A method for cultivating and collecting bezoar using a gel system

Abstract

The present invention belongs to the technical field of gel for cultivating bezoar, and specifically relates to a method for cultivating and collecting bezoar using a gel system. The present invention uses poly (N-isopropylacrylamide-acrylamide) gel solution as the first responsive gel solution, which has temperature sensitivity and realizes the phase change of gel solution to gel within a specific temperature range. Polyvinyl alcohol-cyclodextrin gel solution and polyvinyl alcohol-bile acid gel solution are used as the second responsive gel solution, and the two are host-guest coordination gels, and the two are in the state of gel solution at the beginning, and are mixed together in the state of gel and have self-healing properties. The two gel solutions are mixed together, and the entanglement and cross-linking in the physical space can make the gel have a large pore structure; and the two gel solutions can make the mixed gel have two response characteristics, which can be effectively applied to the cultivation and collection of bezoar.

Description

Technical Field

[0001] The invention belongs to the technical field of gel for cultivating bezoar, and in particular relates to a method for cultivating and collecting bezoar by utilizing a gel system. Background Art

[0002] Bezoar is a gallstone in the liver of cattle, a mammal of the phylum Chordata and class Bovidae. The one produced in the gallbladder is called "bile yellow" or "egg yolk", the one produced in the bile duct is called "tube yellow", and the one produced in the hepatic duct is called "liver yellow". The traditional artificial bezoar has poor formation and low manufacturing quality during the manufacturing process. The main reasons include: no additional injection of E. coli to cause bacteria or parasites to infect the gallbladder or bile duct of the cattle, and it is impossible to form sustainable and effective bezoar small particles; secondly, when the bezoar small particles are formed, there is no corresponding structure to enrich these small particles, resulting in an extremely slow formation speed of the bezoar core, and the final bezoar product is either very little or no. For this reason, it is necessary to use the bezoar bed. The production of the bezoar bed (core) is the matrix for the formation of bezoar and is the key to the cultivation of bezoar.

[0003] The inventor is committed to the technical innovation of collecting bezoars using gel. The hydrogel is placed inside the gallbladder by surgery, and the thermosensitive properties of the gel are used to gel at the temperature of the cow body. Small particles of bezoar need to be enriched to form the initial core of bezoar. With the initial core of bezoar, bezoar will grow further. The gel can stick to the small particles of bezoar in the early stage to enrich them; after the bezoar is fully formed, it is completely degraded, which does not affect the subsequent treatment of bezoar. For example, Chinese patent application CN2024110573910, a method for cultivating bezoars in vivo based on thermosensitive gel, and Chinese patent application CN2024110574519, a thermosensitive gel for cultivating bezoars and its preparation method and application, are all preliminary studies by the inventor, but the gel has poor viscosity and cannot stick to small particles of bezoars. It is difficult to collect bezoars for use. In addition, the gel degradation time is too fast, and the small particles of bezoars are degraded before they are formed, which also increases the difficulty of collecting bezoars. The initial core formation of bezoar requires at least 2-4 months, and the formation of bezoar requires about 10-18 months. The existing polysaccharide gel has no viscosity and no temperature sensitivity. The existing thermosensitive gel has a general gelation speed when bezoar is collected, and is easily dispersed by the flowing bile in the early stage of gelation, which greatly reduces the effect of the final formed gel. The single condition phase change response gel is affected by a single factor and is limited in practical application scenarios. In this application context, the inventors provide a new gel system with temperature sensitivity, degradation characteristics and viscosity that are more compatible with bezoar molding and collection. Summary of the invention

[0004] The technical problem solved by the invention is to provide a new gel system for cultivating and collecting bezoars.

[0005] The gel system of the present invention is divided into a first layer of gel and a second layer of gel, wherein:

[0006] The first layer of gel is composed of poly (N-isopropylacrylamide)-acrylamide gel solution and guest gel solution;

[0007] The second layer of gel is a main coordination gel solution;

[0008] The guest gel solution is a polyvinyl alcohol-cyclodextrin gel solution;

[0009] The main coordination gel solution is one or more of polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution.

[0010] In the above gel system: the weight ratio of each gel solution is calculated based on the solid content:

[0011] For every 150 g of poly (N-isopropylacrylamide)-acrylamide gel, add 10-25 g of poly (vinyl alcohol)-cyclodextrin gel and 15-30 g of the main coordination gel.

[0012] Preferably, for every 150 g of poly (N-isopropylacrylamide)-acrylamide gel, 10-25 g of poly (vinyl alcohol)-cyclodextrin gel and 20-30 g of the main coordination gel are added.

[0013] For every 150 g of poly (N-isopropylacrylamide)-acrylamide gel, add 10-25 g of poly (vinyl alcohol)-cyclodextrin gel and 15-30 g of poly (vinyl alcohol)-bile acid gel (replaced with one or more of polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel).

[0014] Preferably, for every 150 g of poly (N-isopropylacrylamide)-acrylamide gel, 15-25 g of poly (vinyl alcohol)-cyclodextrin gel and 20-30 g of poly (vinyl alcohol)-bile acid gel (replaced with one or more of polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel are added.

[0015] Most preferably, 20 g of polyvinyl alcohol-cyclodextrin gel and 30 g of polyvinyl alcohol-bile acid gel (replaced with one or more of polyethylene glycol-bile acid gel, polyvinyl alcohol-deoxycholic acid gel, polyvinyl alcohol-taurocholic acid gel or polyvinyl alcohol-glycine gel) are added to every 150 g of poly (N-isopropylacrylamide)-acrylamide gel.

[0016] When the gel system is used, the gel is first prepared into a gel solution and then used.

[0017] The concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.1-0.5 g / g; or preferably, the concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.1-0.3 g / g; or most preferably, the concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.3 g / g.

[0018] The concentration of the polyvinyl alcohol-cyclodextrin gel solution is 0.05-0.15 g / g; or preferably, the concentration of the polyvinyl alcohol-cyclodextrin gel solution is 0.05-0.1 g / g; or most preferably, the concentration of the polyvinyl alcohol-cyclodextrin gel solution is 0.1 g / g.

[0019] The main coordination gel solution adopts one or more of polyvinyl alcohol-bile acid, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution or polyvinyl alcohol-glycine gel solution, and its concentration is 0.05-0.15g / g; or preferably, the concentration of the above-mentioned main coordination gel solution is 0.05-0.1g / g; or most preferably, the concentration of the above-mentioned main coordination gel solution is 0.1g / g.

[0020] Preferably, the main coordination gel solution adopts polyvinyl alcohol-bile acid gel solution, and its concentration is 0.05-0.15g / g; or further preferably, the main coordination gel solution adopts polyethylene glycol-bile acid gel solution, and its concentration is 0.05-0.1g / g; or most preferably, the main coordination gel solution adopts polyvinyl alcohol-bile acid gel solution, and its concentration is 0.1g / g.

[0021] The above-mentioned gel system is calculated based on the gel solution, and the ratio of each gel solution is as follows: for every 500g of poly (N-isopropylacrylamide)-acrylamide gel solution with a concentration of 0.1-0.5g / g, 100-250g of poly (vinyl alcohol)-cyclodextrin gel solution with a concentration of 0.05-0.15g / g and 150-300g of main coordination gel solution with a concentration of 0.05-0.15g / g are added; the main coordination gel can be one or more of poly (vinyl alcohol)-cyclodextrin gel, polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel; the main coordination gel preferably uses poly (vinyl alcohol)-cyclodextrin gel.

[0022] Preferably, the ratio of each gel solution is as follows: for every 500g of poly (N-isopropylacrylamide)-acrylamide gel solution with a concentration of 0.3g / g, 100-250g of poly (vinyl alcohol)-cyclodextrin gel solution with a concentration of 0.1g / g and 150-300g of main coordination gel solution with a concentration of 0.1g / g are added; the main coordination gel can be one or more of poly (vinyl alcohol)-cyclodextrin gel, polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel; the main coordination gel preferably uses poly (vinyl alcohol)-cyclodextrin gel.

[0023] Further preferably, the ratio of each gel solution is as follows: for every 500g of poly (N-isopropylacrylamide)-acrylamide gel solution with a concentration of 0.3g / g, 150-250g of poly (Viavinyl alcohol)-cyclodextrin gel solution with a concentration of 0.1g / g and 200-300g of main coordination gel solution with a concentration of 0.1g / g are added; the main coordination gel can be one or more of poly (Viavinyl alcohol)-cyclodextrin gel, polyethylene glycol-bile acid gel, poly (Viavinyl alcohol)-deoxycholic acid gel, poly (Viavinyl alcohol)-taurocholic acid gel or poly (Viavinyl alcohol)-glycine gel; the main coordination gel preferably uses poly (Viavinyl alcohol)-cyclodextrin gel.

[0024] Most preferably, the ratio of each gel solution is as follows: for every 500g of poly (N-isopropylacrylamide)-acrylamide gel solution with a concentration of 0.3g / g, 200g of PVA-CA polyvinyl alcohol-cyclodextrin gel solution with a concentration of 0.1g / g and 300g of main coordination gel solution with a concentration of 0.1g / g are added; the main coordination gel can be one or more of polyvinyl alcohol-cyclodextrin gel, polyethylene glycol-bile acid gel, polyvinyl alcohol-deoxycholic acid gel, polyvinyl alcohol-taurocholic acid gel or polyvinyl alcohol-glycine gel; the main coordination gel preferably uses polyvinyl alcohol-cyclodextrin gel.

[0025] The working principle of the gel system of the present invention is a double gel formation and double response mechanism, specifically, the poly N-isopropylacrylamide-acrylamide gel solution in the first layer of gel is used as the first response gel solution, and the temperature-sensitive characteristics of the gel solution are used to perform phase transition response, so as to achieve the phase transition of the gel solution to the gel within a specific temperature range. The polyvinyl alcohol-cyclodextrin gel solution in the first layer of gel and the polyvinyl alcohol-bile acid gel solution in the second gel are used as the second response gel solution, and the two are host-guest coordination gels, the host coordination gel is the polyvinyl alcohol-bile acid gel solution, and the guest coordination gel is the polyvinyl alcohol-cyclodextrin gel solution. The two are in the state of gel solution at the beginning, and are in the state of gel when mixed together and have self-healing properties. The two gel solutions are mixed together, and the entanglement and cross-linking in the physical space can make the gel have a large pore structure; and the two gel solutions can make the mixed gel have two response characteristics, which can be applied in a wider range of fields.

[0026] In the above gel system:

[0027] The poly (N-isopropylacrylamide)-acrylamide gel solution is prepared by a one-pot method:

[0028] A. Weigh the reaction raw materials N-isopropylacrylamide, acrylamide, carboxyl donor, chloride salt, and polyphenol group donor according to proportion and place them in a reaction device;

[0029] B. After passing inert gas into the reaction device and stirring for a certain period of time, add initiator ammonium persulfate and accelerator tetramethylethylenediamine to obtain gel solution B;

[0030] C. The gel solution obtained in step B is dialyzed to remove impurities, thereby obtaining a poly (N-isopropylacrylamide)-acrylamide gel solution.

[0031] The acrylamide in the reaction raw material of step A can be replaced by one or more of poloxamer and polylactic acid-glycolic acid-ethylene glycol. The LCST value can also be regulated by adjusting the proportion to achieve a temperature sensitive range of 32-45°C.

[0032] The carboxyl donor in the reaction raw material of step A is one or more of acrylic acid, propylene sulfonic acid and itaconic acid.

[0033] In step A, the chloride salt in the reaction raw material is used as a metal ion donor, and the chloride salt that can be used is one or more of aluminum chloride, ferric chloride, and calcium chloride; the above chloride salts are used to provide dynamic metal ions, so that the poly (N-isopropylacrylamide)-acrylamide gel has self-healing properties.

[0034] The polyphenol group donor in the reaction raw materials of step A is one or more of polydopamine, tannic acid and anthocyanin.

[0035] The polydopamine is prepared by the following method: dopamine hydrochloride is used to carry out a self-polymerization reaction under the condition of pH=10-12, and the reaction is considered to be completed when the solution changes from transparent to black, and the polydopamine is obtained.

[0036] Preferably, in the polydopamine preparation method, the pH value is 11-12

[0037] Or most preferably, in the polydopamine preparation method, the pH value is 11.

[0038] Preferably, in the polydopamine preparation method, the self-polymerization reaction time is 20-60 min. Preferably, the self-polymerization reaction time is 30 min.

[0039] The ratio of the reaction materials in step A is:

[0040] In step A, the reaction raw materials are added with 10-80 g of acrylamide or one or more of poloxamer, polylactic acid-glycolic acid-ethylene glycol, 1-50 g of carboxyl donor, 90-450 mg of chloride salt, and 1-120 g of polyphenol group per 500 g of N-isopropylacrylamide.

[0041] Preferably, the reaction raw materials in step A are added with 20-60g acrylamide, 10-30g poloxamer or 20-40g polylactic acid-glycolic acid-ethylene glycol per 500g N-isopropylacrylamide; the carboxyl donor is 1-30g acrylic acid, 1-20g propylene sulfonic acid or 1-20g itaconic acid; the chloride salt is 90-270mg aluminum chloride, 90-270mg ferric chloride or 90-270mg calcium chloride; the polyphenol group is 1-50g polydopamine, 5-30g tannic acid or 1-30g anthocyanin.

[0042] Most preferably, in step A, 30 g of acrylamide is added to the reaction raw materials per 500 g of N-isopropylacrylamide; 20 g of acrylic acid is used as the carboxyl donor; 180 mg of aluminum chloride is used as the chloride salt; and 50 g of polydopamine, 5-30 g of tannic acid, or 1-30 g of anthocyanin is used as the polyphenol group.

[0043] In step B, the amount of the initiator ammonium persulfate and the accelerator tetramethylethylenediamine is calculated based on 1-20 g of initiator ammonium persulfate and 1-20 g of accelerator tetramethylethylenediamine per 500 g of N-isopropylacrylamide.

[0044] Preferably, the amount of the initiator ammonium persulfate and the promoter tetramethylethylenediamine used in step B is based on 15g of initiator ammonium persulfate and 10g of promoter tetramethylethylenediamine per 500g of N-isopropylacrylamide.

[0045] The aeration and stirring for a certain period of time in step B refers to stirring for 0.5-2 hours; preferably, the stirring time in step B is 1 hour.

[0046] The reaction conditions of the reaction in step B are: reaction temperature 40-80°C, reaction time 4-12h. Preferably, the reaction conditions of step B are: reaction temperature 60°C, reaction time 6h.

[0047] The dialysis in step C is water dialysis, and the dialysis time is 5-7 days. Preferably, the dialysis time in step C is 7 days.

[0048] The solution obtained in step C is dried to obtain poly (N-isopropylacrylamide)-acrylamide gel; the drying is carried out by low-temperature oven drying or freeze drying.

[0049] The preparation method of the polyvinyl alcohol-cyclodextrin gel solution comprises the following steps:

[0050] A. Acidification modification of polyvinyl alcohol:

[0051] (1) Polyvinyl alcohol is dissolved in dimethyl sulfoxide;

[0052] (2) adding succinic anhydride and p-toluenesulfonic acid to the solution obtained in step (1) to react;

[0053] (3) adding ether to the reaction solution obtained in step (2), filtering and washing to obtain a precipitate, dialyzing the obtained precipitate with water to remove impurities, and drying to obtain acid-modified polyvinyl alcohol (PVA-Acid).

[0054] B. Cyclodextrin grafting of polyvinyl alcohol

[0055] (1) Acid-modified polyvinyl alcohol is dissolved in water;

[0056] (2) adding aminocyclodextrin (Amino-CD), N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBt) to the solution obtained in step (1), stirring for a certain period of time, reacting, and cooling to terminate the reaction;

[0057] (3) Ether is added to the solution obtained in step (2) to wash out the precipitate, and the precipitate is dialyzed to remove impurities to obtain polyvinyl alcohol grafted cyclodextrin (PVA-CD).

[0058] In the acidification modification of polyvinyl alcohol in step A:

[0059] The polyvinyl alcohol and dimethyl sulfoxide in step (1) are mixed according to the following ratio: 20-50 ml of dimethyl sulfoxide is added for every 2-6 g of polyvinyl alcohol.

[0060] Preferably, the polyvinyl alcohol and dimethyl sulfoxide in step (1) are mixed according to the following ratio: 50 ml of dimethyl sulfoxide is added for every 4 g of polyvinyl alcohol.

[0061] In step (2), the amount of succinic anhydride and p-toluenesulfonic acid added is 200-1000 mg succinic anhydride and 1-50 mg p-toluenesulfonic acid per 2-6 g polyvinyl alcohol.

[0062] Preferably, the amount of succinic anhydride and p-toluenesulfonic acid added in step (2) is 400-500 mg succinic anhydride and 5-25 mg p-toluenesulfonic acid per 4 g polyvinyl alcohol.

[0063] The temperature of the dimethyl sulfoxide (DMSO) in step (1) is 60-90°C, that is, polyvinyl alcohol is dissolved in dimethyl sulfoxide at 60-90°C; preferably, the temperature of the dimethyl sulfoxide (DMSO) in step (1) is 80°C.

[0064] The reaction conditions of step (2) are: reaction temperature 40-80°C, reaction time 36-72h; preferably, the reaction conditions of step (2) are reaction temperature 50°C, reaction time 48h.

[0065] The dialysis conditions in step (3) are: dialysis with water for 5-7 days. Preferably, the dialysis time is 5 days.

[0066] The drying in step (3) is carried out by low-temperature oven drying or freeze drying.

[0067] In the step B of cyclodextrin grafting of polyvinyl alcohol:

[0068] The water in step (1) is ultrapure water.

[0069] The temperature of the water in step (1) is 70-90°C, that is, the acidified modified polyvinyl alcohol is dissolved in water at 70-90°C; preferably, the temperature of the water in step (1) is 80°C.

[0070] The stirring for a certain time in step (2) refers to stirring for 0.5-2 hours. Preferably, the stirring for a certain time in step (2) refers to stirring for 1 hour.

[0071] The reaction conditions of the reaction in step (2) are: reaction temperature 40-80°C, reaction time 8-36h. Preferably, the reaction conditions of the reaction in step (2) are: reaction temperature 70°C, reaction time 24h.

[0072] The step (2) of cooling the reaction to terminate the reaction is to move the reaction solution into an ice water bath to terminate the reaction.

[0073] The dialysis in step (3) is water dialysis, and the dialysis time is 5-7 days. Preferably, the dialysis time in step (3) is 7 days.

[0074] The preparation method of the polyvinyl alcohol-bile acid gel solution comprises the following steps:

[0075] A. Acidification modification of polyvinyl alcohol

[0076] B. Bile acid grafting of polyvinyl alcohol

[0077] (1) Acid-modified polyvinyl alcohol is dissolved in dimethylformamide (DMF);

[0078] (2) adding aminocholic acid (Amino-CA), N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBt) to the solution obtained in step (1), stirring for a certain period of time, reacting, and cooling to terminate the reaction;

[0079] (3) Ether is added to the solution obtained in step (2) to wash out the precipitate, and the precipitate is dialyzed to remove impurities to obtain polyvinyl alcohol grafted bile acid (PVA-CA).

[0080] In the bile acid grafting of polyvinyl alcohol in step B:

[0081] The temperature of the dimethylformamide in step (1) is 70-90°C, that is, the acid-modified polyvinyl alcohol is dissolved in dimethylformamide at 70-90°C; preferably, the temperature of the dimethylformamide in step (1) is 80°C.

[0082] The stirring time in step (2) refers to stirring for 0.5-2 hours. Preferably, the stirring time in step (2) is 1 hour.

[0083] The reaction conditions of the reaction in step (2) are: reaction temperature 40-80°C, reaction time 8-36h. Preferably, the reaction conditions of the reaction in step (2) are: reaction temperature 70°C, reaction time 24h.

[0084] The step (2) of cooling the reaction to terminate the reaction is to move the reaction solution into an ice water bath to terminate the reaction.

[0085] The dialysis in step (3) is water dialysis, and the dialysis time is 5-7 days. Preferably, the dialysis time in step (3) is 7 days.

[0086] In the method for cultivating and collecting bezoars of the present invention, the gel system is cultivated and collected in vivo, that is, the gel system is injected into the gallbladder of the cattle according to the following steps:

[0087] A. First, one or more of the main coordination gel polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution are injected into the bovine gallbladder;

[0088] B. Then, the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and the poly (N-isopropylacrylamide)-acrylamide gel solution are mixed and then injected into the cattle gallbladder; or the two are injected into the cattle gallbladder at the same time without any particular order.

[0089] In the method for cultivating and collecting bezoars of the present invention, the gel system can also be used to cultivate and collect bezoars in vitro.

[0090] A. placing the bezoar crystal core composition into ox bile, heating the ox bile and maintaining the temperature at 38-40° C. (simulating the temperature of ox gallbladder), and then adding one or more of the main coordination gel, which is polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution, into the ox bile;

[0091] B. Then mix the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and poly (N-isopropylacrylamide)-acrylamide gel solution and add them to the cow bile in step A, maintain 38-40°C (simulating the cow gallbladder temperature) and supplement bile (simulating the bile concentration in the cow gallbladder and simulating the cow's body circulation).

[0092] The bezoar crystal core composition is prepared according to the following dosage relationship: for every 1-3g of artificial bezoar powder, add 2-8ml of bacterial solution, 10-40ml of culture medium, 0.5-1g of bilirubin, 0.1-0.5g of bile acid, 0.05-0.1g of deoxycholic acid, and 1-3g of calcium chloride.

[0093] The above-mentioned dual-responsive gel system is used, and poly N-isopropylacrylamide-acrylamide gel solution is used as the first responsive gel solution. The temperature-sensitive characteristics of the gel solution are used for phase transition response, and the phase transition of the gel solution to the gel is realized within a specific temperature range, that is, the temperature-sensitive N-isopropylacrylamide (NIPAM). By introducing the proportion of acrylamide (AM), the LCST value is regulated, and the temperature-sensitive range is controlled at 32-45°C, covering the cow body temperature of 40°C. The polyvinyl alcohol-cyclodextrin gel solution in the first layer of gel and the polyvinyl alcohol-bile acid gel solution in the second gel are used as the second responsive gel solution. The two are host-guest coordination gels, the host coordination gel is the polyvinyl alcohol-bile acid gel solution, and the guest coordination gel is the polyvinyl alcohol-cyclodextrin gel solution. The two are in the state of gel solution at the beginning, and are mixed together in the state of gel and have self-healing properties. When two gel solutions are mixed together, the entanglement and cross-linking in physical space can make the gel have a large pore structure; and the two gel solutions can make the mixed gel have two response characteristics, which can be effectively used in the cultivation and collection of bezoar.

[0094] Specifically, the method of cultivating and collecting bezoar in cattle by using the above-mentioned gel system comprises the following steps:

[0095] 1. Make a 1-20mm incision on the cow's abdomen to expose the cow's gallbladder. Alternatively, insert a catheter into the gallbladder, puncture the gallbladder with a puncture needle, or place the gel directly into the gallbladder through surgery after in vitro cross-linking.

[0096] 2. The gel system of the present invention is sequentially injected into the bovine gallbladder using a syringe.

[0097] 3. Inject bacterial solution.

[0098] 4. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0099] 5. Raise cattle normally. After 10-18 months, the gel has completely degraded and the bezoar can be removed.

[0100] In the above cultivation and collection methods:

[0101] The gel system of the present invention described in step 2 is sequentially injected into the bovine gallbladder using a syringe.

[0102] (1) Use a syringe to first inject the polyvinyl alcohol-bile acid, polyethylene glycol-bile acid, polyvinyl alcohol-deoxycholic acid, polyvinyl alcohol-taurocholic acid, or polyvinyl alcohol-glycine main coordination gel solution into the bovine gallbladder.

[0103] (2) Use a syringe to simultaneously inject the polyvinyl alcohol-cyclodextrin gel guest coordination solution and the poly-N-isopropylacrylamide-acrylamide gel solution of the encapsulated culture medium into the bovine gallbladder to obtain a multi-actuated double-layer hydrogel. Among them, acrylamide can be replaced by one or more of poloxamer and polylactic acid-glycolic acid-ethylene glycol. The purpose of the encapsulated culture medium is to provide nutrients such as carbon source, nitrogen source, phosphorus source, etc. to help the bacterial liquid to grow. The culture medium used can be one or more of the common TB nutrient solution, TSB nutrient solution, and LB nutrient solution.

[0104] The bacterial solution in step 3 is one or more of Escherichia coli, Bacillus subtilis, and Citrobacter rodentium.

[0105] The dosage ratio of the polyvinyl alcohol-bile acid gel solution, the polyvinyl alcohol-cyclodextrin gel solution, the poly (N-isopropylacrylamide)-acrylamide gel solution, the culture medium, and the bacterial solution in step 2 and step 3 is as follows:

[0106] The dosage ratio of the main coordination gel solution, polyvinyl alcohol-cyclodextrin gel solution, poly (N-isopropylacrylamide)-acrylamide gel solution, culture medium, and bacterial solution is calculated based on the dosage of the solid content:

[0107] According to the method, 0.10-0.25 g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.15-0.30 g of main coordination gel, 1-50 ml of culture medium and 1-10 ml of bacterial liquid are added to every 1.5 g of poly (N-isopropylacrylamide)-acrylamide gel; the main coordination gel is one or more of poly (vinyl alcohol)-bile acid, polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel.

[0108] Alternatively, for every 1.5 g of poly (N-isopropylacrylamide)-acrylamide gel, add 0.10-0.25 g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.15-0.30 g of main coordination gel, 10-40 ml of culture medium, and 2-8 ml of bacterial liquid; the main coordination gel is one or more of poly (vinyl alcohol)-bile acid, polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel, or poly (vinyl alcohol)-glycine gel.

[0109] Or preferably, according to every 1.50g of poly (N-isopropylacrylamide)-acrylamide gel, add 0.15-0.25g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.20-0.30g of host coordination gel, 1-50ml of culture medium, and 1-10ml of bacterial solution;

[0110] Most preferably, for every 1.50 g of poly (N-isopropylacrylamide)-acrylamide gel, 0.15-0.25 g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.20-0.30 g of host coordination gel, 10-40 ml of culture medium, and 2-8 ml of bacterial solution are added;

[0111] Furthermore, when the gel system is used, the gel is first prepared into a gel solution before use, and the concentration conditions of each raw material at least meet any one of the following conditions:

[0112] The concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.1-0.5 g / g;

[0113] Or preferably, the concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.3 g / g;

[0114] or the concentration of the PVA-CA guest gel solution is 0.05-0.15 g / g;

[0115] Or preferably, the concentration of the PVA-CA guest gel solution is 0.1 g / g;

[0116] Or the concentration of the main coordination gel solution is 0.05-0.15 g / g;

[0117] Or preferably, the concentration of the main coordination gel solution is 0.1g / g;.

[0118] Or most preferably, the main coordination gel solution adopts polyethylene glycol-bile acid gel solution;

[0119] Or the bacterial content in the bacterial solution is greater than 10 9 / ml;

[0120] Or preferably, the bacterial content in the bacterial solution is 10 9 -10 10 / ml.

[0121] Beneficial effects of the present invention:

[0122] 1. The gel can gently stimulate the gallbladder and induce inflammation in the gallbladder; however, it will not cause serious harm to the cow's body like a metal bezoar bed.

[0123] 2. The gel can self-repair and self-aggregate in the gallbladder. The self-healing gel enhances the strength and integrity of the gel; it can effectively prevent the gel solution from being dispersed by the flowing bile in the initial injection and losing the ability to collect bezoar.

[0124] 3. The gel can be completely degraded in 10-18 months and has high biocompatibility, making it more effective in collecting bezoar. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 In vitro degradation curve of gel (Example 1).

[0126] Figure 2 Flow chart of collecting bezoar by gel in vivo culture.

[0127] Figure 3 Collection of photos of finished bezoar products.

[0128] in, Figure 3 A is a photo of comparative example 2-no gel bezoar was placed. Figure 3 B is a photo of the collection of gel bezoars in Example 1.

[0129] Figure 4 Image of small granules of bezoar collected from in vitro culture. DETAILED DESCRIPTION

[0130] Below in conjunction with embodiment, the scheme of the present invention will be explained and described.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if no specific technology or condition is indicated, it is carried out according to the technology or condition described in the document in this area or according to the product specification.The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

[0131] The following are the definitions of the English abbreviations and main terms used in this invention:

[0132] N-isopropylacrylamide (NIPAM), acrylamide (AM), acrylic acid (AA), polydopamine (DPOA), polyvinyl alcohol (PVA), acidified polyvinyl alcohol (PVA-Acid), polyvinyl alcohol grafted cyclodextrin (PVA-CD), polyvinyl alcohol grafted bile acid (PVA-CA), aminocyclodextrin (Amino-CD), N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), aminocholic acid (Amino-CA).

[0133] In the following examples, the PVA-CA guest gel and the PVA-CD host coordination gel were prepared by the following method:

[0134] 1. Dissolve 2-6 g PVA in 20-50 ml 60-80 ° C dimethyl sulfoxide (DMSO);

[0135] 2. Add 200-1000 mg of succinic anhydride and 1-50 mg of p-toluenesulfonic acid to the solution and react at 40-80°C for 24-72 hours;

[0136] 3. The precipitate was washed out with ether and dialyzed for 5 days to remove impurities, obtaining 2-5 g of PVA-Acid with a yield of 80-90%.

[0137] (II) Cholic acid grafting of polyvinyl alcohol

[0138] 1. Dissolve 200-1000 mg PVA-Acid in 20-50 ml 60-100°C dimethylformamide (DMF);

[0139] 2. Add 50-200 mg of aminocholic acid (Amino-CA), 1-100 mg of N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and 1-20 mg of 1-hydroxybenzotriazole hydrate (HOBt) to the above solution and react at 50-80°C for 6-48 hours;

[0140] 3. Move the solution into an ice water bath to terminate the reaction, wash out the precipitate with ether, dialyze for 5-7 days to remove impurities, and dry to obtain PVA-CA.

[0141] (III) Cyclodextrin grafting of polyvinyl alcohol

[0142] 1. Dissolve 200-1000 mg PVA-Acid in 20-100 ml ultrapure water at 60-100°C;

[0143] 2. Add 100-800 mg aminocyclodextrin (Amino-CD), 20-100 mg EDC, and 1-20 mg HOBt to the above solution, stir for one hour, and react at 50-80°C for 6-48 hours;

[0144] 3. Move the solution into an ice water bath to terminate the reaction, wash out the precipitate with ether, dialyze for 5-7 days to remove impurities, and dry to obtain PVA-CD.

[0145] The PVA-CA and PVA-CD obtained by the above method were respectively added with water to prepare 10wt% (0.1g / g) PVA-CA gel solution and 10wt% (0.1g / g) PVA-CD gel solution, which were used in the preparation of the following examples and comparative examples to examine the beneficial effects of the gel of the present invention for cultivation and collection.

[0146] Example 1

[0147] 1. Dopamine hydrochloride was subjected to self-polymerization reaction at pH = 11 for 30 minutes to obtain polydopamine, at which time the solution changed from transparent to black.

[0148] Prepare poly (N-isopropylacrylamide)-acrylamide gel solution using a one-pot method: 500 g of N-isopropylacrylamide, 30 g of acrylamide, 20 g of acrylic acid, 20 g of aluminum chloride (need to be prepared into a 90 mg / g solution), and 50 g of polydopamine are placed in a three-necked flask;

[0149] 2. After stirring for one hour under nitrogen, add 15g of initiator ammonium persulfate and 10g of initiator tetramethylethylenediamine, and react at 50°C for 7h;

[0150] 3. The gel solution was dialyzed for 7 days to remove impurities and obtain polydopamine-PNIPAM-AM-AA-Al 3+ Gel solution.

[0151] 4. 500g polydopamine-PNIPAM-AM-AA-Al 3+ The gel solution (30 wt %, ie 0.3 g / g) and 200 g of PVA-CA guest gel solution (10 wt %) were mixed to obtain a mixed solution A.

[0152] 5. Inject 300 g of PVA-CD host coordination gel solution (10 wt%) into 500 ml of water;

[0153] 6. Inject the above mixed solution A into 500 ml of water to obtain a gel.

[0154] The adhesion strength is 5.05 kPa, the 90-day degradation rate is 65.2%, and the LCST is 38 °C.

[0155] like Figure 1 , observe the degradation of the gel within 90 days, experimental method: take 5g of gel, record the weight of the gel at this time as W initial, put it into 37℃, 200ml bile to simulate in vivo degradation experiment, take out the gel after a period of time, measure the weight of the gel at this time after 24h of constant humidity and constant weight, and calculate the degradation rate. The results show that compared with the existing gel made of similar raw materials, the degradation time of the gel system of the present invention is significantly improved, and it can maintain a degradation time of at least three months in bile.

[0156] The method for cultivating and collecting bezoars by using a gel system of the present invention comprises cultivating and collecting bezoars in vivo and in vitro:

[0157] The in vitro methods include:

[0158] A. The bezoar crystal core composition (the bezoar crystal core composition is prepared according to the following dosage relationship: according to every 1-3g artificial bezoar powder, add 2-8ml bacterial liquid (bacteria content greater than 10 9 / ml), 10-40ml culture medium, 0.5-1g bilirubin, 0.1-0.5g bile acid, 0.05-0.1g deoxycholic acid, 1-3g calcium chloride. ) are placed in ox bile, the ox bile is heated and maintained at 38-40°C, and then the main coordination gel is added to the ox bile, wherein the main coordination gel is one or more of polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution;

[0159] B. Then, the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and the poly (N-isopropylacrylamide)-acrylamide gel solution are mixed and then added into the ox bile of step A, and the temperature is maintained at 38-40° C. and the bile is supplemented to obtain the ox bile culture system.

[0160] In vivo methods include but are not limited to the following methods. For a flow chart of in vivo culture and collection of bezoar, see Figure 2 :

[0161] (1) The gel system is injected into the cattle gallbladder and the bezoar is collected by in vivo cultivation:

[0162] A. First, inject the main coordination gel into the bovine gallbladder, wherein the main coordination gel is one or more of polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution;

[0163] B. Then, the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and the poly (N-isopropylacrylamide)-acrylamide gel solution are mixed and then injected into the cattle gallbladder; or the two are injected into the cattle gallbladder at the same time in no particular order to obtain a bezoar culture system;

[0164] C. Seal the wound, feed the cattle normally, and take out the bezoar after the bezoar is successfully cultivated and collected;

[0165] (2) mixing the host coordination gel with the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and poly (N-isopropylacrylamide)-acrylamide gel solution and then injecting them into the cattle gallbladder to obtain a bezoar culture system; then sealing the wound, feeding the cattle normally, and taking out the bezoar after the bezoar is successfully cultured and collected; wherein the host coordination gel is one or more of polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution.

[0166] The following examples are examples of the method for cultivating and collecting bezoars using a gel system according to the present invention.

[0167] Example 2

[0168] 1. Select three adult cows of similar weight and age, make a 3mm incision in the abdomen of the cows, and expose the gallbladder.

[0169] 2. Use a syringe to inject 5 g of polyvinyl alcohol-bile acid main coordination gel solution (0.1 g / g) into the bovine gallbladder.

[0170] 3. Use a syringe to simultaneously inject 4 g of polyvinyl alcohol-cyclodextrin gel solution (0.1 g / g) wrapped in 50 ml of TB nutrient solution and 20 g of poly (N-isopropylacrylamide)-acrylamide gel solution (0.3 g / g) into the bovine gallbladder to obtain a multi-actuated double-layer hydrogel.

[0171] 4. Inject 20ml of bacterial solution (E. coli, bacterial content greater than 10 9 / ml).

[0172] 5. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0173] 6. Raise cattle normally, take out the bezoar after 12 months, dry and weigh it, and record the cultured bezoar yield of each cattle, which are 17.1g, 16.1g, and 17.3g respectively, with an average of 16.8g / head. At this time, the gel has been completely degraded.

[0174] Example 3

[0175] 1. Select three adult cows of similar weight and age, make a 2mm incision on the abdomen of the cows, and expose the gallbladder.

[0176] 2. Use a syringe to inject 8 g of polyvinyl alcohol-bile acid main coordination gel solution into the bovine gallbladder.

[0177] 3. Use a syringe to simultaneously inject 6 g of polyvinyl alcohol-cyclodextrin gel solution wrapped in 30 ml of TB nutrient solution and 12 g of poly (N-isopropylacrylamide)-acrylamide-acrylic acid-aluminum ion self-healing gel solution into the bovine gallbladder to obtain a multi-actuated double-layer hydrogel.

[0178] 4. Inject 10ml of bacterial solution (E. coli, bacterial content greater than 10 9 / ml).

[0179] 5. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0180] 6. Raise cattle normally, take out the bezoar after 12 months, dry and weigh it, and record the cultured bezoar yield of each cattle, which are 12.3g, 15.4g, and 14.9g respectively, with an average of 14.2g / head. At this time, the gel has been completely degraded.

[0181] Example 4

[0182] 1. Select three adult cows of similar weight and age, make a 1mm cut in the abdomen of the cows, and expose the gallbladder.

[0183] 2. Use a syringe to inject 6 g of polyvinyl alcohol-bile acid main coordination gel solution into the bovine gallbladder.

[0184] 3. Use a syringe to simultaneously inject 5 g of polyvinyl alcohol-cyclodextrin gel solution wrapped in 40 ml of TB nutrient solution and 15 g of poly (N-isopropylacrylamide)-acrylamide-acrylic acid-aluminum ion self-healing gel solution into the bovine gallbladder to obtain a multi-actuated double-layer hydrogel.

[0185] 4. Inject 15ml of bacterial solution (E. coli, bacterial content greater than 10 9 / ml).

[0186] 5. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0187] 6. Raise cattle normally, take out the bezoar after 12 months, dry and weigh it, and record the cultured bezoar yield of each cattle, which are 14.7g, 16.1g, and 15.2g respectively, with an average of 15.3g / head. At this time, the gel has been completely degraded.

[0188] Example 5 In vitro culture of bezoar experiment

[0189] 1. Take 3 beakers and pour 200ml of bile into 3 250ml beakers respectively.

[0190] 2. Add 8g of bezoar crystal core composition with a total mass. The bezoar crystal core composition is prepared by adding 8ml of bacterial solution (Escherichia coli), 40ml of TB culture medium, 1g of bilirubin, 0.5g of bile acid, 0.1g of deoxycholic acid and 2g of calcium chloride for every 2g of artificial bezoar powder.

[0191] 3. Use a syringe to inject 5 g of polyvinyl alcohol-bile acid main coordination gel solution into the beaker.

[0192] 4. Use a syringe to simultaneously inject 4 g of polyvinyl alcohol-cyclodextrin gel solution wrapped in 50 ml of TB nutrient solution and 20 g of poly (N-isopropylacrylamide)-acrylamide-acrylic acid-aluminum ion self-healing gel solution into a beaker to obtain a multi-actuated double-layer hydrogel.

[0193] 5. Inject 20ml of bacterial solution (E. coli, bacterial content greater than 10 9 / ml).

[0194] 6. To simulate the in vivo environment, use a siphon to drain the bile into the beaker and replenish the bile regularly.

[0195] 7. After 12 months, take out the small particles of bezoar, dry them and weigh them, and record the cultivated bezoar yield of each cow. The collected amounts of bezoar in the three beakers were 6.2g, 7.4g, and 6.9g, respectively, with an average of 6.8g per beaker. At this time, the gel has been completely degraded.

[0196] Note: In vitro experiments do not have the original substance of bezoar in cattle, and can only simulate the circulation in cattle by circulating bile and supplementing corresponding substances. Because the output of in vitro simulation experiments is less than that of in vivo culture, the particles are relatively small, so they are called bezoar small particles or bilirubin calcium mixture.

[0197] Comparative Example 1

[0198] 1. Select three adult cows of similar weight and age, make a 3mm incision in the abdomen of the cows, and expose the gallbladder.

[0199] 2. Use a syringe to inject 5 g of polyvinyl alcohol-bile acid main coordination gel solution into the bovine gallbladder.

[0200] 3. Use a syringe to simultaneously inject 4 g of polyvinyl alcohol-cyclodextrin gel solution wrapped in 50 ml of TB nutrient solution and 20 g of poly (N-isopropylacrylamide)-acrylamide-acrylic acid-aluminum ion self-healing gel solution into the bovine gallbladder to obtain a multi-actuated double-layer hydrogel.

[0201] 4. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0202] 5. The three cows were raised normally and their gallbladders were removed after 12 months. None of the three cows produced yellow urine, indicating that the gel had been completely degraded.

[0203] Comparative Example 2

[0204] 1. Select three adult cows of similar weight and age, make a 3mm incision in the abdomen of the cows, and expose the gallbladder.

[0205] 2. Inject 50ml TB nutrient solution and 20ml bacterial solution (E. coli, bacterial content greater than 10 9 / ml).

[0206] 3. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0207] 4. The cattle were raised normally and their gallbladders were taken out after 12 months. The bile produced by the cattle was in the form of small particles, and there was more bile in the bile duct. The small particles were dried and weighed, and the yield of cultivated bezoar small particles for each cow was recorded. The results were 8.1g, 9.8g, and 7.2g, respectively, with an average of 8.4g per cow. At this time, the gel had been completely degraded.

[0208] Comparative Example 3

[0209] 1. Select three adult cows of similar weight and age, make a 3mm incision in the abdomen of the cows, and expose the gallbladder.

[0210] 2. Use a syringe to inject 5 g of polyvinyl alcohol-bile acid main coordination gel solution into the bovine gallbladder.

[0211] 3. Use a syringe to simultaneously inject 4 g of polyvinyl alcohol-cyclodextrin gel solution and 20 g of poly (N-isopropylacrylamide)-acrylamide-acrylic acid-aluminum ion self-healing gel solution into the bovine gallbladder to obtain a multi-actuated double-layer hydrogel.

[0212] 4. Inject 20ml of bacterial solution (E. coli, bacterial content greater than 10 9 / ml).

[0213] 5. Use sealing gel to seal the needle hole in the gallbladder; use needle and thread to seal the abdominal wound.

[0214] 6. After 12 months of normal feeding of cattle, the bezoar was taken out, dried and weighed, and the cultured bezoar yield of each cattle was recorded, which was 5.9g, 7.6g, and 6.9g, with an average of 6.8g / head. At this time, the gel was completely degraded. The yield of bezoar without culture medium was significantly reduced.

[0215] The inventors studied and applied the gel system to cultivate and collect bezoars by using two methods: in vivo culture and in vitro culture. Figure 3 In vivo culture experiments were conducted to compare the effects of placing gel on the collection of bezoars. The results showed that the gel system of the present invention has the ability to adhere to small particles of bezoars. Bezoars are extremely small particles in the initial period of formation. Only when they are aggregated can the initial bezoar core be formed, and the bezoar core will further grow. The gel of the gel system of the present invention can provide a place for enrichment for the formation of the initial core of bezoars. If no gel is added, the initial bezoar small particles formed in the gallbladder of cattle will be discharged from the cattle body along with the circulation of body fluids, and the enrichment speed and probability are greatly reduced. Figure 4 The results of the in vitro culture experiment show that the gel system of the present invention can also achieve the purpose of enriching and aggregating bezoar substances by simulating the bovine gallbladder environment in vitro. Although the in vitro culture conditions are lower than the in vivo culture conditions, bezoar particles of a certain size can still be obtained by in vitro culture for a certain period of time.

Claims

1. A method for cultivating and collecting bezoars using a gel system, characterized in that: The gel system uses an in vitro method to cultivate and collect bezoars: A. placing the bezoar crystal core composition into cattle bile, heating the cattle bile and maintaining it at 38-40° C., and then adding the main coordination gel into the cattle bile, wherein the main coordination gel is one or more of a polyvinyl alcohol-bile acid gel solution, a polyethylene glycol-bile acid gel solution, a polyvinyl alcohol-deoxycholic acid gel solution, a polyvinyl alcohol-taurocholic acid gel solution, and a polyvinyl alcohol-glycine gel solution; B. Then, the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and the poly (N-isopropylacrylamide)-acrylamide gel solution are mixed and then added to the ox bile of step A, and the mixture is maintained at 38-40° C. and supplemented with bile to obtain a ox bile culture system; Furthermore, the bezoar crystal core composition is prepared according to the following dosage relationship: for every 1-3g of artificial bezoar powder, add 2-8ml of bacterial solution, 10-40ml of culture medium, 0.5-1g of bilirubin, 0.1-0.5g of bile acid, 0.05-0.1g of deoxycholic acid, and 1-3g of calcium chloride.

2. A method for cultivating and collecting bezoars using a gel system, characterized in that: The gel system is injected into the cow gallbladder and the bezoar is collected by in vivo cultivation: A. First, inject the main coordination gel into the bovine gallbladder, wherein the main coordination gel is one or more of polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution; B. Then, the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and the poly (N-isopropylacrylamide)-acrylamide gel solution are mixed and then injected into the cattle gallbladder; or the two are injected into the cattle gallbladder at the same time in no particular order to obtain a bezoar culture system; C. Seal the wound, feed the cattle normally, and take out the bezoar after the bezoar is successfully cultivated and collected; Alternatively, the host coordination gel and the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and poly (N-isopropylacrylamide-acrylamide) gel solution are mixed and then injected into the cattle gallbladder to obtain a bezoar culture system; then the wound is sealed, the cattle are fed normally, and the bezoar is taken out after the bezoar is successfully cultivated and collected; wherein the host coordination gel is one or more of polyvinyl alcohol-bile acid gel solution, polyethylene glycol-bile acid gel solution, polyvinyl alcohol-deoxycholic acid gel solution, polyvinyl alcohol-taurocholic acid gel solution, and polyvinyl alcohol-glycine gel solution.

3. The method for cultivating and collecting bezoars by utilizing a gel system according to claim 2, characterized in that: The method of injecting the gel system into the bovine gallbladder satisfies at least any one of the following conditions: Method (1) a 1-20 mm wound is cut in the cow's abdomen to expose the cow's gallbladder, and then the gel system is injected into the cow's gallbladder; Method (2) inserting a catheter into the bovine gallbladder and then injecting the gel system through the catheter; Method (3) using a puncture needle to puncture into the bovine gallbladder and then injecting the gel system through a catheter; Method (4) The host coordination gel and the guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and poly (N-isopropylacrylamide-acrylamide) gel solution are mixed and cross-linked, and then the cross-linked gel is placed into the bovine gallbladder by surgical means.

4. The method for cultivating and collecting bezoars by using a gel system according to claim 1 or 2, characterized in that: The guest coordination gel polyvinyl alcohol-cyclodextrin gel solution and poly (N-isopropylacrylamide)-acrylamide gel solution are injected into the bovine gallbladder after using culture medium; Preferably, the culture medium is one or more of TB nutrient solution, TSB nutrient solution, and LB nutrient solution.

5. The method for cultivating and collecting bezoars by using a gel system according to claim 1 or 2, characterized in that: Injecting the bacterial solution into the bezoar culture system; Preferably, the bacterial liquid is one or more of Enterobacter, Bacillus subtilis, and Citrobacter rodi.

6. The method for cultivating and collecting bezoars by using a gel system according to claim 2, characterized in that: The wound sealing in step C is to seal the gallbladder needle hole with a plugging gel or to seal the abdominal wound with a needle and thread.

7. The method for cultivating and collecting bezoars by using a gel system according to claim 1 or 2, characterized in that: The poly (N-isopropylacrylamide)-acrylamide gel solution is prepared by a one-pot method: A. Weigh the reaction raw materials N-isopropylacrylamide, acrylamide, carboxyl donor, chloride salt, and polyphenol group donor according to proportion and place them in a reaction device; B. After passing inert gas into the reaction device and stirring for a certain period of time, add initiator ammonium persulfate and accelerator tetramethylethylenediamine to obtain gel solution B; C. dialyzing the gel solution obtained in step B to remove impurities, thereby obtaining a poly (N-isopropylacrylamide)-acrylamide gel solution; The preparation method of the polyvinyl alcohol-cyclodextrin gel solution comprises the following steps: A. Acidification modification of polyvinyl alcohol: (1) Polyvinyl alcohol is dissolved in dimethyl sulfoxide; (2) adding succinic anhydride and p-toluenesulfonic acid to the solution obtained in step (1) to react; (3) adding ether to the reaction solution obtained in step (2), filtering and washing to obtain a precipitate, dialyzing the precipitate with water to remove impurities, and drying to obtain acid-modified polyvinyl alcohol; B. Cyclodextrin grafting of polyvinyl alcohol (1) Acid-modified polyvinyl alcohol is dissolved in water; (2) adding aminocyclodextrin, N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and 1-hydroxybenzotriazole hydrate to the solution obtained in step (1), stirring for a certain period of time, reacting, and cooling to terminate the reaction; (3) adding ether to the solution obtained in step (2) to wash out the precipitate, and dialyzing the precipitate to remove impurities to obtain polyvinyl alcohol grafted cyclodextrin; The preparation method of the polyvinyl alcohol-bile acid gel solution comprises the following steps: A. Acidification modification of polyvinyl alcohol (1) Polyvinyl alcohol is dissolved in dimethyl sulfoxide; (2) adding succinic anhydride and p-toluenesulfonic acid to the solution obtained in step (1) to react; (3) adding ether to the reaction solution obtained in step (2), filtering and washing to obtain a precipitate, dialyzing the precipitate with water to remove impurities, and drying to obtain acid-modified polyvinyl alcohol; B. Bile acid grafting of polyvinyl alcohol (1) Acid-modified polyvinyl alcohol is dissolved in dimethylformamide; (2) adding aminocholic acid, N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and 1-hydroxybenzotriazole hydrate to the solution obtained in step (1), stirring for a certain period of time, reacting, and cooling to terminate the reaction; (3) Ether is added to the solution obtained in step (2) to wash out the precipitate, and the precipitate is dialyzed to remove impurities to obtain polyvinyl alcohol-grafted bile acid.

8. The method for cultivating and collecting bezoars by using a gel system according to claim 7, characterized in that: The poly (N-isopropylacrylamide)-acrylamide gel solution is prepared by a one-pot method and satisfies at least any one of the following conditions: The acrylamide in the reaction raw material of step A can be replaced by one or more of poloxamer and polylactic acid-glycolic acid-ethylene glycol; Or, the carboxyl donor in the reaction raw material of step A is one or more of acrylic acid, propylene sulfonic acid, and itaconic acid; Or, the chloride salt in the reaction raw material of step A is used as the metal ion donor, and the chloride salt that can be used is one or more of aluminum chloride, ferric chloride, and calcium chloride; Or, the polyphenol group donor in the reaction raw material of step A is one or more of polydopamine, tannic acid, and anthocyanin; Alternatively, the polydopamine is prepared by the following method: using dopamine hydrochloride, a self-polymerization reaction is carried out at a pH value of 10-12, and when the solution changes from transparent to black, the reaction is considered to be complete, and polydopamine is obtained; Or preferably, in the polydopamine preparation method, the pH value is 11-12 Or most preferably, in the polydopamine preparation method, the pH value is 11; Or preferably, in the polydopamine preparation method, the self-polymerization reaction time is 20-60 min; preferably, the self-polymerization reaction time is 30-60 min; most preferably, the self-polymerization reaction time is 30 min; Or the reaction raw material ratio relationship described in step A: The reaction raw materials in step A are added with 10-80 g of acrylamide or one or more of poloxamer, polylactic acid-glycolic acid-ethylene glycol, 1-50 g of carboxyl donor, 90-450 mg of chloride salt, and 1-120 g of polyphenol group per 500 g of N-isopropylacrylamide; Or preferably, the reaction raw materials in step A are added with 20-60g acrylamide, 10-30g poloxamer or 20-40g polylactic acid-glycolic acid-ethylene glycol per 500g N-isopropylacrylamide; the carboxyl donor is 1-30g acrylic acid, 1-20g propylene sulfonic acid or 1-20g itaconic acid; the chloride salt is 90-270mg aluminum chloride, 90-270mg ferric chloride or 90-270mg calcium chloride; the polyphenol group is 1-50g polydopamine, 5-30g tannic acid or 1-30g anthocyanin; Or most preferably, in step A, 30 g of acrylamide is added to the reaction raw materials per 500 g of N-isopropylacrylamide; 20 g of acrylic acid is used as the carboxyl donor; 180 mg of aluminum chloride is used as the chloride salt; and 50 g of polydopamine, 5-30 g of tannic acid, or 1-30 g of anthocyanin is used as the polyphenol group; In step B, the amount of the initiator ammonium persulfate and the accelerator tetramethylethylenediamine is calculated based on 1-20g of initiator ammonium persulfate and 1-20g of accelerator tetramethylethylenediamine per 500g of N-isopropylacrylamide; Or preferably, the amount of the initiator ammonium persulfate and the accelerator tetramethylethylenediamine in step B is calculated based on 15g of initiator ammonium persulfate and 10g of accelerator tetramethylethylenediamine per 500g of N-isopropylacrylamide; Alternatively, the aeration and stirring for a certain period of time in step B refers to stirring for 0.5-2 hours; preferably, the stirring time in step B is 1 hour; Alternatively, the reaction conditions of the reaction in step B are: reaction temperature 40-80°C, reaction time 4-12h; preferably, the reaction conditions of step B are: reaction temperature 60°C, reaction time 6h; Or, the dialysis in step C is water dialysis, and the dialysis time is 5-7 days; Or preferably, the dialysis time in step C is 7 days; Alternatively, the solution obtained in step C is dried to obtain poly (N-isopropylacrylamide)-acrylamide gel; Or preferably, the drying in step C is carried out by low-temperature oven drying or freeze drying.

9. The method for cultivating and collecting bezoars by using a gel system according to claim 7, characterized in that: The preparation method of the polyvinyl alcohol-cyclodextrin gel solution satisfies at least any one of the following: Wherein, in the acidification modification of polyvinyl alcohol in step A: The polyvinyl alcohol and dimethyl sulfoxide in step (1) are mixed according to the following ratio: 20-50 ml of dimethyl sulfoxide is added for every 2-6 g of polyvinyl alcohol; Preferably, the polyvinyl alcohol and dimethyl sulfoxide in step (1) are mixed according to the following ratio: 50 ml of dimethyl sulfoxide is added for every 4 g of polyvinyl alcohol; The amount of succinic anhydride and p-toluenesulfonic acid added in step (2) is 200-1000 mg succinic anhydride and 1-50 mg p-toluenesulfonic acid per 2-6 g polyvinyl alcohol; Preferably, the amount of succinic anhydride and p-toluenesulfonic acid added in step (2) is 400-500 mg succinic anhydride and 5-25 mg p-toluenesulfonic acid per 4 g polyvinyl alcohol; The temperature of the dimethyl sulfoxide (DMSO) in step (1) is 60-90°C, that is, polyvinyl alcohol is dissolved in dimethyl sulfoxide at 60-90°C; preferably, the temperature of the dimethyl sulfoxide (DMSO) in step (1) is 80°C; The reaction conditions of step (2) are: reaction temperature 40-80°C, reaction time 36-72h; preferably, the reaction conditions of step (2) are reaction temperature 50°C, reaction time 48h; The dialysis conditions in step (3) are: dialysis with water for 5-7 days; preferably, the dialysis time is 5 days; The drying in step (3) is carried out by low temperature oven drying or freeze drying; In the step B of cyclodextrin grafting of polyvinyl alcohol: The water in step (1) is ultrapure water; The temperature of the water in step (1) is 70-90° C., that is, the acidified modified polyvinyl alcohol is dissolved in water at 70-90° C.; preferably, the temperature of the water in step (1) is 80° C.; The stirring for a certain time in step (2) refers to stirring for 0.5-2 hours; preferably, the stirring for a certain time in step (2) refers to stirring for 1 hour; The reaction conditions of the reaction in step (2) are: reaction temperature 40-80°C, reaction time 8-36h; preferably, the reaction conditions of the reaction in step (2) are: reaction temperature 70°C, reaction time 24h; The step (2) of cooling the reaction to terminate the reaction is to move the reaction solution into an ice water bath to terminate the reaction; The dialysis in step (3) is water dialysis, and the dialysis time is 5-7 days; preferably, the dialysis time in step (3) is 7 days; The preparation method of the polyvinyl alcohol-bile acid gel solution satisfies at least any one of the following conditions: In the bile acid grafting of polyvinyl alcohol in step B: The temperature of the dimethylformamide in step (1) is 70-90° C., that is, the acidified modified polyvinyl alcohol is dissolved in dimethylformamide at 70-90° C.; preferably, the temperature of the dimethylformamide in step (1) is 80° C.; The stirring time in step (2) refers to stirring for 0.5-2 hours; preferably, the stirring time in step (2) is 1 hour; The reaction conditions of the reaction in step (2) are: reaction temperature 40-80°C, reaction time 8-36h; preferably, the reaction conditions of the reaction in step (2) are: reaction temperature 70°C, reaction time 24h; The step (2) of cooling the reaction to terminate the reaction is to move the reaction solution into an ice water bath to terminate the reaction; The dialysis in step (3) is water dialysis, and the dialysis time is 5-7 days; preferably, the dialysis time in step (3) is 7 days.

10. The method for cultivating and collecting bezoars using a gel system according to claim 1 or 2, characterized in that: The dosage ratio of the main coordination gel solution, polyvinyl alcohol-cyclodextrin gel solution, poly (N-isopropylacrylamide)-acrylamide gel solution, culture medium, and bacterial solution is calculated based on the dosage of the solid content: According to the ratio of 0.10-0.25g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.15-0.30g of main coordination gel, 1-50ml of culture medium and 1-10ml of bacterial liquid to every 1.5g of poly (N-isopropylacrylamide)-acrylamide gel; the main coordination gel is one or more of poly (vinyl alcohol)-bile acid, polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel; Or, according to every 1.5g of poly (N-isopropylacrylamide)-acrylamide gel, add 0.10-0.25g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.15-0.30g of main coordination gel, 10-40ml of culture medium, and 2-8ml of bacterial liquid; the main coordination gel is one or more of poly (vinyl alcohol)-bile acid, polyethylene glycol-bile acid gel, poly (vinyl alcohol)-deoxycholic acid gel, poly (vinyl alcohol)-taurocholic acid gel or poly (vinyl alcohol)-glycine gel; Or preferably, according to every 1.50g of poly (N-isopropylacrylamide)-acrylamide gel, add 0.15-0.25g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.20-0.30g of host coordination gel, 1-50ml of culture medium, and 1-10ml of bacterial solution; Most preferably, for every 1.50 g of poly (N-isopropylacrylamide)-acrylamide gel, 0.15-0.25 g of poly (vinyl alcohol)-cyclodextrin guest gel, 0.20-0.30 g of host coordination gel, 10-40 ml of culture medium, and 2-8 ml of bacterial solution are added; Furthermore, when the gel system is used, the gel is first prepared into a gel solution before use, and the concentration conditions of each raw material at least meet any one of the following conditions: The concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.1-0.5 g / g; Or preferably, the concentration of the poly (N-isopropylacrylamide)-acrylamide gel solution is 0.3 g / g; or the concentration of the PVA-CA guest gel solution is 0.05-0.15 g / g; Or preferably, the concentration of the PVA-CA guest gel solution is 0.1 g / g; Or the concentration of the main coordination gel solution is 0.05-0.15 g / g; Or preferably, the concentration of the host coordination gel solution is 0.1 g / g; Or most preferably, the main coordination gel solution adopts polyethylene glycol-bile acid gel solution; Or the bacterial content in the bacterial solution is greater than 10 9 / ml; Or preferably, the bacterial content in the bacterial solution is 10 9 -10 10 / ml.

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