.

[0114] The mixture was homogenized to obtain an injectable gel aqueous solution;

[0115] d. Then, the prepared injectable gel solution is introduced into the bovine gallbladder via a minimally invasive implantation.

[0116] 2) Closely observe the diet, defecation and urination of the transplanted cattle within 1 day after surgery to help them recover to normal diet and growth as soon as possible and to carry out normal feeding.

[0117] The bezoar testing method was the same as in Example 1. The results showed that the average bezoar production of the experimental cattle was 11.82±0.61g / head / year. The cultivated bezoar was of poor quality, with a bilirubin content of less than 35%, which did not meet the standard for high-quality bezoar.

[0118] Comparative Example 2:

[0119] A method for in vivo culture of bezoar involves implanting a compressed bezoar bed into the bovine gallbladder via a minimally invasive procedure, with the bezoar bed inside the gallbladder in an expanded state. The diet, defecation, and urination of the transplanted cattle are closely monitored within one day post-surgery to help them quickly return to a normal diet and growth state for normal feeding.

[0120] The bezoar testing method was the same as in Example 1. The results showed that the average bezoar production of the experimental cattle was 3.09±0.08g / head / year. The cultivated bezoar was of poor quality, with a bilirubin content of less than 35%, which did not meet the standard for high-quality bezoar.

[0121] As shown in Table 1, comparing Example 1 with Example 2 and Example 3 with Example 4, it can be found that the yield of bezoar is higher when bezoar transforming bacteria are injected alone into the bezoar bed. This is because a large amount of bezoar transforming bacteria are initially released into the bovine gallbladder. The high concentration of bezoar transforming bacteria will quickly cause bacterial infection in the bovine gallbladder or bile duct, initially promoting the formation of a large number of small bezoar particles, resulting in a larger quality of the final enriched bezoar.

[0122] In Examples 1 and 2, polyacrylamide hydrogels in a common cross-linked state were prepared. In Examples 3 and 4, polyacrylamide hydrogels with an interpenetrating network structure were prepared, exhibiting advantages such as faster gelation, greater adhesion strength, and lower degradation rate. Compared with Example 3, Example 2 resulted in a significantly higher yield of bezoar, increasing the yield by up to 30%, and the quality of the bezoar met the standards for high-quality bezoar.

[0123] Comparing Example 1 with Comparative Examples 1 and 2, it was found that implanting the gel into a bezoar bed for in vivo culture significantly improved the yield and quality of bezoar, increasing the yield by up to 6 times compared to traditional bezoar beds. Traditional bezoar beds had lower yields and poorer bezoar quality. While using polyacrylamide gel as a bezoar bed increased the bezoar yield relatively, the gel's adhesion and degradation were unstable due to direct exposure to the bovine gallbladder, resulting in unfiltered adhered particles and a lower bezoar yield than in Example 1, with poorer bezoar quality.

[0124] Table 1

[0125]

[0126]

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for in vivo culture of bovine gallstones based on thermosensitive gel, characterized in that, Includes the following steps: Step S1: Prepare a room-temperature injectable thermosensitive polyacrylamide hydrogel; Step S2: Insert the bezoar bed into the bovine gallbladder; Step S3: Mix the thermosensitive polyacrylamide hydrogel prepared in step S1 with bezoar transforming bacteria and inject it into the bezoar bed; Alternatively, the thermosensitive polyacrylamide hydrogel prepared in step S1 can be injected into the bezoar bed first, and then the bezoar transforming bacteria can be injected into the bezoar bed. Step S4: Proceed with normal feeding; In step S1, N-isopropylacrylamide, acrylamide, and N,N'-methylenebisacrylamide are polymerized to obtain a room-temperature injectable thermosensitive polyacrylamide hydrogel; the molar ratio of N-isopropylacrylamide to acrylamide is 10-3:1; the content of N,N'-methylenebisacrylamide is 1%-2% of the total molar amount of monomers. In step S2, the bezoar bed is a compressible structure. The compressed bezoar bed is implanted into the bovine gallbladder, and the bezoar bed inside the bovine gallbladder is in an expanded state. The bezoar bed includes a bezoar bed body (1), a cover (2), and an elastic skeleton (3). The two ends of the bezoar bed body (1) are respectively provided with wave-shaped elastic skeletons (3) along the circumference. The bezoar bed body (1) is a hollow structure, and the two ends of the bezoar bed body (1) are respectively provided with flexible covers (2). The middle part of the cover (2) is provided with flow holes (4).

2. The method for in vivo culture of bovine gallstones based on thermosensitive gel according to claim 1, characterized in that, Step S1 includes the following steps: Step S11: Add N-isopropylacrylamide, acrylamide, N,N'-methylenebisacrylamide and surfactant to the reactor and stir to dissolve; Step S12: Heat to 40-80℃ in an inert gas environment, then add an initiator to the reactor to react and obtain a gel dispersion aqueous solution.

3. The method for in vivo culture of bovine gallstones based on thermosensitive gel according to claim 2, characterized in that, Step S1 further includes step S13: sodium alginate and compatible monomers and / or compatibilizers are added to the gel dispersion aqueous solution prepared in step S12 and mixed and stirred evenly; then, mucin and nutrients of bezoar transforming bacteria are added and mixed and stirred evenly to obtain an injectable gel aqueous solution; in step S3, the injectable gel aqueous solution prepared in step S13 is first introduced into the bezoar bed, and then the bezoar transforming bacteria and calcium salt solution are injected into the bezoar bed.

4. The method for in vivo culture of bovine gallstones based on thermosensitive gel according to claim 2, characterized in that, Step S1 further includes step S13: sodium alginate and compatible monomers and / or compatibilizers are added to the gel dispersion aqueous solution prepared in step S12 and mixed and stirred evenly; then, bezoar transforming bacteria and mucin and nutrients of bezoar transforming bacteria are added and mixed and stirred evenly to obtain an injectable gel aqueous solution; in step S3, the injectable gel aqueous solution prepared in step S13 is first introduced into the bezoar bed, and then the calcium salt solution is injected into the bezoar bed.

5. A method for in vivo culture of bovine gallstones based on a thermosensitive gel according to claim 3 or 4, characterized in that, In step S13, the molar ratio of the compatible monomer, compatibilizer, sodium alginate, and calcium salt is 0.01-0.1:0.015-0.2:1-20:0.5-5; the compatible monomer is any one or more of dopamine, tannic acid, anthocyanin, catechin, and lignin; and the compatibilizer is any one or more of polyvinyl alcohol, polyethylene glycol, starch, and cellulose.

6. The method for in vivo culture of bovine gallstones based on thermosensitive gel according to claim 1, characterized in that, The main body (1) of the yellow bed is made of mesh, the cover (2) is made of PET mesh, and the elastic skeleton (3) is made of shape memory alloy.

7. A method for in vivo culture of bovine gallstones based on a thermosensitive gel according to claim 1 or 6, characterized in that, A compressed bezoar bed was implanted into the bovine gallbladder using a minimally invasive procedure.

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