A production system for in vitro cultivation of bezoar

Abstract

The present invention discloses a production system for in vitro cultivation of bezoar, including a strain fermentation tank, a bile supply tank, a feeding tank and a fermentation tank; the output ends of the strain fermentation tank, the bile supply tank and the feeding tank are respectively connected to the fermentation tank, the fermentation tank includes a tank body and an elastic bionic liner, the tank body is provided with a spindle-shaped bionic liner, and the tank body and the bionic liner are sealed with a chamber, and the sealed chamber is connected to a vacuum pump; the upper and lower sides of the tank body are respectively provided with a feed inlet and a discharge port corresponding to the bionic liner. The present invention realizes continuous feeding of the fermentation tank through a strain fermentation tank, a bile supply tank and a feeding tank, simulates the forming mechanism of bezoar inside the cattle body, realizes accurate control of the configuration of each raw material, and promotes the efficiency and quality of in vitro cultivation of bezoar. Secondly, the present invention simulates the gallbladder in the cattle body based on the bionic liner, realizes the regular contraction control of the bionic liner by controlling the vacuum degree of the sealed chamber, simulates the generation environment of natural bezoar to the greatest extent, and is conducive to the generation of in vitro cultivated bezoar with the same efficacy as natural bezoar.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro bezoar production, and in particular relates to a production system for in vitro cultivation of bezoar. 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. In 1972, the State Food and Drug Administration approved three alternatives to natural bezoar, namely cultivated bezoar, in vitro cultivated bezoar and artificial bezoar. Among them, cultivated bezoar is equivalent to natural bezoar, but it requires surgical implantation of bezoar beds and raising 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, and it 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 bezoar production process uses cattle bile as raw material, after bacterial fermentation, a certain amount of calcium hydroxide, bilirubin, bile acid and other substances are added, and according to the formation mechanism of natural bezoar, biochemistry, physical chemistry, fluid mechanics and other technical means are used to cultivate it in a simulated gallbladder, and it is produced by 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 gallbladder is wrapped in the body of the cattle, and the environment it is in changes with the changes in the physiological activities of the cattle. In order to make the in vitro cultivated bezoar closer to the 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.

[0004] However, the fermentation tanks commonly used in industrial fermentation are stainless steel fermentation tanks, which are completely different from the microenvironment of the cow's body. The fermentation process of existing industrial equipment is continuous and single, with relatively few control variables and links. It cannot accurately simulate the microenvironment in the cow's gallbladder, and cannot better form high-quality bezoar with the same efficacy as natural bezoar. For example, Chinese patent CN117582456A discloses a cultivation tank and cultivation system, which includes a tank body, a tank cover and a membrane filtration stirring mechanism. The tank body includes a cavity and a membrane filtration water storage tank and a circulating water tank wrapped around the outside of the cavity; the tank cover is connected to the open end of the cavity; the membrane filtration stirring mechanism is arranged in the cavity, including a scraper agitator and a membrane filtration interlayer, and the membrane filtration interlayer is wrapped between the scraper agitator and the membrane filtration water storage tank. Chinese patent CN118477340 A discloses a crystallization forming device and method for preparing bovine gallstones by biological enzymes, comprising a crystallization tank body, a stirring assembly installed inside the crystallization tank body, a lifting assembly for driving the stirring assembly to rise and fall installed on the inner top of the crystallization tank body, a recovery assembly for collecting bezoar solution attached to its inner wall installed in the crystallization tank body, and a direction adjustment assembly for changing the position of flushing the inner wall of the crystallization tank body installed at the bottom of the stirring assembly; the recovery assembly comprises an insulating barrel with an opening at the bottom, the top of the insulating barrel is an arc surface, both sides of the insulating barrel are fixedly connected to the inner wall of the crystallization tank body by transverse blocks, a plurality of arc plates are fixedly connected to the inner top of the insulating barrel, and a condensation water barrel with an opening at the top is fixedly connected between the plurality of arc plates. Summary of the invention

[0005] The object of the present invention is to provide a production system for cultivating bezoar in vitro, aiming to solve the above-mentioned problems.

[0006] The present invention is mainly achieved through the following technical solutions:

[0007] A production system for in vitro cultivation of bezoars, comprising a strain fermentation tank, a bile supply tank, a feed tank and a fermentation tank; the output ends of the strain fermentation tank, the bile supply tank and the feed tank are respectively connected to the fermentation tank, the fermentation tank comprises a tank body and an elastic bionic liner, a spindle-shaped bionic liner is arranged inside the tank body, and a sealed cavity is formed between the tank body and the bionic liner, and the sealed cavity is connected to a vacuum pump; a feed inlet and a discharge port are respectively arranged on the upper and lower sides of the tank body corresponding to the bionic liner;

[0008] The strain fermentation tank is used to ferment and store strain seed liquid, and output it to the bionic liner inside the fermentation tank; the bile supply tank is used to store bile and output it to the bionic liner inside the fermentation tank; the feed tank is used to store feed liquid and output it to the bionic liner inside the fermentation tank.

[0009] In order to better realize the present invention, further, the sealed cavity between the tank body and the bionic liner is provided with a temperature control mechanism, a dissolved oxygen regulating mechanism and a pH control mechanism, and the interior of the bionic liner is respectively connected to the dissolved oxygen regulating mechanism and the pH control mechanism.

[0010] In order to better realize the present invention, further, the interior of the bionic liner is respectively provided with filter layers corresponding to the feed inlet and the discharge outlet; the interior of the bionic liner is provided with folds and serves as a fermentation space.

[0011] In order to better realize the present invention, it further includes a strain adaptation tank, a strain breeding tank, a bezoar formation tank and a bezoar collection tank, and the strain adaptation tank, strain breeding tank, fermentation tank, bezoar formation tank and bezoar collection tank are connected in sequence from front to back; the strain adaptation tank is respectively connected to the strain fermentation tank and the bile supply tank.

[0012] In order to better realize the present invention, further, the control parameters of the strain adaptation tank are: pH 6-9, temperature 30-40°C, dissolved oxygen 10-30%, and rotation speed 60-180RPM;

[0013] The control parameters of the bacterial breeding tank are: pH 6-8, temperature 30-40°C, dissolved oxygen 20-45%, and rotation speed 100-600RPM;

[0014] The control parameters of the fermentation tank are: pH 6.2-7.8, temperature 35-55°C, dissolved oxygen 10-20%, oscillation frequency 2-20HZ, and pressure difference between the bionic liner and the tank body of the fermentation tank 50-100KPa;

[0015] The control parameters of the bezoar forming tank are: pH 10-12, temperature 100-140° C., dissolved oxygen 15-20%, and rotation speed 300-900 RPM;

[0016] The control parameters of the bezoar collection tank are: pH 6-9, temperature 25-40° C., dissolved oxygen 20-30%, and rotation speed 1-60 RPM.

[0017] In order to better realize the present invention, further, the feeding tank stores 20wt%-40wt% bilirubin, 1wt%-10wt% bile 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.

[0018] In order to better realize the present invention, further, the strain fermentation tank, strain adaptation tank, strain breeding tank, bezoar formation tank and bezoar collection tank respectively include a tank body, a stirring mechanism, a temperature control mechanism, a dissolved oxygen regulation mechanism and a pH control mechanism; the interior of the tank body is provided with a stirring mechanism and a temperature control mechanism, and the interior of the tank body is respectively connected to the dissolved oxygen regulation mechanism and the pH control mechanism.

[0019] In order to better realize the present invention, further, the bile supply tank and the feed tank respectively include a tank body, a stirring mechanism, a temperature control mechanism, a filtering mechanism and a pH detection mechanism, the interior of the tank body is provided with a stirring mechanism, a temperature control mechanism and a pH detection mechanism, and the output end of the tank body is provided with a filtering mechanism.

[0020] In order to better realize the present invention, further, the bionic inner liner of the fermentation tank sequentially undergoes a strain adaptation period, a strain reproduction period, a fermentation period, a bezoar formation period and a bezoar collection period; and the time ratio of the strain adaptation period, the strain reproduction period, the fermentation period, the bezoar formation period and the 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 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 of the fermentation tank is 50-80KPa;

[0022] 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 fermentation tank is 40-90KPa;

[0023] 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 of the fermentation tank is adjusted to 50-100KPa;

[0024] During the bezoar formation period, 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 fermentation tank is adjusted to 20-50 KPa;

[0025] 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 fermentation tank body is 50-80KPa.

[0026] The beneficial effects of the present invention are as follows:

[0027] (1) The present invention realizes continuous feeding of the fermentation tank through the strain fermentation tank, the bile supply tank, and the feed tank, simulates the formation mechanism of bezoar inside the cattle body, realizes accurate control of the configuration of each raw material, and promotes the efficiency and quality of in vitro cultivation of bezoar. Secondly, the present invention simulates the gallbladder in the cattle body based on the bionic liner, 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 the generation of in vitro cultured bezoar with the same efficacy as natural bezoar.

[0028] (2) The present invention divides the production cycle of in vitro cultivation of bezoar into a strain adaptation period, a strain breeding period, a fermentation period, a bezoar formation period and a bezoar collection period by sequentially arranging a strain adaptation tank, a strain breeding tank, a fermentation tank, a bezoar formation tank and a bezoar collection tank, which is highly consistent with the biological change cycle in the cattle body during the natural bezoar production process, and accurately controls the environmental conditions of the strain adaptation tank, the strain breeding tank, the fermentation tank, the bezoar formation tank and the bezoar collection tank, effectively promoting the generation of in vitro bezoar with the same efficacy as natural bezoar. The present invention can accelerate the production efficiency of bezoar formation by setting the stage classification tank body, so that the production process can be carried out continuously and with high yield, greatly reducing the cost of industrial production.

[0029] (3) The present invention provides a fermentation tank to simulate a live bovine gallbladder, and then uses industrial control means to quickly regulate the strain adaptation tank, strain breeding tank, bezoar formation tank and bezoar collection tank, so as to simulate the internal environment of the bovine body during the key reaction period, accelerate the regulation of the environment during the early and forming periods, and while simulating the favorable conditions inside the bovine gallbladder, overcome the limitations of the organism and accelerate the in vitro production of high-quality bezoar.

[0030] (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.

[0031] (4) 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 movement 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 movement 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

[0032] Figure 1 It is a schematic diagram of the structure of the production system for in vitro cultivation of bezoar in Example 1;

[0033] Figure 2 This is a schematic diagram of the structure of the production system for cultivating bezoar in vitro in Example 2.

[0034] Among them: 1- strain fermentation tank, 2- bile supply tank, 3- feed tank, 4- strain adaptation tank, 5- strain breeding tank, 6- fermentation tank, 7- bezoar formation tank, 8- bezoar collection tank, 9-bionic liner. DETAILED DESCRIPTION

[0035] Embodiment 1:

[0036] A production system for in vitro cultivation of bezoars, such as Figure 1 As shown, it includes a strain fermentation tank 1, a bile supply tank 2, a feed tank 3 and a fermentation tank 6; the output ends of the strain fermentation tank 1, the bile supply tank 2 and the feed tank 3 are respectively connected to the fermentation tank 6. The strain fermentation tank 1 is used to ferment and store strain seed liquid, and output it to the bionic liner 9 inside the fermentation tank 6; the bile supply tank 2 is used to store bile and output it to the bionic liner 9 inside the fermentation tank 6; the feed tank 3 is used to store feed liquid and output it to the bionic liner 9 inside the fermentation tank 6.

[0037] The fermentation tank 6 includes a tank body and an elastic bionic liner 9, wherein a spindle-shaped bionic liner 9 is disposed inside the tank body, and a sealed cavity is formed between the tank body and the bionic liner 9, and the sealed cavity is connected to a vacuum pump; a feed port and a discharge port are disposed on the upper and lower sides of the tank body respectively corresponding to the bionic liner 9. Filter layers are disposed inside the bionic liner 9 corresponding to the feed port and the discharge port respectively; pleats are disposed inside the bionic liner 9 and serve as a fermentation space. Specifically, a temperature control mechanism, a dissolved oxygen regulating mechanism and a pH control mechanism are disposed in the sealed cavity between the tank body and the bionic liner 9, and the inside of the bionic liner 9 is respectively connected to the dissolved oxygen regulating mechanism and the pH control mechanism.

[0038] During use, as shown in Table 1, the present invention can sequentially undergo a bacterial strain adaptation period, a bacterial strain reproduction period, a fermentation period, a bezoar formation period, and a bezoar collection period in the bionic inner liner 9 of the fermentation tank 6. The specific steps are:

[0039] ① During the strain adaptation period, 1-15wt% strain seed solution, 5-30wt% fresh culture medium, and 50-100wt% bile are added to the bionic liner 9 in the fermentation tank 6, wherein the fermentation OD value of the strain seed solution is ≥1.0; the pH inside the bionic liner 9 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 9 and the tank body to 50-80KPa;

[0040] ② During the bacterial reproduction period, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 3000-5000U / L; the pH inside the bionic liner 9 is adjusted to 6-8, the temperature is adjusted to 30-40°C, the dissolved oxygen is adjusted to 20-45%, the oscillation frequency is adjusted to 1-10HZ, and the pressure difference between the bionic liner 9 and the tank body is adjusted to 40-90KPa;

[0041] ③ During the fermentation period, 20wt%-40wt% bilirubin, 1wt%-10wt% bile 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 stored in the feed tank 3 are added to the bionic liner 9 in the fermentation tank 6; the pH inside the bionic liner 9 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 9 and the tank body is adjusted to 50-100KPa;

[0042] ④ During the bezoar formation period, the pH inside the bionic liner 9 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 9 and the tank body is 20-50 KPa;

[0043] ⑤ During the bezoar collection period, the pH inside the bionic liner 9 is adjusted to 6-9, the temperature is 25-40° C., the dissolved oxygen is 20-30%, the oscillation frequency is 0-1 Hz, and the pressure difference between the bionic liner 9 and the tank body is 50-80 KPa;

[0044] ⑥ Drain 10vol%-95vol% of the fermented liquid and retain the formed bezoar in the bionic liner 9, repeat the above steps 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.

[0045] Table 1

[0046] parameter Bacteria adaptation period Bacteria reproduction period Fermentation period Bezoar formation period Bezoar Collection Period pH 6-9 6-8 6.2-7.8 10-12 6-9 Temperature(℃) 30-40℃ 30-40℃ 35-55℃ 100-140℃ 25-40℃ Dissolved oxygen (%) 10-30 20-45 10-20 15-20 20-30 Holding time (h) 0.1-2 10-14 0.5-2 1-3 3-5 Pressure(KPa) 50-80 40-90 50-100 20-50 50-80 Oscillation frequency(HZ) 1-6 1-10 2-20 5-15 0-1

[0047] In actual production and life, the cow's body can provide support for the gallbladder and ensure that the gallbladder is not harmed 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 the 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, helping the cow's body to 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 9, which are the same as the functions of the cow's body; the flexible bionic liner 9 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 place where high-quality bezoar is produced and collected.

[0048] 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 6 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 terms of 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 output 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 form small bezoar particles, and provides favorable conditions for improving the quality and output of bezoar.

[0049] Preferably, the above system is used to set up experimental groups 1 to 7 and comparative example 1, and the reaction results are characterized by the β-glucuronidase activity of the bacterial liquid during the bacterial reproduction period, the final bezoar collection rate, and the bilirubin content in the bezoar.

[0050] Experimental Group 1:

[0051] A production method for in vitro cultivation of bezoar comprises the following steps:

[0052] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 is adjusted to 6, the temperature to 30°C, the dissolved oxygen to 10%, the oscillation frequency to 1HZ, and the pressure difference between the inner tank and the tank body to 50-Kpa, and the duration is 0.1;

[0053] 2) Bacteria breeding period: adjust the pH inside the bionic liner 9 to 6, the temperature to 30° C., the dissolved oxygen to 20%, the oscillation frequency to 1 Hz, and the pressure difference between the liner and the tank to 40 KPa, and the duration is 10 hours;

[0054] 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 are added to the bionic liner 9 in the fermentation tank 6, and the pH inside the bionic liner 9 is 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 is 0.5h;

[0055] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0056] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0057] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0058] Experimental Group 2:

[0059] A production method for in vitro cultivation of bezoar comprises the following steps:

[0060] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 is adjusted to 9, the temperature to 40°C, the dissolved oxygen to 30%, the oscillation frequency to 6HZ, and the pressure difference between the inner tank and the tank body to 80KPa, and the duration is 2h;

[0061] 2) Bacteria reproduction period: adjust the pH inside the bionic liner 9 to 8, the temperature to 40° C., the dissolved oxygen to 45%, the oscillation frequency to 10 Hz, and the pressure difference between the liner and the tank to 90 KPa, and the duration is 14 hours;

[0062] 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 are added to the bionic liner 9 in the fermentation tank 6, and the pH inside the bionic liner 9 is 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 is 2h;

[0063] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0064] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0065] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0066] Experimental Group 3:

[0067] A production method for in vitro cultivation of bezoar comprises the following steps:

[0068] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 is adjusted to 7.5, the temperature to 37°C, the dissolved oxygen to 25%, the oscillation frequency to 3HZ, and the pressure difference between the inner tank and the tank body to 60KPa, and the duration is 1h;

[0069] 2) Bacteria breeding period: adjust the pH inside the bionic liner 9 to 7.0, the temperature to 37° C., the dissolved oxygen to 30%, the oscillation frequency to 5 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 12 hours;

[0070] 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 are added to the bionic liner 9 in the fermentation tank 6, and the pH inside the bionic liner 9 is 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 is 1h;

[0071] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0072] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0073] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0074] Experimental Group 4:

[0075] A production method for in vitro cultivation of bezoar comprises the following steps:

[0076] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 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;

[0077] 2) Bacteria breeding period: adjust the pH inside the bionic liner 9 to 10, the temperature to 55° C., the dissolved oxygen to 60%, the oscillation frequency to 30 Hz, and the pressure difference between the liner and the tank to 20 KPa, and the duration is 2 hours;

[0078] 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 are added to the bionic liner 9 in the fermentation tank 6, and the pH inside the bionic liner 9 is 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 is 7h;

[0079] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0080] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0081] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0082] Experimental Group 5:

[0083] A production method for in vitro cultivation of bezoar comprises the following steps:

[0084] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 is adjusted to 7.5, the temperature to 37°C, the dissolved oxygen to 25%, the oscillation frequency to 3HZ, and the pressure difference between the inner tank and the tank body to 60KPa, and the duration is 1h;

[0085] 2) Bacteria reproduction period: None;

[0086] 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 are added to the bionic liner 9 in the fermentation tank 6, and the pH inside the bionic liner 9 is 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 is 1h;

[0087] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0088] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0089] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0090] Experimental Group 6:

[0091] A production method for in vitro cultivation of bezoar comprises the following steps:

[0092] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 is adjusted to 7.5, the temperature to 37°C, the dissolved oxygen to 25%, the oscillation frequency to 3HZ, and the pressure difference between the inner tank and the tank body to 60KPa, and the duration is 1h;

[0093] 2) Bacteria breeding period: adjust the pH inside the bionic liner 9 to 7.0, the temperature to 37° C., the dissolved oxygen to 30%, the oscillation frequency to 5 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 12 hours;

[0094] 3) Fermentation period: None;

[0095] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0096] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0097] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0098] Experimental Group 7:

[0099] A production method for in vitro cultivation of bezoar comprises the following steps:

[0100] 1) Bacteria adaptation period: 5wt% bacteria seed solution, 10wt% fresh culture medium, and 85wt% bile are added to the bionic inner tank 9 in the fermentation tank 6, wherein the fermentation OD value of the bacteria seed solution is ≥1.0. The pH inside the bionic inner tank 9 is adjusted to 7.5, the temperature to 37°C, the dissolved oxygen to 25%, the oscillation frequency to 3HZ, and the pressure difference between the inner tank and the tank body to 60KPa, and the duration is 1h;

[0101] 2) Bacteria breeding period: adjust the pH inside the bionic liner 9 to 7.0, the temperature to 37° C., the dissolved oxygen to 30%, the oscillation frequency to 5 Hz, and the pressure difference between the liner and the tank to 65 KPa, and the duration is 12 hours;

[0102] 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 are added to the bionic liner 9 in the fermentation tank 6, and the pH inside the bionic liner 9 is 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 is 1h;

[0103] 4) Bezoar formation period: adjust the pH inside the bionic liner 9 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;

[0104] 5) Bezoar collection period: adjust the pH inside the bionic liner 9 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.

[0105] 6) Drain 75 vol% of the fermented liquid and keep the formed bezoar inside the bionic liner 9, repeat steps 1) to 5), iterate 8 times, and finally drain all the liquid, keep it warm and let it stand for a while before collecting the bezoar.

[0106] Comparative Example 1:

[0107] A production method for in vitro cultivation of bezoar comprises the following steps:

[0108] (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;

[0109] (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.

[0110] (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.

[0111] 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 2, 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.

[0112] 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.

[0113] Table 2

[0114] 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％

[0115] Embodiment 2:

[0116] A production system for in vitro cultivation of bezoars, such as Figure 2 As shown, it includes a strain fermentation tank 1, a bile supply tank 2, a feed tank 3, and a strain adaptation tank 4, a strain breeding tank 5, a fermentation tank 6, a bezoar formation tank 7, and a bezoar collection tank 8 arranged in sequence from front to back. The strain adaptation tank 4 is respectively connected to the strain fermentation tank 1 and the bile supply tank 2. The output ends of the strain fermentation tank 1, the bile supply tank 2, and the feed tank 3 are respectively connected to the fermentation tank 6.

[0117] The fermentation tank 6 includes a tank body and an elastic bionic liner 9, wherein a spindle-shaped bionic liner 9 is arranged inside the tank body, and a sealed cavity is formed between the tank body and the bionic liner 9, and the sealed cavity is connected to a vacuum pump; a feed inlet and a discharge port are arranged on the upper and lower sides of the tank body respectively corresponding to the bionic liner 9. A filter layer is arranged inside the bionic liner 9 corresponding to the feed inlet and the discharge port respectively; the bionic liner 9 is provided with folds inside and serves as a fermentation space. A temperature control mechanism, a dissolved oxygen control mechanism and a pH control mechanism are arranged in the sealed cavity between the tank body and the bionic liner 9, and the inside of the bionic liner 9 is connected to the dissolved oxygen control mechanism and the pH control mechanism respectively.

[0118] The strain fermentation tank 1, strain adaptation tank 4, strain breeding tank 5, bezoar formation tank 7 and bezoar collection tank 8 respectively include a tank body, a stirring mechanism, a temperature control mechanism, a dissolved oxygen regulating mechanism and a pH control mechanism; the interior of the tank body is provided with a stirring mechanism and a temperature control mechanism, and the interior of the tank body is respectively connected to the dissolved oxygen regulating mechanism and the pH control mechanism.

[0119] The bile supply tank 2 and the feed tank 3 respectively include a tank body, a stirring mechanism, a temperature control mechanism, a filtering mechanism and a pH detection mechanism. The interior of the tank body is provided with a stirring mechanism, a temperature control mechanism and a pH detection mechanism, and the output end of the tank body is provided with a filtering mechanism.

[0120] During use of the present invention, the specific steps are:

[0121] 1) In the strain adaptation tank 4, 5wt% of the strain seed solution and 10wt% of the fresh culture medium in the strain fermentation tank 1 and 85wt% of the bile in the bile supply tank 2 are added to the strain adaptation tank 4, wherein the fermentation OD value of the strain seed solution is ≥1.0; the pH inside the strain adaptation tank 4 is adjusted to 6-9, the temperature to 30-40°C, the dissolved oxygen to 10-30%, and the stirring speed to 60-180RPM;

[0122] 2) In the bacterial culture propagation tank 5, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 3000-5000 U / L; the pH inside the bacterial culture propagation tank 5 is adjusted to 6-8, the temperature is adjusted to 30-40° C., the dissolved oxygen is adjusted to 20-45%, and the stirring speed is adjusted to 100-600 RPM;

[0123] 3) In the fermenter 6, 20wt%-40wt% bilirubin, 1wt%-10wt% bile 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 stored in the feed tank 3 are added to the bionic liner 9 in the fermenter 6; the pH inside the bionic liner 9 is adjusted to 6.2-7.8, the temperature to 35-55°C, the dissolved oxygen to 10-20%, the oscillation frequency to 2-20HZ, and the pressure difference between the bionic liner 9 and the tank body to 50-100KPa;

[0124] 4) In the bezoar forming tank 7, the pH inside the bezoar forming tank 7 is adjusted to 10-12, the temperature is 100-140° C., the dissolved oxygen is 15-20%, and the stirring speed is 300-900 RPM;

[0125] 5) In the bezoar collecting tank 8, the pH inside the bezoar collecting tank 8 is adjusted to 6-9, the temperature is 25-40° C., the dissolved oxygen is 20-30%, and the stirring speed is 1-30 RPM;

[0126] 6) 10vol%-95vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bezoar collection tank 8, and the above steps are repeated until the set number of iterations is reached or the quality of the bezoar reaches the set standard; finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand. Preferably, 50vol%-90vol% of the fermented liquid is discharged under an internal pressure of 10-300KPa; finally, all the liquid is discharged under an internal pressure of 80-200KPa, and the bezoar is collected after being kept warm for 12-48h.

[0127] Preferably, the above system is used to set up experimental groups T1 to T3, and the reaction results are characterized by the β-glucuronidase activity of the bacterial liquid during the bacterial growth period, the final bezoar collection rate, and the bilirubin content in the bezoar.

[0128] Experimental group T1:

[0129] A production method for in vitro cultivation of bezoar comprises the following steps:

[0130] 1) In the strain adaptation tank 4, 5wt% of the strain seed solution and 10wt% of the fresh culture medium in the strain fermentation tank 1 and 85wt% of the bile in the bile supply tank 2 are added to the strain adaptation tank 4, wherein the fermentation OD value of the strain seed solution is ≥1.0; the pH inside the strain adaptation tank 4 is adjusted to 6, the temperature to 30°C, the dissolved oxygen to 10%, and the rotation speed to 60RPM;

[0131] 2) In the bacterial culture propagation tank 5, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 3000-5000 U / L; the pH inside the bacterial culture propagation tank 5 is adjusted to 6, the temperature is adjusted to 30° C., the dissolved oxygen is adjusted to 20%, and the rotation speed is adjusted to 100 RPM;

[0132] 3) In the fermentation tank 6, 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium metabisulfite stored in the feed tank 3 are added to the bionic liner 9 in the fermentation tank 6; the pH inside the bionic liner 9 is 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;

[0133] 4) In the bezoar forming tank 7, the pH inside the bezoar forming tank 7 is adjusted to 10, the temperature is 90° C., the dissolved oxygen is 15%, and the rotation speed is 300 RPM;

[0134] 5) In the bezoar collecting tank 8, the pH inside the bezoar collecting tank 8 is adjusted to 6, the temperature is 25° C., and the dissolved oxygen is 20% and 1 RPM;

[0135] 6) 75vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bezoar collection tank 8, and the above steps are repeated for 8 iterations; finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand. Preferably, 50vol%-90vol% of the fermented liquid is discharged under an internal pressure of 10-300KPa; finally, all the liquid is discharged under an internal pressure of 80-200KPa, and the bezoar is collected after being kept warm for 12-48h.

[0136] Experimental group T2:

[0137] A production method for in vitro cultivation of bezoar comprises the following steps:

[0138] 1) In the strain adaptation tank 4, 5wt% of the strain seed solution and 10wt% of the fresh culture medium in the strain fermentation tank 1 and 85wt% of the bile in the bile supply tank 2 are added to the strain adaptation tank 4, wherein the fermentation OD value of the strain seed solution is ≥1.0; the pH inside the strain adaptation tank 4 is adjusted to 9, the temperature to 40°C, the dissolved oxygen to 30%, and the rotation speed to 180RPM;

[0139] 2) In the bacterial culture propagation tank 5, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 3000-5000 U / L; the pH inside the bacterial culture propagation tank 5 is adjusted to 8, the temperature is adjusted to 40° C., the dissolved oxygen is adjusted to 45%, and the rotation speed is adjusted to 600 RPM;

[0140] 3) In the fermentation tank 6, 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium pyrosulfite stored in the feed tank 3 are added to the bionic liner 9 in the fermentation tank 6; the pH inside the bionic liner 9 is 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;

[0141] 4) In the bezoar forming tank 7, the pH inside the bezoar forming tank 7 is adjusted to 12, the temperature is 130° C., the dissolved oxygen is 20%, and the rotation speed is 900 RPM;

[0142] 5) In the bezoar collection tank 8, the pH inside the bezoar collection tank 8 is adjusted to 9, the temperature is 40° C., and the dissolved oxygen is 30% and 30 RPM;

[0143] 6) 75vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bezoar collection tank 8, and the above steps are repeated for 8 iterations; finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand. Preferably, 50vol%-90vol% of the fermented liquid is discharged under an internal pressure of 10-300KPa; finally, all the liquid is discharged under an internal pressure of 80-200KPa, and the bezoar is collected after being kept warm for 12-48h.

[0144] Experimental group T3:

[0145] 1) In the strain adaptation tank 4, 0.01-30wt% strain seed liquid and 0-50wt% fresh culture medium in the strain fermentation tank 1 and 20-10wt% bile in the bile supply tank 2 are added to the strain adaptation tank 4, wherein the fermentation OD value of the strain seed liquid is ≥1.0; the pH inside the strain adaptation tank 4 is adjusted to 7.5, the temperature to 37°C, the dissolved oxygen to 25%, and the rotation speed to 120RPM;

[0146] 2) In the bacterial culture propagation tank 5, the activity of β-glucuronidase in the bacterial solution is finally adjusted to 3000-5000 U / L; the pH inside the bacterial culture propagation tank 5 is adjusted to 7.0, the temperature is adjusted to 37° C., the dissolved oxygen is adjusted to 30%, and the rotation speed is adjusted to 300 RPM;

[0147] 3) In the fermentation tank 6, 30wt% bilirubin, 5wt% bile acid, 200wt% saturated calcium hydroxide, 1.5wt% zinc sulfate, 1wt% magnesium sulfate, 2wt% deoxycholic acid, and 3wt% sodium pyrosulfite stored in the feed tank 3 are added to the bionic liner 9 in the fermentation tank 6; the pH inside the bionic liner 9 is 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;

[0148] 4) In the bezoar forming tank 7, the pH inside the bezoar forming tank 7 is adjusted to 11, the temperature is 100° C., the dissolved oxygen is 17.5%, and the rotation speed is 600 RPM;

[0149] 5) In the bezoar collection tank 8, the pH inside the bezoar collection tank 8 is adjusted to 7.5, the temperature is 37° C., and the dissolved oxygen is 25% and 5 RPM;

[0150] 6) 75vol% of the fermented liquid is discharged, and the formed bezoar is retained in the bezoar collection tank 8, and the above steps are repeated for 8 iterations; finally, all the liquid is discharged, and the bezoar is collected after being kept warm and allowed to stand. Preferably, 50vol%-90vol% of the fermented liquid is discharged under an internal pressure of 10-300KPa; finally, all the liquid is discharged under an internal pressure of 80-200KPa, and the bezoar is collected after being kept warm for 12-48h.

[0151] As shown in Table 3, the in vitro cultured bezoars prepared by the experimental groups T1 to T3 of the present invention all meet the standards specified in the pharmacopoeia, wherein the bezoars of the experimental group T3 have the best bilirubin and bezoar collection rates, and are of higher quality than the bezoars of the experimental group 3. The present invention is based on the reaction environment of the bovine gallbladder simulated by the fermentation tank 6, and industrial control is adopted in other stages, which avoids the influence of antibody obstruction in the bovine body, accelerates industrial quality control, and effectively improves the reaction rate and reaction quality.

[0152] Table 3

[0153] Experimental Group β-glucuronidase activity of bacterial broth before fermentation Bilirubin content in bezoar Bezoar collection rate T1 3328U / L 36.92％ 95.98％ T2 3092U / L 38.37％ 91.22％ T3 4302U / L 43.85％ 96.57％

[0154] 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 production system for in vitro cultivation of bezoars, characterized in that: The invention comprises a strain fermentation tank (1), a bile supply tank (2), a feed tank (3) and a fermentation tank (6); the output ends of the strain fermentation tank (1), the bile supply tank and the feed tank (3) are respectively connected to the fermentation tank (6); the fermentation tank (6) comprises a tank body and an elastic bionic liner (9); a spindle-shaped bionic liner (9) is arranged inside the tank body; a sealed cavity is formed between the tank body and the bionic liner (9), and the sealed cavity is connected to a vacuum pump; a feed inlet and a discharge outlet are respectively arranged on the upper and lower sides of the tank body corresponding to the bionic liner (9); The bacterial seed fermentation tank (1) is used to ferment and store bacterial seed liquid, and output it to the bionic inner tank (9) inside the fermentation tank (6); the bile supply tank (2) is used to store bile and output it to the bionic inner tank (9) inside the fermentation tank (6); and the feed tank (3) is used to store feed liquid and output it to the bionic inner tank (9) inside the fermentation tank (6).

2. A production system for in vitro cultivation of bezoar according to claim 1, characterized in that, The sealed cavity between the tank body and the bionic liner (9) is provided with a temperature control mechanism, a dissolved oxygen control mechanism and a pH control mechanism, and the interior of the bionic liner (9) is connected to the dissolved oxygen control mechanism and the pH control mechanism respectively.

3. A production system for in vitro cultivation of bezoar according to claim 2, characterized in that, The interior of the bionic liner (9) is provided with filter layers corresponding to the feed inlet and the discharge outlet, respectively; the interior of the bionic liner (9) is provided with folds and serves as a fermentation space.

4. A production system for in vitro cultivation of bezoar according to any one of claims 1 to 3, characterized in that: It also comprises a strain adaptation tank (4), a strain breeding tank (5), a bezoar formation tank (7) and a bezoar collection tank (8), wherein the strain adaptation tank (4), the strain breeding tank (5), the fermentation tank (6), the bezoar formation tank (7) and the bezoar collection tank (8) are connected in sequence from front to back; the strain adaptation tank (4) is respectively connected to the strain fermentation tank (1) and the bile supply tank (2).

5. A production system for in vitro cultivation of bezoar according to claim 4, characterized in that, The control parameters of the strain adaptation tank (4) are: pH 6-9, temperature 30-40°C, dissolved oxygen 10-30%, and rotation speed 60-180RPM; The control parameters of the bacterial breeding tank (5) are: pH 6-8, temperature 30-40°C, dissolved oxygen 20-45%, and rotation speed 180-600RPM; The control parameters of the fermentation tank (6) are: pH 6.2-7.8, temperature 35-55°C, dissolved oxygen 10-20%, oscillation frequency 2-20 Hz, and a pressure difference of 50-100 KPa between the bionic liner (9) and the tank body of the fermentation tank (6); The control parameters of the bezoar forming tank (7) are: pH 10-12, temperature 100-140°C, dissolved oxygen 15-20%, and rotation speed 300-900RPM; The control parameters of the bezoar collection tank (8) are: pH 6-9, temperature 25-40° C., dissolved oxygen 20-30%, and rotation speed 1-30 RPM.

6. A production system for in vitro cultivation of bezoar according to claim 5, characterized in that, The feed tank (3) stores 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.

7. A production system for in vitro cultivation of bezoar according to claim 4, characterized in that, The strain fermentation tank (1), strain adaptation tank (4), strain breeding tank (5), bezoar formation tank (7) and bezoar collection tank (8) respectively comprise a tank body, a stirring mechanism, a temperature control mechanism, a dissolved oxygen control mechanism and a pH control mechanism; the interior of the tank body is provided with a stirring mechanism and a temperature control mechanism, and the interior of the tank body is respectively connected to the dissolved oxygen control mechanism and the pH control mechanism.

8. A production system for in vitro cultivation of bezoar according to claim 1, characterized in that, The bile supply tank (2) and the feed tank (3) respectively comprise a tank body, a stirring mechanism, a temperature control mechanism, a filtering mechanism and a pH detection mechanism. The interior of the tank body is provided with a stirring mechanism, a temperature control mechanism and a pH detection mechanism, and the output end of the tank body is provided with a filtering mechanism.

9. A production system for in vitro cultivation of bezoar according to any one of claims 1 to 3, characterized in that: The bionic inner liner (9) of the fermentation tank (6) sequentially undergoes a strain adaptation period, a strain reproduction period, a fermentation period, a bezoar formation period, and a bezoar collection period; and the time ratio of the strain adaptation period, the strain reproduction period, the fermentation period, the bezoar formation period, and the bezoar collection period is 0.1-2:10-14:0.5-2:1-3:3-5.

10. A production system for in vitro cultivation of bezoar according to claim 9, characterized in that: During the strain adaptation period, the pH inside the bionic liner (9) is adjusted to 6-9, the temperature is adjusted to 30-40°C, the dissolved oxygen is adjusted to 10-30%, the oscillation frequency is adjusted to 1-6 Hz, and the pressure difference between the bionic liner (9) and the tank body of the fermentation tank (6) is adjusted to 50-80 KPa; During the bacterial reproduction period, the pH inside the bionic liner (9) is adjusted to 6-8, the temperature to 30-40°C, the dissolved oxygen to 20-45%, the oscillation frequency to 1-10 Hz, and the pressure difference between the bionic liner (9) and the tank body of the fermentation tank (6) to 40-90 KPa; During the fermentation period, the pH inside the bionic liner (9) 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 2-20 Hz, and the pressure difference between the bionic liner (9) and the tank body of the fermentation tank (6) is adjusted to 50-100 KPa; During the bezoar formation period, the pH inside the bionic liner (9) is adjusted to 10-12, the temperature is adjusted to 100-140° C., the dissolved oxygen is adjusted to 15-20%, the oscillation frequency is adjusted to 5-15 Hz, and the pressure difference between the bionic liner (9) and the tank body of the fermentation tank (6) is adjusted to 20-50 KPa; During the bezoar collection period, the pH inside the bionic liner (9) is adjusted to 6-9, the temperature is adjusted to 25-40°C, the dissolved oxygen is adjusted to 20-30%, the oscillation frequency is adjusted to 0-1 Hz, and the pressure difference between the bionic liner (9) and the tank body of the fermentation tank (6) is adjusted to 50-80 KPa.

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