Coagulation method for in-vitro enzyme-cultured calculus bovis

Abstract

The invention discloses a coagulation method for in-vitro enzyme-bred calculus bovis, and belongs to the technical field of preparation of traditional Chinese medicine raw materials. According to the method, after repeated research and testing, the key point of'core coagulation 'is implanted into'enzyme breeding', so that the yellowing time of the in-vitro enzyme-bred calculus bovis is shortened, complete transfer of components in bile is ensured, and the yield is greatly improved and can reach 1.5-2.0%. The technical problems that in the prior art, the settling speed is low, the enzyme breeding time is long, the yield is low, and effective components are not transferred thoroughly are solved. The invention aims to ensure the completeness of components contained in the in-vitro enzyme-cultured calculus bovis, so that the medicine property and the medicine effect of the calculus bovis are closer to those of natural calculus bovis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine raw material preparation, and particularly relates to a method for in-vitro enzyme breeding of bovine bezoar. BACKGROUND

[0002] Bovine bezoar (Calculus bovis) is derived from the gallbladder or bile duct of Bos taurus domesticus Gmelin and is a dry calculus in the gallbladder or bile duct of Bos taurus domesticus Gmelin.

[0003] Bovine bezoar is a traditional and precious Chinese medicine in China and has a history of more than 2,000 years. According to the first volume of the 2020 edition of the People's Republic of China Pharmacopoeia, bovine bezoar has the functions of clearing the heart, resolving phlegm, opening the orifices, cooling the liver, calming wind, and detoxifying, and is clinically used for treating heat disease with coma, stroke with phlegm, convulsions, epilepsy, throat swelling and pain, and sores.

[0004] To solve the resource problem, domestic scholars have been exploring since the 1950s and have made some achievements. For example, based on the analysis of the components of natural bovine bezoar, artificial bovine bezoar was developed in the 1960s; based on the pathological process of bovine bezoar formation in the body, bovine bezoar was developed in the 1980s (see 1. New Technology of Bovine Bezoar Cultivation, edited by Xu Yiyu, Gu Daixian, and Zhang Zhen, Agricultural Press, 1989; 2. Artificial Cultivation of Bovine Bezoar Technology, edited by Duan Wenzeng, Tao Liansheng, and Zhao Bingshou, Heilongjiang Science and Technology Press, 1990). However, both of them have limitations. Artificial bovine bezoar cannot completely replace natural bovine bezoar because of the lack of completeness of its components; bovine bezoar developed in the body is equivalent to natural bovine bezoar in terms of components and efficacy, but it needs to be surgically implanted into the cow, which directly hinders the promotion of the technology in pastoral areas, and the yield of bovine bezoar is also limited.

[0005] Therefore, since the mid-1990s, in-vitro cultivation of bovine bezoar has emerged as the mainstream research direction. After decades of exploration, corresponding achievements have been made in research and production, which has to some extent alleviated the demand pressure on the bovine bezoar market.

[0006] Bovine bezoar is a pathological product of cattle, which comes from bile and exists in bile, so it almost contains all the components of original bile, but it is a pathological change product of bile, and the content of each component is different from that of bile.

[0007] The first question about "in vitro cultivation of cowbezoar" was seen in the patent "preparation method of enzyme-cultivated cowbezoar" (CN101474209A). It is stated in the text that "in vitro cultivation of cowbezoar" widely promoted in February 2004 can replace natural cowbezoar, but in fact it is not the case. It is a mixture of seven components such as calcium bilirubin added to bovine bile, which is a formula. Due to the lack of effective components of cowbezoar, it cannot replace natural cowbezoar. Therefore, the formula was abandoned in the second half of that year, and the enzyme method was copied. The "in vitro cultivation of cowbezoar" mentioned above is a name with no actual existence.

[0008] The above-mentioned patent mentions the method of producing cowbezoar by combining "formula" with "enzyme method", which seems to be consistent with the "in vitro cultivation of cowbezoar" in the 2020 edition of "Chinese Pharmacopoeia". The "in vitro cultivation of cowbezoar" in the pharmacopoeia should be an improved formula, as it is clear that several raw materials are listed under it. By checking the patent "method for cultivating medicinal bovine gallstones in vitro by using natural bovine gallbladder" (CN1164276C) granted in September 2004, we can see the obvious defects in the current production of in vitro cultivation of cowbezoar.

[0009] Referring to the components contained in natural cowbezoar: bile pigments, bile acids, amino acids and proteins, cholesterols, other components (phospholipids, carotenoids, etc.) (Zou Qinwen, Shi Yan, Wei Feng, et al. Comparison of chemical components of cowbezoar series medicinal materials and research overview of their pharmacological effects [J]. Chinese Pharmaceutical Affairs, 2014, 28(6): 646-650). By analyzing the process of in vitro enzyme cultivation of cowbezoar patents (CN1041105A; CN1563393A; CN101474209A) and comparing the formation process of natural cowbezoar, it can be seen that the "glycoprotein" in the necessary bile mucus (see "Bile, Cowbezoar and Animal Stones" edited by Yu Changfang, China Medical Science and Technology Press, 1991, 100-101) is missing in the existing enzyme cultivation patents.

[0010] CN 1041105A discloses that the natural bovine bile is cultured in a special incubator by simulating the pathological conditions of bovine gallbladder stone formation with bovine bile or a certain proportion of bovine and pig mixed bile as raw material. The product meets the requirements of Chinese Pharmacopoeia (1985 edition), and its properties, ingredients and pharmacological effects are similar to those of natural bovine bile. The economic benefits of in-vitro culture of natural bovine bile are 24.4 times of in-vivo cultivation of natural bovine bile, and the yield is 1-3.85‰. CN1563393A discloses that according to the causes of bovine bile (bovine gallstone), Escherichia coli is cultured to prepare β-GA enzyme (Escherichia coli β-glucuronidase). The β-GA enzyme solution is added to bovine bile, and incubated at room temperature for several hours. The effective components of bovine bile catalyzed by β-GA enzyme, such as bilirubin, cholic acid, taurine, cholesterol and inorganic salts, react to produce brown (red) color and precipitate of calcium bilirubinate, etc. After drying and pressing into spherical or ellipsoidal shape of bovine bile, the product is "enzymatic natural bovine bile". In addition to bovine bile, pig and poultry bile can also be used as raw material for the preparation of bovine bile, which is very rich, but must be fresh, and the yield is 2-3‰.

[0011] As for the "core" of "an enzymatic production process for in-vitro cultivation of bovine bile" (CN 103316046 B), from the changes of components in the process of bovine bile formation, the presence of acid will displace calcium ions from calcium hydroxide, free calcium ions, and then form components such as bilirubin calcium, cholic acid calcium and calcium stearate. In principle, it is no different from the previous patent. According to the yield and bilirubin content of the seven examples listed in the patent, the yield of bilirubin in the product is 2.89-3.8‰, which is questionable. Literature (Han Shenglan, Liu Yingpeng, Qiu Lihe. Study on extraction of pig and bovine bile bilirubin by resin method [J]. China Veterinary Science and Technology, 1990, (6): 36) reported that the bilirubin content in bile of various animals is different, and the physiological content of bilirubin in pig bile is 0.4-0.6 g / L, and the physiological content of bilirubin in bovine bile is 0.13-0.3 g / L. In other words, the content of bilirubin in bile is in the order of ten thousandth (bilirubin content: 0.4-0.6‰ in pig bile and 0.13-0.3‰ in bovine bile), and the yield of bilirubin in the patent (2.89-3.8‰, thousandth) has exceeded the conventional content of bilirubin in bovine bile by an order of magnitude. It is known that the yield cannot reach 100% of the content in the raw material.

[0012] So far, in-vitro cultivation of bovine bile, even if it involves a fermentation process, is still a formula, and the type and amount of functional components in the fermented bovine bile that migrate to the bovine bile product will directly affect the clinical efficacy of the end product. SUMMARY

[0013] The present application aims to provide a method for in-vitro enzyme breeding of bovine bezoar, which implants the key point of "core precipitation" into "enzyme breeding" after repeated research and test, shortens the time for in-vitro enzyme breeding of bovine bezoar, ensures the complete transfer of components in bile, greatly improves the yield, and solves the technical problems of slow precipitation speed, long enzyme breeding time, low yield and incomplete transfer of effective components in the prior art. Moreover, the present application aims to ensure the integrity of components contained in in-vitro enzyme breeding of bovine bezoar, and makes the drug property and efficacy more similar to natural bovine bezoar.

[0014] The present application is implemented by the following technical solutions:

[0015] A method for in-vitro enzyme breeding of bovine bezoar, comprising the following steps:

[0016] S1, obtaining of bile-resistant fermentation bacteria enzyme:

[0017] After selecting bile-resistant bacteria and culturing the bacteria in a culture medium, the bacteria are separated, and the bacteria bodies are broken by ultrasonic wave to obtain bile-resistant fermentation bacteria enzyme;

[0018] Or,

[0019] After selecting bile-resistant bacteria and directly inoculating the bacteria into fresh bovine bile for co-fermentation, the fermentation bacteria are cultured to obtain bile-resistant fermentation bacteria enzyme;

[0020] S2, enzyme breeding of bovine bile:

[0021] The bile-resistant fermentation bacteria enzyme obtained in S1 is mixed with fresh bovine bile for fermentation and enzyme breeding, and then sterilized to obtain fermented bile;

[0022] S3, selecting a natural high-molecular compound and its derivative with no toxicity, strong biocompatibility and biodegradability as a precipitation agent, slowly adding the fermented bile under low-speed stirring, stopping stirring after mixing uniformly, and cold storage and standing;

[0023] S4, filtering out the precipitate, freeze-drying, and crushing to obtain artificial bovine bezoar.

[0024] As a preferred, the bile-resistant bacteria include any one of Escherichia coli, Enterococcus, Bacillus subtilis, Bacillus coagulans, Bifidobacterium and lactic acid bacteria.

[0025] As a preferred, in S2, the fresh bovine bile is pretreated, specifically: the fresh bovine bile is centrifuged at 3000-5000 rpm for 10-15 min until the bovine bile concentration is 10-30% (w / v) and the pH is 6.0-7.5.

[0026] As preferably, the added amount of the bile-fermentation-resistant bacterial enzyme is 1:50-20 of the fresh bovine bile dry weight;

[0027] The enzyme activity of the bile-fermentation-resistant bacterial enzyme is ≥50 U / g.

[0028] As preferably, the temperature of the enzyme incubation is 30-45℃, the time length is 12-48h, and the pH is 6.5-7.0.

[0029] Stirring is carried out at 100-200rpm during the enzyme incubation.

[0030] As preferably, the sterilization is carried out at 110-121℃ for 15-20min.

[0031] As preferably, in the S3, the flocculating agent is any one of ammonium-type cationic starch, chitosan, chitosan quaternary ammonium salt, basic protein and basic protein peptide.

[0032] The concentration of the flocculating agent is 0.5-5% (w / v), and the pH is 5.0-7.5.

[0033] As preferably, in the S3, the flocculating agent is mixed with the fermented bile at a weight ratio of 0.5%-1%, the stirring speed is 50-80rpm, and the time length is 15-30min.

[0034] The adding speed of the fermented bile is 10-20ml / min.

[0035] The refrigeration is carried out at 3-4℃ for 2-5h.

[0036] An artificial in-vitro enzyme-incubated bovine bezoar is prepared by the flocculation method.

[0037] Compared with the prior art, the present application has at least the following technical effects:

[0038] The present application provides a flocculation method for in-vitro enzyme-incubated bovine bezoar. After repeated research and test, the key point of "core flocculation" is implanted into "enzyme incubation", which not only shortens the time for forming the bovine bezoar in the in-vitro enzyme incubation, but also ensures the complete transfer of the components in the bile, greatly improves the yield, and can reach 1.5-2.0%. The technical problems of slow settling speed, long enzyme incubation time, low yield and incomplete transfer of effective components in the prior art are solved. The present application aims to ensure the integrity of the components contained in the in-vitro enzyme-incubated bovine bezoar, so that the drug properties and efficacy are more close to the natural bovine bezoar. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The specific implementation flowchart of Example 1 is shown. DETAILED DESCRIPTION

[0040] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application, the specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0041] The technical solution of one specific embodiment of the present application is:

[0042] To achieve the above-mentioned purpose of the present application, the inventors have designed a scheme by observing the component changes of natural bovine bezoar and fermented bovine bile. Based on the component structure characteristics, the "core" substance capable of rapidly and effectively aggregating and precipitating the effective components is selected to complete the new technology and preparation of in vitro enzyme-cultured bovine bezoar.

[0043] The specific screening method and basis are as follows:

[0044] 1. Classification and structure characteristics of components contained in natural bovine bezoar and fermented bovine bile:

[0045] Molecular structure of bilirubin:

[0046]

[0047] In normal bile, most of the bilirubin is water-soluble conjugated bilirubin, i.e. bilirubin glucuronide, which has strong affinity with phospholipids and is often combined together. After degradation by enzyme culture, the conjugated bilirubin is decomposed into bilirubin and glucuronide, and the affinity with phospholipids is reduced.

[0048] 2. Molecular structure of cholic acid in bile

[0049]

[0050] Cholic acid in bile is combined with glycine and taurine through the carboxyl group to form conjugated bile acid. After fermentation by intestinal bacteria, it can be dissociated into cholic acid, glycine and taurine.

[0051] 3. Amino acids, peptides and proteins

[0052]

[0053] 4. Phospholipids, cholesterols and other lipid-soluble components

[0054] Phospholipids have affinity with conjugated bilirubin and form a complex colloid with bile acid salt to maintain the uniform and stable state of bile. When phospholipids are subjected to enzymatic hydrolysis to generate stearic acid, the colloid stability is broken.

[0055] The above component structure, bilirubin, bile acid contains carboxyl; amino acid, peptide, protein also contains carboxyl. During the formation of bovine bezoar or after the fermentation of bovine bile, the PH is 7.2-7.8, under this environment, the carboxyl dissociates and carries negative charge; in addition to the carboxyl, the molecular structure also contains polar groups such as carbonyl, hydroxyl, amino, acyl, amide, etc.

[0056] 5. The general principle of the selection of the coagulation agent is: natural, non-toxic, strong biocompatibility, biodegradable polymer and its derivatives;

[0057] 6. The coagulation agent carries positive charge and polar group, and achieves the best coagulation effect through multiple actions of electric neutralization and complexation, adsorption and bridging;

[0058] 7. The coagulation of the corresponding coexisting components makes the bile uniform system unbalanced. Lipid-soluble components such as cholesterol are attached to phospholipid-bile salt complex colloids and dissolved in bile as 2-10 nm small colloidal particles. When bile acid is coagulated, the steady colloids are broken, and phospholipids are degraded into soft fatty acids by phospholipase produced by bacteria, and then cholesterol is coagulated together to form a core, achieving co-coagulation.

[0059] The structure of the coagulation agent is shown in the figure, which takes chitosan molecule as an example:

[0060]

[0061] Example 1:

[0062] A coagulation method of in vitro enzyme breeding bovine bezoar, comprising the following steps: the specific process is shown in Figure 1 .

[0063] S1, obtaining bile-resistant bacterial enzyme:

[0064] After selecting bile-resistant strains and culturing them in culture medium, the strains are separated, and the bacterial bodies are broken by ultrasonic wave to obtain bile-resistant bacterial enzyme for fermentation;

[0065] S2, enzyme breeding of bovine bile:

[0066] The bile-resistant bacterial enzyme obtained in S1 is mixed with fresh bovine bile for fermentation and enzyme breeding, and then sterilized to obtain fermented bile;

[0067] S3, selecting natural polymer compounds and their derivatives with no toxicity, strong biocompatibility and biodegradability as coagulation agents, slowly adding the fermented bile under low-speed stirring, stopping stirring after mixing uniformly, and cold storage and standing;

[0068] S4, filtering out the precipitate, freeze-drying, and crushing to obtain artificial bovine bezoar.

[0069] The bile-resistant strain is Escherichia coli.

[0070] The fresh bovine bile is pretreated in S2, specifically: selecting fresh bovine bile, centrifuging at 3000-5000 rpm for 10 min to obtain bovine bile with a concentration of 10% (w / v) and a pH of 6.0-7.5.

[0071] The added amount of the bile-resistant fermenting bacteria enzyme accounts for 1:50 of the dry weight of the fresh bovine bile.

[0072] The enzyme activity of the bile-resistant fermenting bacteria enzyme is ≥50 U / g.

[0073] The temperature of the enzyme incubation is 45°C, the time length is 48 h, and the pH is 6.5-7.0.

[0074] Stirring is performed at 100-200 rpm during the enzyme incubation.

[0075] The sterilization is performed at 121°C for 20 min.

[0076] In S3, the polymeric precipitant is ammonium cationic starch, specifically starch-based dimethyl diallyl ammonium chloride.

[0077] Fermented bovine bile 5L, starch-based dimethyl diallyl ammonium chloride is added in an amount of 0.5% (polymeric precipitant / bile w / w), stirring is performed at a speed of 50 rpm during the addition, after uniform mixing, cold storage is performed for 5 h, filtration is performed, freeze-drying is performed, and crushing is performed to obtain the product, with a weight of 85.9 g and a yield of 1.72%.

[0078] Example 2:

[0079] A polymeric precipitation method for in-vitro enzyme incubation of bovine bezoar includes the following steps:

[0080] S1, obtaining of bile-resistant fermenting bacteria enzyme:

[0081] A bile-resistant strain is directly inoculated into fresh bovine bile for co-fermentation, and then the fermented strain is cultured to obtain a bile-resistant fermenting bacteria enzyme;

[0082] S2, enzyme incubation of bovine bile:

[0083] The bile-resistant fermenting bacteria enzyme obtained in S1 is mixed with fresh bovine bile for fermentation and enzyme incubation, sterilization is performed, and fermented bile is obtained;

[0084] S3, a natural high-molecular compound and its derivative with no toxicity, strong biocompatibility and biodegradability are selected as a polymeric precipitant, the fermented bile is slowly added under low-speed stirring, stirring is stopped after uniform mixing, and cold storage is performed;

[0085] S4, the precipitate is filtered out, freeze-drying is performed, and crushing is performed to obtain artificial bovine bezoar.

[0086] The bile-resistant strain is Escherichia coli.

[0087] The fresh bovine bile is pretreated in S2, specifically: selecting fresh bovine bile, centrifuging at 3000-5000 rpm for 15 min to obtain bovine bile with a concentration of 30% (w / v) and a pH of 6.0-7.5.

[0088] The added amount of the bile-resistant fermentation enzyme is 1:20 of the dry weight of the fresh bovine bile.

[0089] The enzyme activity of the bile-resistant fermentation enzyme is ≥50 U / g.

[0090] The temperature of the enzyme incubation is 30°C, the time length is 12 h, and the pH is 6.5-7.0.

[0091] Stirring is performed at 200 rpm during the enzyme incubation.

[0092] The sterilization is performed at 121°C for 15 min.

[0093] In S3, the polymeric precipitant is chitosan quaternary ammonium salt, specifically hydroxypropyl trimethyl ammonium chloride chitosan.

[0094] Fermented bovine bile 2.5L, 0.3% (polymeric precipitant / bile w / w) hydroxypropyl trimethyl ammonium chloride chitosan is added, stirring is performed at a speed of 60 rpm during the addition, after uniform mixing, cold storage is performed for 2.5 h, filtration is performed, freeze-drying is performed, and crushing is performed to obtain the product, with a weight of 50.2 g and a yield of 2.01%.

[0095] Example 3:

[0096] An in-vitro enzyme incubation bovine bezoar polymeric precipitation method, comprising the following steps:

[0097] S1, obtaining of bile-resistant fermentation enzyme:

[0098] After selecting bile-resistant strains and culturing the strains in a culture medium, strain separation and ultrasonic crushing of the bacterial bodies are performed to obtain bile-resistant fermentation enzyme.

[0099] S2, bovine bile enzyme incubation:

[0100] The bile-resistant fermentation enzyme obtained in S1 is mixed with fresh bovine bile for fermentation and enzyme incubation, sterilization is performed, and fermented bile is obtained.

[0101] S3, selecting a natural high-molecular compound and its derivatives with no toxicity, strong biocompatibility, and biodegradability as a polymeric precipitant, slowly adding the fermented bile under low-speed stirring, stopping stirring after uniform mixing, and cold storage is performed.

[0102] S4, filtering out the precipitate, performing freeze-drying, and crushing to obtain artificial bovine bezoar.

[0103] The bile-resistant bacterial strain is Escherichia coli.

[0104] In the S2, the fresh bovine bile is pretreated, specifically: fresh bovine bile is selected and centrifuged at 3000-5000 rpm for 10 min to obtain bovine bile with a concentration of 10% (w / v) and a pH of 6.0-7.5.

[0105] The addition amount of the bile-resistant fermentation bacterial enzyme accounts for 1:50 of the dry weight of the fresh bovine bile.

[0106] The enzyme activity of the bile-resistant fermentation bacterial enzyme is ≥50 U / g.

[0107] The temperature of the enzyme incubation is 45°C, the time length is 48 h, and the pH is 6.5-7.0.

[0108] In the enzyme incubation process, stirring is performed at 100-200 rpm.

[0109] The sterilization is performed at 121°C for 20 min.

[0110] In the S3, the polymeric precipitant is chitosan (degree of deacetylation > 85%)

[0111] The fermentation bovine bile 4L is added with 1% acetic acid solution of chitosan at an amount of 0.8% (polymeric precipitant / bile w / w), and stirring is performed at a rotating speed of 60 rpm during the adding process; after uniform mixing, cold storage and standing for 3 h, filtration, freeze-drying, and crushing are performed to obtain the product, with a weight of 78.7 g and a yield of 1.97%.

[0112] Finally, it should be noted that: the above only describes the preferred embodiments of the present application and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for in vitro enzyme breeding of calculus bovis by aggregation, characterized in that, It comprises the following steps: S1, obtaining bile-resistant fermentation bacteria enzyme: The bile-resistant strains are selected and cultured in the culture medium, and then the strains are separated and the bacterial bodies are broken by ultrasonic wave to obtain the bile-resistant fermentation bacteria enzyme; Or, The bile-resistant strains are directly inoculated in fresh bovine bile for co-fermentation, and then the fermentation strains are cultured to obtain the bile-resistant fermentation bacteria enzyme; S2, bovine bile enzyme breeding: The bile-resistant fermentation bacteria enzyme obtained in S1 is mixed with fresh bovine bile for fermentation and enzyme breeding, and then sterilized to obtain fermented bile; S3, a natural high molecular compound and its derivative with non-toxic, strong biocompatibility and biodegradability are selected as a polymeric precipitant, and the fermented bile is slowly added under low-speed stirring, and after mixing uniformly, the stirring is stopped and the mixture is refrigerated and placed; S4, the precipitate is filtered out, freeze-dried and crushed to obtain artificial bovine bezoar.

2. The in-vitro enzyme rearing calculus bovis method according to claim 1, characterized by, The bile-resistant strains include any one of Escherichia coli, Enterococcus, Bacillus subtilis, Bacillus coagulans, Bifidobacterium and lactic acid bacteria.

3. The in-vitro enzyme rearing calculus bovis method according to claim 1, characterized by, In S2, the fresh bovine bile is pretreated, specifically: the fresh bovine bile is centrifuged at 3000-5000 rpm for 10-15 min to obtain bovine bile with a concentration of 10-30% (w / v) and a pH of 6.0-7.

5.

4. The in-vitro enzyme rearing calculus bovis method according to claim 3, characterized by, The addition amount of the bile-resistant fermentation bacteria enzyme is 1:50-20 of the dry weight of fresh bovine bile. The enzyme activity of the bile-resistant fermentation bacteria enzyme is ≥50 U / g.

5. The in-vitro enzyme rearing calculus bovis method according to claim 3, wherein, The enzyme breeding temperature is 30-45℃, the time length is 12-48h, and the pH is 6.5-7.

0. Stirring is performed at 100-200 rpm during the enzyme breeding.

6. The in-vitro enzyme rearing calculus bovis method according to claim 3, wherein, The sterilization is performed at 110-121℃ for 15-20 min.

7. The in-vitro enzyme rearing calculus bovis method according to claim 1, wherein, In S3, the polymeric precipitant is any one of ammonium cationic starch, chitosan, chitosan quaternary ammonium salt, basic protein and basic protein peptide; The concentration of the polymeric precipitant is 0.5-5% (w / v), and the pH is 5.0-7.

5.

8. The in-vitro enzyme rearing calculus bovis method according to claim 7, wherein, In S3, the polymeric precipitant and the fermented bile are mixed at a weight ratio of 0.5%-1%, the stirring speed is 50-80 rpm, and the time length is 15-30 min. The addition speed of the fermented bile is 10-20 ml / min; Refrigeration is performed at 3-4℃ for 2-5h.

9. An artificial in-vitro enzyme rearing calculus bovis, characterized by, It is prepared by the polymeric precipitation method according to any one of claims 1-8.

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