A production method for in vitro cultivation of bezoar

Abstract

The invention discloses a production method for in vitro cultivation of bezoar, wherein a spindle-shaped elastic bionic liner is arranged inside a fermenter, a sealed cavity is formed between the tank body of the fermenter and the bionic liner, and the bionic liner is contracted and moved by controlling the vacuum degree of the sealed cavity, so that the liquid inside the bionic liner oscillates. The whole fermentation cycle is successively divided into a strain adaptation period, a strain breeding period, a fermentation period, a bezoar formation period and a bezoar collection period. After the bezoar collection period, the liquid after 10vol%-95vol% fermentation is discharged, and the formed bezoar is retained inside the bionic liner, and the production cycle is repeated until the set number of iterations is reached or the bezoar quality reaches the set standard; finally, all liquids are discharged, and the bezoar is collected after being kept warm and left to stand. The present invention realizes the regular contraction control of the bionic liner by controlling the vacuum degree of the sealed cavity, simulates the generation environment of natural bezoar to the greatest extent, and is conducive to generating in vitro cultivated bezoar with the effect equivalent to that of natural bezoar.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro cultivation of bezoars, and in particular relates to a production method for in vitro cultivation of bezoars. Background Art

[0002] Bezoar is a precious Chinese medicinal material. The probability of natural bezoar forming in cattle gallbladder is extremely low, resulting in an extreme shortage of high-quality natural bezoar, which is difficult to meet clinical needs. The state has approved three substitutes for natural bezoar, namely cultivated bezoar, in vitro cultivated bezoar and artificial bezoar. Among them, cultivated bezoar is equivalent to natural bezoar. It requires surgery to implant a bezoar bed and raise a large number of cattle to cultivate bezoar. The operation is complicated, the cost is high and the yield is extremely low, which is difficult to meet market demand. The efficacy of artificial bezoar is very different from that of natural bezoar, which is very limited in practical application; in vitro cultivated bezoar is equivalent to 90% of the efficacy of natural bezoar, with a wide range of applications, high market acceptance, and suitable for industrial production.

[0003] The existing in vitro cultivation of bezoar production process is to use cattle bile as raw material, after bacterial fermentation, add a certain amount of calcium hydroxide, bilirubin, bile acid and other substances, according to the formation mechanism of natural bezoar, use biochemistry, physical chemistry, fluid mechanics and other technical means, cultivate in a simulated gallbladder, and produce it through industrial equipment. Bezoar is the product of cattle gallbladder under pathological conditions, and its production is bound to be accompanied by changes in cattle body temperature, pH, dissolved oxygen, hormones and some trace elements. The environment of the gallbladder in cattle changes with the changes in the physiological activities of cattle. In order to make the in vitro cultivated bezoar closer to natural bezoar in terms of various components, it is necessary to simulate the micro-reactions and micro-changes in the natural cattle gallbladder as much as possible in terms of fermentation conditions and components. However, the fermentation tank commonly used in industrial fermentation is a stainless steel fermentation tank, which is completely different from the microenvironment of the cattle itself. For example, the body temperature of cattle can change with the external environment, the endocrine system of cattle can change with the mood of cattle, and the pH and dissolved oxygen in cattle can change with the physiological health status of cattle (inflammation, fever, etc.). These changes will affect the secretion of hormones and trace substances in cattle. However, the fermentation process of existing industrial equipment is continuous and single, with relatively few control variables and links, and it cannot accurately simulate the microenvironment in the gallbladder of cattle, and cannot better form high-quality bezoar with the same efficacy as natural bezoar. Summary of the invention

[0004] The purpose of the present invention is to provide a method for in vitro cultivation of bezoar, which aims to more closely simulate the microenvironmental changes of bovine gallbladder, and to precisely control pH, temperature, dissolved oxygen, bilirubin, Ca 2+ By studying the periodic changes of bezoar, we can cultivate in vitro cultured bezoar with efficacy closer to that of natural bezoar, and improve the efficiency of in vitro production of bezoar.

[0005] The present invention is mainly achieved through the following technical solutions:

[0006] A production method for in vitro cultivation of bezoar comprises the following steps:

[0007] Step S1: a spindle-shaped elastic bionic liner is arranged inside a fermentation tank, wherein the interior of the bionic liner is provided with folds and serves as a fermentation space; a sealed cavity is formed between the tank body of the fermentation tank and the bionic liner, and the bionic liner is contracted and moved by controlling the vacuum degree of the sealed cavity, so that the liquid inside the bionic liner oscillates;

[0008] Step S2: dividing the entire fermentation cycle into a strain adaptation period, a strain reproduction period, a fermentation period, a bezoar formation period and a bezoar collection period in sequence, and the time ratio is 0-3:5-15:0-3:1-5:2-10;

[0009] Step S3: During the strain adaptation period, 1-15wt% strain seed solution, 5-30wt% fresh culture medium, and 50-100wt% bile are added to the bionic inner tank in the fermentation tank, wherein the fermentation OD value of the strain seed solution is ≥1.0; the pH inside the bionic inner tank is adjusted to 3-10, the temperature to 20-50°C, the dissolved oxygen to 5-40%, the oscillation frequency to 0-10HZ, and the pressure difference between the bionic inner tank and the tank body to 20-80KPa;

[0010] Step S4: During the bacterial reproduction period, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 500-5000 U / L; the pH inside the bionic liner is adjusted to 4-9.5, the temperature is adjusted to 20-50° C., the dissolved oxygen is adjusted to 10-50%, the oscillation frequency is adjusted to 1-20 Hz, and the pressure difference between the bionic liner and the tank body is adjusted to 30-100 KPa;

[0011] Step S5: During the fermentation period, 10wt%-50wt% bilirubin, 1wt%-20wt% bile acid, 50wt%-1000wt% saturated calcium hydroxide, 0.1wt%-5wt% zinc sulfate, 0.1wt%-5wt% magnesium sulfate, 0.1wt%-10wt% deoxycholic acid and 0.1wt%-10wt% sodium metabisulfite are added to the bionic liner in the fermentation tank; the pH inside the bionic liner is adjusted to 5-10, the temperature is adjusted to 25-60°C, the dissolved oxygen is adjusted to 5-50%, the oscillation frequency is adjusted to 1-30HZ, and the pressure difference between the bionic liner and the tank body is adjusted to 30-100KPa;

[0012] Step S6: During the bezoar formation period, the pH inside the bionic liner is adjusted to 5-14, the temperature is adjusted to 70-140° C., the dissolved oxygen is adjusted to 5-30%, the oscillation frequency is adjusted to 1-30 Hz, and the pressure difference between the bionic liner and the tank body is adjusted to 10-100 KPa;

[0013] Step S7: During the bezoar collection period, the pH inside the bionic liner is adjusted to 5-14, the temperature is adjusted to 25-50° C., the dissolved oxygen is adjusted to 5-40%, the oscillation frequency is adjusted to 0-5 Hz, and the pressure difference between the bionic liner and the tank body is adjusted to 30-100 KPa;

[0014] Step S8: draining 10 vol%-95 vol% of the fermented liquid, and retaining the formed bezoar in the bionic liner, repeating steps S3-S7 until the set number of iterations is reached or the quality of the bezoar reaches the set standard; finally, draining all the liquid, keeping the bezoar warm and collecting it.

[0015] In order to better implement the present invention, further, during the strain adaptation period, the pH inside the bionic liner is adjusted to 6-9, the temperature to 30-40°C, the dissolved oxygen to 10-30%, the oscillation frequency to 1-6HZ, and the pressure difference between the bionic liner and the tank body to 50-80KPa.

[0016] In order to better realize the present invention, further, during the bacterial reproduction period, the pH inside the bionic liner is adjusted to 6-8, the temperature to 30-40°C, the dissolved oxygen to 20-45%, the oscillation frequency to 1-10HZ, and the pressure difference between the bionic liner and the tank body to 40-90KPa.

[0017] In order to better realize the present invention, further, during the fermentation period, the pH inside the bionic liner is adjusted to 6.2-7.8, the temperature is 35-55°C, the dissolved oxygen is 10-20%, the oscillation frequency is 2-20HZ, and the pressure difference between the bionic liner and the tank body is 50-100KPa.

[0018] In order to better realize the present invention, further, during the bezoar formation period, the pH inside the bionic liner is adjusted to 10-12, the temperature to 100-140°C, the dissolved oxygen to 15-20%, the oscillation frequency to 5-15HZ, and the pressure difference between the bionic liner and the tank body to 20-50KPa.

[0019] In order to better implement the present invention, further, during the bezoar collection period, the pH inside the bionic liner is adjusted to 6-9, the temperature to 25-40°C, the dissolved oxygen to 20-30%, the oscillation frequency to 0-1HZ, and the pressure difference between the bionic liner and the tank body to 50-80KPa.

[0020] In order to better realize the present invention, further, the time ratio of the strain adaptation period, strain reproduction period, fermentation period, bezoar formation period and bezoar collection period is 0.1-2:10-14:0.5-2:1-3:3-5.

[0021] In order to better realize the present invention, further, during the bacterial reproduction period, the β-glucuronidase activity of the bacterial liquid is finally made to be 3000-5000 U / L.

[0022] In order to better realize the present invention, further, during the fermentation period, 20wt%-40wt% bilirubin, 1wt%-10wt% cholic acid, 100wt%-400wt% saturated calcium hydroxide, 0.1wt%-3wt% zinc sulfate, 0.1wt%-3wt% magnesium sulfate, 0.1wt%-8wt% deoxycholic acid and 0.1wt%-5wt% sodium metabisulfite are added.

[0023] In order to better implement the present invention, further, in step S8, a pressure of 10-300 KPa is applied to the inside of the bionic liner to discharge 50 vol%-90 vol% of the fermented liquid; finally, a pressure of 80-200 KPa is applied to the inside of the bionic liner to discharge all the liquid, the temperature of the fermentation tank is set at 40-50°C, and the bezoar is collected after keeping warm for 12-48 hours.

[0024] In actual production and life, the cow's body can provide support for the gallbladder to ensure that the gallbladder is not damaged by the outside world; secondly, the cow's body can provide the gallbladder with the temperature required for its normal physiological activities; in addition, the gallbladder renews bile from the cow's body secretion. The gallbladder is in the cow's body, soft and tough, and the gallbladder wall can perform rhythmic contraction movements according to the cow's physiological life to help the cow receive and discharge fresh bile. The tank body of the present invention mainly provides support, protection, heat preservation and rhythmic contraction functions for the bionic liner, which are the same as the functions of the cow's body; the flexible bionic liner is the main place for the yellowing reaction, and it mainly plays the functions of receiving fresh bile, culturing bacteria, enzymatic reactions, and discharging waste liquid. Its function is the same as that of the gallbladder, and it is the formation and collection place of high-quality bezoar.

[0025] Secondly, the gallbladder wall is composed of three layers: mucosa, muscular layer and outer membrane, and its inner surface is covered with mucosa and has developed folds. When the gallbladder contracts and empties, the folds are tall and branched; when the gallbladder is full, the folds are reduced and shortened; the present invention uses bionic materials to simulate the physiological structure of the gallbladder, and when it is full of fermentation liquid for fermentation, the folds are reduced and shortened, and when the fermentation is finished and part of the waste liquid is emptied, the folds are tall and branched. At the same time, during the formation of natural bezoar, the movement of bile is the vibration generated by the movement of the cattle body and the contraction of the gallbladder. This oscillation mode is sufficient and gentle, which can ensure the full mixing of the substances in the gallbladder, including bile and bezoar small particles, bezoar small particles and bezoar small particles, bezoar core and bezoar small particles, and can also play a good protective role on the formed bezoar small particles / bezoar core, so that it can be further enlarged to form larger bezoar particles. The present invention realizes the oscillation and stillness of the liquid in the fermentation tank by controlling the vacuum degree between the flexible material liner and the tank body, and effectively simulates the contraction mode of the cattle gallbladder in both form and actual effect, and well avoids the shear force caused by mechanical stirring to damage the formed bezoar small particles / bezoar core, and effectively improves the yield and quality of the produced bezoar. The present invention adopts the change of the vacuum degree between the tank body and the flexible material liner to produce a rhythmic contraction to make the internal fermentation liquid surge, and well avoids the shear force of mechanical stirring to damage the formed bezoar small particles, which provides favorable conditions for improving the quality and yield of bezoar.

[0026] In the reaction process, bile is the only source of natural bezoar bilirubin, and its formation must be catalyzed by β-glucuronidase. Therefore, in the production process of natural bezoar, the content of β-glucuronidase in bovine bile and the size of its enzyme activity directly determine the quality and yield of the formed bezoar. There are mainly two sources of bilirubin in in vitro cultured bezoar: one is the bile added during the fermentation process, and the other is the addition of commercial free bilirubin during the formation period of bezoar. Since commercial free bilirubin is expensive and the amount added is limited. The content of bilirubin in natural bile is only 0.03%, so how to effectively obtain more high-quality natural free bilirubin from bile is an important link to improve the quality and yield of in vitro cultured bezoar and reduce production costs.

[0027] The bilirubin in the bile must be converted by the β-glucuronidase produced by the strains before it can participate in the formation of bezoar. The present invention provides a continuous source of bile. Therefore, the content of β-glucuronidase produced during the growth period of the strains directly determines the quality and yield of bezoar in the subsequent fermentation process with its enzyme activity. First, in terms of the amount of enzyme, the β-glucuronidase in the fermentation broth comes from the added enzyme-producing strains, and its amount directly depends on the degree of reproduction of the strains. In the same time, the more vigorous the strains are, the more they reproduce, and the more enzymes they produce; on the contrary, the smaller the strain activity, the less it reproduces, and the less enzymes it produces. The factors that affect the degree of reproduction of the strains during in vitro fermentation are mainly the strains themselves, followed by the culture medium, temperature, pH, dissolved oxygen, rotation speed, etc. Secondly, from the perspective of enzyme activity, the enzyme is a biologically active protein with a multidimensional spatial structure, and the catalytic effect of the enzyme is highly dependent on the integrity of the spatial structure. The factors that affect the spatial structure of the enzyme are mainly pH, temperature, inhibitors, and activators.

[0028] During the bacterial reproduction period, the reaction characteristics in the bionic liner are mainly: the bacteria reproduce in large numbers, releasing precursor substances such as β-glucuronidase and mucin, which are the precursors of bezoar. At this time, the most important thing in the bionic liner is to maintain a stable pH and temperature range that is conducive to the growth and reproduction of the bacteria. Since the entire bionic liner is sealed, if there is a lack of oxygen, anaerobic respiration will occur, a large amount of acidic substances will be produced, and the pH of the fermentation liquid will be reduced, which is not conducive to the reproduction of the bacteria and the maintenance of enzyme activity; at the same time, the increase in temperature will also reduce the dissolved oxygen. Therefore, on the one hand, we increase the oxygen content in the bionic liner by providing sterile air, and at the same time increase the dissolved oxygen in the fermentation liquid by accelerating the stirring, reduce the degree of anaerobic respiration of the bacteria, and avoid the production of acidic metabolites; on the other hand, by accurately controlling the temperature of the bionic liner (the temperature changes from 35℃ to 37℃, and the dissolved oxygen changes ≤0.3ppm), the change of dissolved oxygen in the fermentation liquid is reduced, and at the same time, the pH is adjusted to 6-8 by hydrochloric acid and ammonia water to ensure that the environment in the bionic liner is conducive to the reproduction of bacteria and the release of β-glucuronidase.

[0029] During the fermentation period, the reaction characteristics in the bionic liner are mainly: β-glucuronidase converts bilirubin in bile. At this stage, the bacterial reproduction enters the decline period, and the enzyme content reaches the highest, providing the prerequisite for the formation of bezoar in the later stage. At this time, the most important thing in the bionic liner is to maintain a stable environment that is conducive to the active conformation of β-glucuronidase, so as to facilitate the full release of its catalytic activity. Through many experiments, we found that β-glucuronidase has the highest conversion efficiency at 45-55℃. The dissolved oxygen will decrease with the increase of temperature, and the decrease of dissolved oxygen may cause the change of pH. Therefore, in order to ensure the stability of the structure of β-glucuronidase, we control the pH of the enzymatic reaction stage of the fermentation period at 6.2-7.8 to ensure the smooth progress of the enzymatic reaction; in addition, since the growth of the bacterial strain enters the decline period at this stage, the demand for dissolved oxygen is reduced, and there is no need to introduce a large amount of dissolved oxygen through sterile air and rapid stirring. Therefore, strict control of dissolved oxygen and contraction frequency control at this stage is conducive to the full mixing of conjugated bilirubin and β-glucuronidase in bile, accelerating the progress of enzymatic reaction, and improving the conversion efficiency of conjugated bilirubin in bile.

[0030] The formation of both natural bezoar and in vitro cultured bezoar mainly involves the enzymatic reaction of β-glucuronidase, the reaction of bilirubin and Ca 2+ The formation of bilirubin calcium precipitation reaction, the formation of stone bile with the participation of deoxycholic acid, and the precipitation and aggregation reaction of bezoar small particles. Among them, the enzymatic reaction involving β-glucuronidase, the reaction of bilirubin and Ca 2+ The formation of the stone bile formation reaction in which calcium bilirubin precipitation reaction and deoxycholic acid etc. participate all requires the full mixing of the reactants, so the fermentation liquid should be given a certain disturbance depending on the severity of the reaction. And the sedimentation and aggregation reaction of the bezoar small particles, especially when the bezoar core is not formed in the early stage, the particle size of the bezoar small particles initially formed may be only hundreds of microns, and it is very easy to flow with the fermentation liquid in a slight disturbance. If the fermentation liquid is excessively disturbed at this time, it is not conducive to the formation of the bezoar core. And the formation speed of the bezoar core affects the decisive factor of the final bezoar collection efficiency. Therefore, bezoar collection, especially the sedimentation and aggregation reaction of the bezoar small particles needs to be carried out under static state.

[0031] During the formation of natural bezoar, the cow is in an active state during most of the day. Frequent eating causes bile emptying and secretion, physiological or pathological gallbladder contraction, etc., which causes the bile and its substances in the gallbladder to be in motion. The enzymatic reaction involving β-glucuronidase, bilirubin and Ca 2+ The stone bile formation reaction involving calcium bilirubin precipitation reaction and deoxycholic acid can proceed smoothly; when the cow lies down to rest at night, the eating activity is reduced, and the bile emptying and secretion activities are reduced. The bile and the substances in its gallbladder are in a relatively static state, which is conducive to the sedimentation and aggregation of small bezoar particles and accelerates the formation of the bezoar core.

[0032] During the in vitro culture process, the strain adaptation period, strain reproduction period, fermentation period and bezoar formation period are the enzymatic reactions involving β-glucuronidase, bilirubin and Ca 2+ The main link of the stone bile formation reaction in which calcium bilirubin precipitation reaction and deoxycholic acid etc. participate is formed. During this part, the present invention gives the fermentation liquid different degrees of disturbance according to the reaction needs, ensures the full mixing of the reactants, and ensures the output of the bezoar small particles, which is very similar to the bile environment in the gallbladder of cattle when the cattle body is active during the day. The formation of the bezoar core and the collection of the bezoar small particles mainly occur in the bezoar collection period. The present invention gives the fermentation liquid a slight disturbance or keeps it absolutely still, so that the bezoar small particles produced in the early stage of fermentation can be settled, accelerates the formation of the bezoar core, and thus improves the collection efficiency of the bezoar, which is very similar to the bile environment in the gallbladder when the cattle lie still at night.

[0033] The beneficial effects of the present invention are as follows:

[0034] (1) The present invention simulates the gallbladder in the body of cattle based on the bionic liner, and controls the regular contraction of the bionic liner by controlling the vacuum degree of the sealed cavity, simulating the generation environment of natural bezoar to the greatest extent, which is conducive to the generation of in vitro cultivated bezoar with the same efficacy as natural bezoar. Secondly, the present invention divides the production cycle of in vitro cultivated bezoar into strain adaptation period, strain reproduction period, fermentation period, bezoar formation period and bezoar collection period, which is highly consistent with the biological change cycle of cattle in the production process of natural bezoar, accurately controls the environmental conditions of the bionic liner at each stage, and effectively promotes the generation of the final bezoar.

[0035] (2) The present invention combines the bacterial species propagation period and the fermentation period, and the bile supply is sufficient, the content of β-glucuronidase produced in the bacterial species propagation period is high, the enzyme activity remains high, the bilirubin content converted in the fermentation period is high, and the bilirubin content that needs to be added in the bezoar formation period is low, and the formed bezoar is of good quality and low cost. In addition, the present invention converts bilirubin at a temperature higher than the body temperature of cattle, which improves the conversion efficiency of natural bilirubin and is conducive to the biomimetic formation of bezoar in the body, which is consistent with the state that the gallbladder temperature rises under bacterial infection and the enzymatic reaction is accelerated.

[0036] (3) The present invention uses a flexible bionic liner to produce bezoars, and drives the fermentation liquid disturbance and bile discharge by controlling the vacuum between the tank body and the flexible liner, thereby avoiding the shear force generated by mechanical stirring from destroying the formed bezoar small particles / bezoar core, and is highly consistent with the actual bile emptying, bile secretion, and pathological contraction of the bovine gallbladder in terms of structure and function. In addition, the present invention reacts to form bezoar small particles in the state of fermentation liquid disturbance, forms bezoar small particles and collects bezoars in the state of relative static state of fermentation liquid; this fermentation mode combining movement and stillness allows the bezoar small particles generated when the fermentation liquid moves to be accumulated and adsorbed to form bezoar cores when stationary, accelerating the formation of bezoar cores and improving the collection efficiency of bezoar small particles.

[0037] (4) In the process of iterative production, the present invention recycles part of the fermentation liquid and the formed bezoar small particles in the previous round of products to the next round, accelerates the formation of bezoar core, and improves the collection efficiency and yield of in vitro fermented bezoar. 1 / 3 of the total volume of the previous round of fermentation liquid is left as the mother liquor of the next round of fermentation, which contains excess Ca 2+ , which is an activator of many enzymes, has an activating effect on the enzymatic activity of the second round of β-glucuronidase, accelerates the enzymatic reaction, and improves the conversion efficiency of bilirubin; secondly, a certain volume of bezoar core has been formed in the product of the previous round of fermentation, and the bezoar small particles produced by the second round of fermentation can adhere to it faster to form larger bezoar small particles. During the entire cycle fermentation process, the bezoar small particles (bezoar core) are always present in the fermentation liquid until the yellow is taken after the fermentation is completed. This method can greatly increase the yield of bezoar cultivated in vitro.

[0038] (5) The in vivo formation process of bezoar is essentially the process in which the cattle gallbladder becomes diseased under bacterial infection and then undergoes a series of changes to become solid. Under natural conditions, the pH value in the cattle gallbladder will fluctuate due to different types of food, physical discomfort (cold, fever, various diseases, inflammation), and emotional excitement, which will affect the secretion of bile and the activity of the gallbladder; the temperature in the cattle gallbladder will fluctuate due to different exercise states, such as low body temperature at rest and high body temperature during exercise; body temperature rises when physical discomfort (cold, fever, various diseases, inflammation), and emotional excitement, which will cause the species and abundance of bacteria in the gallbladder to fluctuate, affecting the conversion of bilirubin and the production of bezoar; the dissolved oxygen in the cattle gallbladder will fluctuate due to different exercise states, such as low dissolved oxygen at rest and high dissolved oxygen during exercise (such as running) and emotional excitement, which will affect the species and abundance of bacteria in the gallbladder, and finally affect the secretion of bile and the formation of bezoar. The present invention divides the production cycle of in vitro cultivated bezoar into a strain adaptation period, a strain reproduction period, a fermentation period, a bezoar formation period and a bezoar collection period, and mainly accurately controls the bezoar production process of the simulated bovine body microenvironment from the changes in pH, dissolved oxygen and temperature, which is conducive to the generation of in vitro cultivated bezoar with the same efficacy as natural bezoar. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention is a flow chart of the production method of in vitro cultivation of bezoar. DETAILED DESCRIPTION

[0040] Embodiment 1:

[0041] A production method for in vitro cultivation of bezoars, such as Figure 1 As shown, the following steps are included:

[0042] 1. Ferment the seed liquid in the seed fermentation tank to an OD value ≥ 1.0;

[0043] 2. Add seed solution (1-15wt%), fresh culture medium (5-30wt%), and bile into the bionic liner in a ratio of (50-100wt%);

[0044] 3. With a 24-hour cycle, adjust the periodic changes of pH, temperature, dissolved oxygen, pressure difference between the inner liner and the tank body, oscillation frequency, etc. in the bionic liner. As shown in Tables 1 and 2, the entire fermentation cycle is divided into strain adaptation period, strain reproduction period, fermentation period, bezoar formation period and bezoar collection period, and the time ratio is 0-3: 5-15: 0-3: 1-5: 2-10. Preferably, the time ratio is 0.1-2: 10-14: 0.5-2: 1-3: 3-5.

[0045] (1) During the strain adaptation period, the pH inside the bionic liner is adjusted to 3-10, the temperature is 20-50°C, the dissolved oxygen is 5-40%, the oscillation frequency is 0-10HZ, and the pressure difference between the bionic liner and the tank is 20-80KPa. Preferably, the pH inside the bionic liner is adjusted to 6-9, the temperature is 30-40°C, the dissolved oxygen is 10-30%, the oscillation frequency is 1-6HZ, and the pressure difference between the bionic liner and the tank is 50-80KPa.

[0046] (2) During the bacterial reproduction period, the pH inside the bionic liner is adjusted to 4-9.5, the temperature is 20-50°C, the dissolved oxygen is 10-50%, the oscillation frequency is 1-20HZ, and the pressure difference between the bionic liner and the tank is 30-100KPa. Preferably, the pH inside the bionic liner is adjusted to 6-8, the temperature is 30-40°C, the dissolved oxygen is 20-45%, the oscillation frequency is 1-10HZ, and the pressure difference between the bionic liner and the tank is 40-90KPa.

[0047] The sign of the end of the bacterial reproduction period is that the β-glucuronidase activity of the bacterial liquid is 500-5000U / L, preferably 3000-5000U / L.

[0048] (3) During the fermentation period, 10wt%-50wt% bilirubin, 1wt%-20wt% cholic acid, 50wt%-1000wt% saturated calcium hydroxide, 0.1wt%-5wt% zinc sulfate, 0.1wt%-5wt% magnesium sulfate, 0.1wt%-10wt% deoxycholic acid and 0.1wt%-10wt% sodium pyrosulfite are added to the bionic inner tank in the fermentation tank. Preferably, 20wt%-40wt% bilirubin, 1wt%-10wt% cholic acid, 100wt%-400wt% saturated calcium hydroxide, 0.1wt%-3wt% zinc sulfate, 0.1wt%-3wt% magnesium sulfate, 0.1wt%-8wt% deoxycholic acid and 0.1wt%-5wt% sodium pyrosulfite are added.

[0049] During the fermentation period, the pH inside the bionic liner is adjusted to 5-10, the temperature is 25-60°C, the dissolved oxygen is 5-50%, the oscillation frequency is 1-30HZ, and the pressure difference between the bionic liner and the tank is 30-100KPa. Preferably, the pH inside the bionic liner is adjusted to 6.2-7.8, the temperature is 35-55°C, the dissolved oxygen is 10-20%, the oscillation frequency is 2-20HZ, and the pressure difference between the bionic liner and the tank is 50-100KPa.

[0050] (4) During the bezoar formation period, the pH inside the bionic liner is adjusted to 5-14, the temperature is 70-140°C, the dissolved oxygen is 5-30%, the oscillation frequency is 1-30 Hz, and the pressure difference between the bionic liner and the tank body is 10-100 KPa. Preferably, the pH inside the bionic liner is adjusted to 10-12, the temperature is 100-140°C, the dissolved oxygen is 15-20%, the oscillation frequency is 5-15 Hz, and the pressure difference between the bionic liner and the tank body is 20-50 KPa.

[0051] (5) During the bezoar collection period, the pH inside the bionic liner is adjusted to 5-14, the temperature is 25-50°C, the dissolved oxygen is 5-40%, the oscillation frequency is 0-5HZ, and the pressure difference between the bionic liner and the tank is 30-100KPa. Preferably, the pH inside the bionic liner is adjusted to 6-9, the temperature is 25-40°C, the dissolved oxygen is 20-30%, the oscillation frequency is 0-1HZ, and the pressure difference between the bionic liner and the tank is 50-80KPa.

[0052] After the bezoar formation period is over, the waste discharge port at the lower end of the fermentation tank is opened to apply a pressure of 10-300 KPa to discharge the fermentation liquid filtered by the filter membrane, and the discharge volume is 10%-95% of the total fermentation volume, preferably 50-90%.

[0053] 4. Add fresh bile and bacterial fermentation liquid to the bionic liner again to make the total volume the same as that of the first fermentation (the ratio of bacterial strain to fresh bile added is exactly the same as the first time), and carry out the second cycle of fermentation under the same conditions as the first cycle of fermentation;

[0054] 5. After multiple rounds of fermentation, apply a pressure of 80-200 KPa, discharge all waste liquid, set the temperature of the bionic liner at 40-50°C, maintain for 12-48 hours, open the fermentation tank, collect the produced brown-red product, and obtain in vitro cultured bezoar.

[0055] 6. The quality of the obtained in vitro cultured bezoar products is inspected according to the standards of in vitro cultured bezoar in the 2020 edition of the Chinese Pharmacopoeia, and the quality control standard is higher than that of the Pharmacopoeia. LC-MS can be used to accurately detect the content of bilirubin, bile acid and other components.

[0056] 7. Store the finished bezoar products that meet the quality inspection requirements of the 2020 edition of the "Chinese Pharmacopoeia" in a sealed and light-proof place.

[0057] Table 1

[0058] parameter Bacteria adaptation period Bacteria reproduction period Fermentation period Bezoar formation period Bezoar Collection Period pH 3-10 4-9.5 5-10 5-14 5-14 Temperature(℃) 20-50℃ 20-50℃ 25-60℃ 70-140℃ 25-50℃ Dissolved oxygen (%) 5-40 10-50 5-50 5-30 5-40 Holding time (h) 0-3 5-15 0-3 1-5 2-10 Pressure(KPa) 20-80 30-100 30-100 10-100 30-100 Oscillation frequency(HZ) 0-10 1-20 1-30 1-30 0-5

[0059] Table 2

[0060]

[0061]

[0062] Embodiment 2:

[0063] A production method for cultivating bezoar in vitro, using the above method to set experimental groups 1 to 7 and comparative example 1, and characterizing the reaction results from the bezoar β-glucuronidase activity of the bacterial liquid during the bacterial reproduction period, the final bezoar collection rate, and the bilirubin content in the bezoar.

[0064] Experimental Group 1:

[0065] A production method for in vitro cultivation of bezoar comprises the following steps:

[0066] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 6, the temperature is 30°C, the dissolved oxygen is 10%, the oscillation frequency is 1HZ, and the pressure difference between the inner tank and the tank body is 50-Kpa, and the duration is 0.1;

[0067] 2) Bacteria reproduction period: adjust the pH inside the bionic liner to 6, the temperature to 30°C, the dissolved oxygen to 20%, the oscillation frequency to 1HZ, and the pressure difference between the liner and the tank to 40KPa, and the duration is 10h;

[0068] 3) Fermentation period: 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium metabisulfite were added to the bionic liner in the fermentation tank, and the pH inside the bionic liner was adjusted to 6.2, the temperature to 35°C, the dissolved oxygen to 10%, the oscillation frequency to 2HZ, and the pressure difference between the liner and the tank body to 50KPa, and the duration was 0.5h;

[0069] 4) Bezoar formation period: adjust the pH inside the bionic liner to 10, the temperature to 90° C., the dissolved oxygen to 15%, the oscillation frequency to 5 Hz, and the pressure difference between the liner and the tank to 20 KPa, and the duration is 1 hour;

[0070] 5) Bezoar collection period: adjust the pH inside the bionic liner to 6, the temperature to 25° C., the dissolved oxygen to 20%, the oscillation frequency to 0 Hz, and the pressure difference between the liner and the tank to 50 KPa, and the duration is 8 hours.

[0071] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0072] Experimental Group 2:

[0073] A production method for in vitro cultivation of bezoar comprises the following steps:

[0074] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 9, the temperature is 40°C, the dissolved oxygen is 30%, the oscillation frequency is 6HZ, and the pressure difference between the inner tank and the tank body is 80KPa, and the duration is 2h;

[0075] 2) Bacteria reproduction period: adjust the pH inside the bionic liner to 8, the temperature to 40°C, the dissolved oxygen to 45%, the oscillation frequency to 10HZ, and the pressure difference between the liner and the tank to 90KPa, and the duration is 14h;

[0076] 3) Fermentation period: 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium metabisulfite were added to the bionic liner in the fermentation tank, and the pH inside the bionic liner was adjusted to 7.8, the temperature to 55°C, the dissolved oxygen to 20%, the oscillation frequency to 20HZ, and the pressure difference between the liner and the tank body to 100KPa, and the duration was 2h;

[0077] 4) Bezoar formation period: adjust the pH inside the bionic liner to 12, the temperature to 130° C., the dissolved oxygen to 20%, the oscillation frequency to 15 Hz, and the pressure difference between the liner and the tank to 50 KPa, and the duration is 3 hours;

[0078] 5) Bezoar collection period: adjust the pH inside the bionic liner to 9, the temperature to 40° C., the dissolved oxygen to 30%, the oscillation frequency to 1 Hz, and the pressure difference between the liner and the tank to 80 KPa, and the duration is 3 hours.

[0079] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0080] Experimental Group 3:

[0081] A production method for in vitro cultivation of bezoar comprises the following steps:

[0082] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 7.5, the temperature is 37°C, the dissolved oxygen is 25%, the oscillation frequency is 3HZ, and the pressure difference between the inner tank and the tank body is 60KPa, and the duration is 1h;

[0083] 2) Bacteria reproduction period: adjust the pH inside the bionic liner to 7.0, the temperature to 37°C, the dissolved oxygen to 30%, the oscillation frequency to 5HZ, and the pressure difference between the liner and the tank to 65KPa, and the duration is 12h;

[0084] 3) Fermentation period: 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium pyrosulfite were added to the bionic liner in the fermentation tank, and the pH inside the bionic liner was adjusted to 7.3, the temperature to 50°C, the dissolved oxygen to 15%, the oscillation frequency to 10HZ, and the pressure difference between the liner and the tank body to 75KPa, and the duration was 1h;

[0085] 4) Bezoar formation period: adjust the pH inside the bionic liner to 11, the temperature to 100° C., the dissolved oxygen to 17.5%, the oscillation frequency to 8 Hz, and the pressure difference between the liner and the tank to 30 KPa, and the duration is 2 hours;

[0086] 5) Bezoar collection period: adjust the pH inside the bionic liner to 7.5, the temperature to 37° C., the dissolved oxygen to 25%, the oscillation frequency to 0.5 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 4 hours.

[0087] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0088] Experimental Group 4:

[0089] A production method for in vitro cultivation of bezoar comprises the following steps:

[0090] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 2.9, the temperature is 51°C, the dissolved oxygen is 41%, the oscillation frequency is 10HZ, and the pressure difference between the inner tank and the tank body is 10KPa, and the duration is 6h;

[0091] 2) Bacteria reproduction period: adjust the pH inside the bionic liner to 10, the temperature to 55°C, the dissolved oxygen to 60%, the oscillation frequency to 30HZ, and the pressure difference between the liner and the tank to 20KPa, and the duration is 2h;

[0092] 3) Fermentation period: 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium metabisulfite were added to the bionic liner in the fermentation tank, and the pH inside the bionic liner was adjusted to 4, the temperature to 20°C, the dissolved oxygen to 64%, the oscillation frequency to 45HZ, and the pressure difference between the liner and the tank body to 120KPa, and the duration was 7h;

[0093] 4) Bezoar formation period: adjust the pH inside the bionic liner to 2, the temperature to 40° C., the dissolved oxygen to 10%, the oscillation frequency to 50 Hz, and the pressure difference between the liner and the tank to 2 KPa, and the duration is 5 hours;

[0094] 5) Bezoar collection period: adjust the pH inside the bionic liner to 3, the temperature to 65° C., the dissolved oxygen to 70%, the oscillation frequency to 10 Hz, and the pressure difference between the liner and the tank to 110 KPa, and the duration to 0 h.

[0095] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0096] Experimental Group 5:

[0097] A production method for in vitro cultivation of bezoar comprises the following steps:

[0098] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 7.5, the temperature is 37°C, the dissolved oxygen is 25%, the oscillation frequency is 3HZ, and the pressure difference between the inner tank and the tank body is 60KPa, and the duration is 1h;

[0099] 2) Bacteria reproduction period: None;

[0100] 3) Fermentation period: 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium pyrosulfite were added to the bionic liner in the fermentation tank, and the pH inside the bionic liner was adjusted to 7.3, the temperature to 50°C, the dissolved oxygen to 15%, the oscillation frequency to 10HZ, and the pressure difference between the liner and the tank body to 75KPa, and the duration was 1h;

[0101] 4) Bezoar formation period: adjust the pH inside the bionic liner to 11, the temperature to 100° C., the dissolved oxygen to 17.5%, the oscillation frequency to 8 Hz, and the pressure difference between the liner and the tank to 30 KPa, and the duration is 2 hours;

[0102] 5) Bezoar collection period: adjust the pH inside the bionic liner to 7.5, the temperature to 37° C., the dissolved oxygen to 25%, the oscillation frequency to 0.5 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 4 hours.

[0103] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0104] Experimental Group 6:

[0105] A production method for in vitro cultivation of bezoar comprises the following steps:

[0106] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 7.5, the temperature is 37°C, the dissolved oxygen is 25%, the oscillation frequency is 3HZ, and the pressure difference between the inner tank and the tank body is 60KPa, and the duration is 1h;

[0107] 2) Bacteria reproduction period: adjust the pH inside the bionic liner to 7.0, the temperature to 37°C, the dissolved oxygen to 30%, the oscillation frequency to 5HZ, and the pressure difference between the liner and the tank to 65KPa, and the duration is 12h;

[0108] 3) Fermentation period: None;

[0109] 4) Bezoar formation period: adjust the pH inside the bionic liner to 11, the temperature to 100° C., the dissolved oxygen to 17.5%, the oscillation frequency to 8 Hz, and the pressure difference between the liner and the tank to 30 KPa, and the duration is 2 hours;

[0110] 5) Bezoar collection period: adjust the pH inside the bionic liner to 7.5, the temperature to 37° C., the dissolved oxygen to 25%, the oscillation frequency to 0.5 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 4 hours.

[0111] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0112] Experimental Group 7:

[0113] A production method for in vitro cultivation of bezoar comprises the following steps:

[0114] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium and 85wt% bile are added to the bionic inner tank, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank is adjusted to 7.5, the temperature is 37°C, the dissolved oxygen is 25%, the oscillation frequency is 3HZ, and the pressure difference between the inner tank and the tank body is 60KPa, and the duration is 1h;

[0115] 2) Bacteria reproduction period: adjust the pH inside the bionic liner to 7.0, the temperature to 37°C, the dissolved oxygen to 30%, the oscillation frequency to 5HZ, and the pressure difference between the liner and the tank to 65KPa, and the duration is 12h;

[0116] 3) Fermentation period: 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium pyrosulfite were added to the bionic liner in the fermentation tank, and the pH inside the bionic liner was adjusted to 7.3, the temperature to 50°C, the dissolved oxygen to 15%, the oscillation frequency to 10HZ, and the pressure difference between the liner and the tank body to 75KPa, and the duration was 1h;

[0117] 4) Bezoar formation period: adjust the pH inside the bionic liner to 11, the temperature to 100° C., the dissolved oxygen to 17.5%, the oscillation frequency to 8 Hz, and the pressure difference between the liner and the tank to 30 KPa, and the duration is 2 hours;

[0118] 5) Bezoar collection period: adjust the pH inside the bionic liner to 7.5, the temperature to 37° C., the dissolved oxygen to 25%, the oscillation frequency to 20 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 4 hours.

[0119] 6) 75 vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bionic liner, and steps 1) to 5) are repeated 8 times. Finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand.

[0120] Comparative Example 1:

[0121] A production method for in vitro cultivation of bezoar comprises the following steps:

[0122] (1) adding a seed liquid with an OD value ≥ 1.0 at a ratio of 10% to a common fermenter, and adjusting the internal pH of the common fermenter to 7-7.5, the temperature to 37° C., the dissolved oxygen to 30%, and the rotation speed to 150 rpm / min for 24 h;

[0123] (2) 30 wt% bilirubin, 5 wt% cholic acid, 200 wt% saturated calcium hydroxide, 1.5 wt% zinc sulfate, 1 wt% magnesium sulfate, 2 wt% deoxycholic acid, and 3 wt% sodium pyrosulfite were added to a common fermentation tank. The pH inside the common fermentation tank was adjusted to 12, the temperature was 100° C., the dissolved oxygen was 10%, the rotation speed was 100 rpm / min, and the duration was 1 hour.

[0124] (3) The prepared bile is pumped into a rotary evaporator, and an appropriate amount of zinc acetate is added. The mixture is rotary evaporated at 30-50°C and 50-100 rpm / min for 48-72 hours to obtain bezoar.

[0125] The most important index from bile in bezoar is exactly bilirubin, and it is the content of combined bilirubin to be exact, and the pharmacopoeia stipulates that the combined bilirubin in the finished product of in vitro cultivation bezoar must be ≥35%.As shown in Table 3, compared with experimental group 1-experimental group 4 and comparative example 1, it can be seen that the in vitro cultivation bezoar prepared by experimental group 1-experimental group 3 of the present invention all meets the standard stipulated in the pharmacopoeia, wherein the quality of experimental group 3 is the best, and in experimental group 1, static state is adopted to collect in bezoar collection period, and collection time is extended, and in experimental group 3, low-speed stirring state is adopted to collect in bezoar collection period, and collection time is shortened, relatively speaking, the bezoar collection rate of experimental group 1 is slightly high, but, time efficiency is lower.Compared with experimental group 7 simultaneously, it can be found that, in bezoar collection period, high-speed stirring is adopted, which is unfavorable for the aggregation and forming of bezoar, and small particles are caused to be more, and serious loss is made when high-pressure drainage, so that final bezoar collection rate is lower. Compared with comparative example 1, the present invention promotes the β-glucuronidase activity of the bacterial liquid before fermentation, and the efficacy and collection rate of the generated bezoar are higher, which has achieved significant progress and made the industrial production of in vitro cultivated bezoar controllable and highly reliable.

[0126] Comparison of experimental group 3, experimental group 5 and experimental group 6 shows that the bacterial species reproduction period is crucial to the bacterial liquid β-glucuronidase activity. After removing the bacterial species reproduction period in experimental group 5, the bacterial liquid β-glucuronidase activity is relatively low, which ultimately results in a lower efficacy and collection rate of bezoar. And experimental group 6 without a fermentation period weakens the centralized reaction of generating bezoar, which ultimately results in the worst efficacy and collection rate of bezoar. It has been proved that the bacterial species reproduction period and the fermentation period in the present invention have produced a synergistically promoted effect.

[0127] Table 3

[0128] Experimental Group β-glucuronidase activity of bacterial broth before fermentation Bilirubin content in bezoar Bezoar collection rate 1 3123U / L 35.83％ 95.13％ 2 3093U / L 36.29％ 90.42％ 3 4230U / L 40.19％ 93.32％ 4 324U / L 28.31％ 33.14％ 5 125U / L 22.42％ 39.32％ 6 4125U / L 19.12％ 27.68％ 7 3919U / L 39.42％ 23.55％ Comparative Example 1 1334U / L 35.22％ 50.15％

[0129] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for producing bezoars in vitro, characterized in that: The following steps are involved: Step S1: a spindle-shaped elastic bionic liner is arranged inside a fermentation tank, wherein the interior of the bionic liner is provided with folds and serves as a fermentation space; a sealed cavity is formed between the tank body of the fermentation tank and the bionic liner, and the bionic liner is contracted and moved by controlling the vacuum degree of the sealed cavity, so that the liquid inside the bionic liner oscillates; Step S2: dividing the entire fermentation cycle into a strain adaptation period, a strain reproduction period, a fermentation period, a bezoar formation period and a bezoar collection period in sequence, and the time ratio is 0-3:5-15:0-3:1-5:2-10; Step S3: During the strain adaptation period, 1-15wt% strain seed solution, 5-30wt% fresh culture medium, and 50-100wt% bile are added to the bionic inner tank in the fermentation tank, wherein the fermentation OD value of the strain seed solution is ≥1.0; the pH inside the bionic inner tank is adjusted to 3-10, the temperature to 20-50°C, the dissolved oxygen to 5-40%, the oscillation frequency to 0-10HZ, and the pressure difference between the bionic inner tank and the tank body to 20-80KPa; Step S4: During the bacterial reproduction period, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 500-5000 U / L; the pH inside the bionic liner is adjusted to 4-9.5, the temperature is adjusted to 20-50°C, the dissolved oxygen is adjusted to 10-50%, the oscillation frequency is adjusted to 1-20 Hz, and the pressure difference between the bionic liner and the tank body is adjusted to 30-100 KPa; Step S5: During the fermentation period, 10wt%-50wt% bilirubin, 1wt%-20wt% bile acid, 50wt%-1000wt% saturated calcium hydroxide, 0.1wt%-5wt% zinc sulfate, 0.1wt%-5wt% magnesium sulfate, 0.1wt%-10wt% deoxycholic acid and 0.1wt%-10wt% sodium pyrosulfite are added to the bionic liner in the fermentation tank; the pH inside the bionic liner is adjusted to 5-10, the temperature is adjusted to 25-60°C, the dissolved oxygen is adjusted to 5-50%, the oscillation frequency is adjusted to 1-30HZ, and the pressure difference between the bionic liner and the tank body is adjusted to 30-100KPa; Step S6: During the bezoar formation period, the pH inside the bionic liner is adjusted to 5-14, the temperature is adjusted to 70-140° C., the dissolved oxygen is adjusted to 5-30%, the oscillation frequency is adjusted to 1-30 Hz, and the pressure difference between the bionic liner and the tank body is adjusted to 10-100 KPa; Step S7: During the bezoar collection period, the pH inside the bionic liner is adjusted to 5-14, the temperature is adjusted to 25-50° C., the dissolved oxygen is adjusted to 5-40%, the oscillation frequency is adjusted to 0-5 Hz, and the pressure difference between the bionic liner and the tank body is adjusted to 30-100 KPa; Step S8: drain 10vol%-95vol% of the fermented liquid, and retain the formed bezoar inside the bionic liner, repeat steps S3-S7 until the set number of iterations is reached or the quality of the bezoar reaches the set standard; finally, drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

2. A method for producing bezoars in vitro according to claim 1, characterized in that, During the strain adaptation period, the pH inside the bionic liner is adjusted to 6-9, the temperature is 30-40°C, the dissolved oxygen is 10-30%, the oscillation frequency is 1-6HZ, and the pressure difference between the bionic liner and the tank body is 50-80KPa.

3. A method for producing bezoars in vitro according to claim 1, characterized in that, During the bacterial reproduction period, the pH inside the bionic liner is adjusted to 6-8, the temperature is 30-40°C, the dissolved oxygen is 20-45%, the oscillation frequency is 1-10HZ, and the pressure difference between the bionic liner and the tank body is 40-90KPa.

4. A method for producing bezoars in vitro according to claim 1, characterized in that, During the fermentation period, the pH inside the bionic liner is adjusted to 6.2-7.8, the temperature is adjusted to 35-55°C, the dissolved oxygen is adjusted to 10-20%, the oscillation frequency is adjusted to 2-20HZ, and the pressure difference between the bionic liner and the tank body is adjusted to 50-100KPa.

5. A method for producing bezoars in vitro according to claim 1, characterized in that, During the bezoar formation period, the pH inside the bionic liner is adjusted to 10-12, the temperature to 100-140°C, the dissolved oxygen to 15-20%, the oscillation frequency to 5-15HZ, and the pressure difference between the bionic liner and the tank body to 20-50KPa.

6. A method for producing bezoars in vitro according to claim 1, characterized in that: During the bezoar collection period, the pH inside the bionic liner is adjusted to 6-9, the temperature is 25-40°C, the dissolved oxygen is 20-30%, the oscillation frequency is 0-1HZ, and the pressure difference between the bionic liner and the tank body is 50-80KPa.

7. A method for producing bezoar in vitro according to any one of claims 1 to 6, characterized in that: The time ratio of the strain adaptation period, strain reproduction period, fermentation period, bezoar formation period and bezoar collection period is 0.1-2:10-14:0.5-2:1-3:3-5.

8. A method for producing bezoar in vitro according to any one of claims 1 to 6, characterized in that: During the bacterial reproduction period, the β-glucuronidase activity of the bacterial solution is finally made to 3000-5000U / L.

9. A method for producing bezoars in vitro according to claim 1, characterized in that: During the fermentation period, 20wt%-40wt% bilirubin, 1wt%-10wt% cholic acid, 100wt%-400wt% saturated calcium hydroxide, 0.1wt%-3wt% zinc sulfate, 0.1wt%-3wt% magnesium sulfate, 0.1wt%-8wt% deoxycholic acid and 0.1wt%-5wt% sodium metabisulfite are added.

10. The method for producing bezoar in vitro according to claim 1, characterized in that: In step S8, a pressure of 10-300 KPa is applied to the interior of the bionic liner to discharge 50 vol%-90 vol% of the fermented liquid; finally, a pressure of 80-200 KPa is applied to the interior of the bionic liner to discharge all the liquid, and the temperature of the fermentation tank is set at 40-50° C. and kept warm for 12-48 hours before collecting the bezoar.

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