A method for preparing high-bionic bovine bezoar by using 3D printing technology and application

Abstract

The application relates to the technical field of biological pharmacy, and discloses a method for preparing high-bionic bovine bezoar by using a 3D printing technology and application. The preparation raw material of the high-bionic bovine bezoar comprises the following components: fermented bovine bile, saturated calcium hydroxide, hydroxypropyl methyl cellulose, bilirubin, cholic acid, deoxycholic acid and taurine; wherein the fermented bovine bile is obtained by fermenting bovine bile by using escherichia coli. The high-bionic bovine bezoar prepared by using the preparation raw material to perform 3D printing can be comparable to natural bovine bezoar in terms of components, effects and appearance, the main pharmaceutical components can be adjusted according to actual requirements, the production cost of the bovine bezoar can be greatly reduced, and the demand of people for the bovine bezoar can be met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to a method for preparing highly bionic bovine bezoar by using 3D printing technology and application. BACKGROUND

[0002] 3D printing technology (Three-dimensional printing, 3DP) is a technology that produces three-dimensional entities through the way of 'layered printing, layer-by-layer stacking' based on the digital model built by computer. Its digital and personalized manufacturing method has attracted much attention in the pharmaceutical field. The concept of 3D printing originated from the photographic sculpture and topographic shaping technology in the United States in the late 19th century. In 1986, the researcher Charles Hull applied the stereolithography appearance (SLA) to invent the first 3D printer. In 1996, Professor Michael J. Cima of Massachusetts Institute of Technology first reported that powder bonding 3D printing technology could be applied to the pharmaceutical field. In the same year, the first drug 3D printing company Therics was established in the United States, and the drug 3D printing industry thus started.

[0003] Bovine bezoar is a kind of precious traditional Chinese medicine, which can be used for antipyretic, detoxification, and calming. It is used for treating high fever, coma, epilepsy, infantile convulsion, and convulsion, etc. when taken internally, and for treating sore throat, aphthous ulcer, uremia, etc. when used externally. However, natural bovine bezoar is very precious, and its international price is higher than gold. Most of the bovine bezoar used is artificially cultured in vitro, which has the defects of long cycle and uneven quality. In related technologies, most of the 3D printing technologies are based on extrusion mechanism, that is, the semi-solid material is extruded through the nozzle by compressed air, syringe plunger or screw, and is deposited into the final three-dimensional object layer by layer. Usually, these semi-solid materials can be gel, paste or molten composition of drugs and carriers, but the controlled flow of semi-solid materials through the nozzle is challenging, and there are many influencing factors, so various factors need to be considered in the preparation process to achieve satisfactory printing effect.

[0004] Therefore, the present application provides a method for preparing highly bionic bovine bezoar by using 3D printing technology, which can highly restore the structure of natural bovine bezoar itself, and the effective components are closer to natural bovine bezoar. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a high-bionic calculus bovis and a method for preparing the high-bionic calculus bovis by using 3D printing technology. The high-bionic calculus bovis prepared by the method of the present application can be comparable to natural calculus bovis in composition, efficacy and appearance, and the main effective components can be adjusted according to actual needs, which can greatly reduce the production cost of calculus bovis and meet the demand of people for calculus bovis.

[0006] In a first aspect of the present application, a high-bionic calculus bovis is provided, and the preparation raw material comprises the following components:

[0007] fermented bovine bile, saturated calcium hydroxide, hydroxypropyl methyl cellulose, bilirubin, cholic acid, deoxycholic acid and taurine; wherein the fermented bovine bile is obtained by fermenting bovine bile by Escherichia coli.

[0008] The high-bionic calculus bovis according to the embodiments of the present application has at least the following beneficial effects: the calculus bovis prepared by the high-bionic calculus bovis preparation raw material of the present application has good forming effect, moderate hardness, and presents similar concentric layer structure and color as natural calculus bovis, and can be comparable to natural calculus bovis.

[0009] In some embodiments of the present application, the Escherichia coli is selected from at least one of the Escherichia coli with the accession numbers CCTCC AB 2012885, CCTCC AB 2013330 and CCTCC AB 2013342.

[0010] In some embodiments of the present application, the inoculation concentration of the Escherichia coli is 1×10 6 ~ 1×10 8 / mL. Preferably, the inoculation concentration of the Escherichia coli is 1×10 7 ~ 5×10 7 / mL.

[0011] It can be understood that the inoculation concentration refers to the number of inoculated Escherichia coli per mL of fresh bovine bile.

[0012] In some embodiments of the present application, the fermentation temperature is 37±2℃.

[0013] In some embodiments of the present application, the fermentation time is 48-96h.

[0014] In some embodiments of the present application, the preparation raw material comprises, in terms of weight fraction:

[0015]

[0016] In some embodiments of the present application, the preparation raw material further comprises at least one of zinc sulfate, ferrous sulfate and magnesium sulfate.

[0017] In some embodiments of the present application, the preparation raw materials further comprise, by weight fraction:

[0018] Zinc sulfate 0.1-1 parts;

[0019] Ferrous sulfate 0.1-1 parts;

[0020] Magnesium sulfate 0.1-1 parts.

[0021] In a second aspect of the present application, a 3D printing method of the high-bionic calculus bovis of any one of the first aspect is provided, comprising the following steps:

[0022] S1, constructing a 3D printing digital model;

[0023] S2, mixing the preparation raw materials as a bio-ink for 3D printing forming, wherein the printing method is layer-by-layer printing, the printing speed is 40-60 mm / s, and the interval time of the layer-by-layer printing is 30-90 s;

[0024] S3, cultivating the printing sample obtained in step S2, and obtaining the high-bionic calculus bovis.

[0025] According to the 3D printing method of the embodiments of the present application, at least the following beneficial effects are achieved:

[0026] (1) The high-bionic calculus bovis prepared by the 3D printing method of the present application has good stability. Bilirubin in the calculus bovis is a main effective component, which is a substance with strong heat and light sensitivity and is very unstable. The oxidation of bilirubin will be accelerated by increasing temperature and light. The high-bionic calculus bovis prepared by the 3D printing method of the present application can greatly avoid the oxidation.

[0027] (2) The high-bionic calculus bovis prepared by the method of the present application can be comparable to natural calculus bovis in composition, efficacy and appearance. The main effective components can be adjusted according to actual needs, which can greatly reduce the production cost of calculus bovis and meet the demand of people for calculus bovis.

[0028] In some embodiments of the present application, the construction of the 3D printing digital model specifically comprises: using a drawing software to establish a pre-printing model and generate a digital model, then using a slicing software to calculate, obtaining G code and importing into a printer.

[0029] In some embodiments of the present application, the 3D printing is selected from double-nozzle printing or single-nozzle printing.

[0030] In some embodiments of the present application, when the 3D printing is double-nozzle printing, the main nozzle prints the precipitate of the fermented bovine bile by 90-95% weight fraction, and the auxiliary nozzle prints the mixture of the remaining preparation raw materials.

[0031] It should be noted that "90% to 95% by weight of the fermented bovine bile" refers to 90% to 95% of the total fermented bovine bile content in the preparation raw material.

[0032] In some embodiments of the present application, when the 3D printing is double-nozzle printing, the viscosity of the main nozzle printing raw material is 3000 to 10000 centipoise, that is, the viscosity of the precipitate of the fermented bovine bile is 3000 to 10000 centipoise.

[0033] In some embodiments of the present application, when the 3D printing is double-nozzle printing, the viscosity of the main nozzle printing raw material is 3000 to 10000 centipoise, that is, the viscosity of the precipitate of the fermented bovine bile is 3000 to 10000 centipoise.

[0034] In some embodiments of the present application, when the 3D printing is double-nozzle printing, the diameter of the nozzle is 0.15 to 0.3 mm.

[0035] In some embodiments of the present application, the preparation method of 90 to 95% by weight of the precipitate of the fermented bovine bile comprises: centrifuging 90 to 95% by weight of the fermented bovine bile, and then removing the supernatant, to obtain the precipitate.

[0036] In some embodiments of the present application, when the 3D printing is single-nozzle printing, the preparation method of the bio-ink comprises:

[0037] S11, 90% to 95% by weight of the fermented bovine bile is mixed with the saturated calcium hydroxide, heated, centrifuged, and the precipitate is taken out, then the bilirubin, cholic acid, deoxycholic acid, taurine, ferrous sulfate, zinc sulfate and magnesium sulfate are mixed, and after freeze-drying, a composite bilirubin calcium is obtained;

[0038] S12, the composite bilirubin calcium, the hydroxypropyl methyl cellulose and the remaining 5 to 10% by weight of the fermented bovine bile are mixed to obtain the bio-ink.

[0039] In some embodiments of the present application, when the 3D printing is single-nozzle printing, the viscosity of the bio-ink is 2000 to 5000 centipoise.

[0040] In some embodiments of the present application, the heating temperature is 90 to 105°C.

[0041] In some embodiments of the present application, the heating time is 15 to 20 min.

[0042] In some embodiments of the present application, the incubation temperature is 25 to 38°C.

[0043] In some embodiments of the present application, the incubation time is 2 to 3 h.

[0044] In a third aspect of the present application, the high-bionic calculus or the 3D printing method of any one of the first aspect or the second aspect is used for preparing a medicine for clearing heat and detoxifying or improving heart function.

[0045] Other features and advantages of the present application will be set forth in the following description. DETAILED DESCRIPTION

[0046] The concept and the technical effects of the present application will be described below in conjunction with the embodiments so as to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative effort fall within the scope of the present application.

[0047] The words "preferably", "more preferably" and the like in the present application refer to the embodiments of the present application which can provide certain beneficial effects in certain cases. However, other embodiments can also be preferred in the same or other cases. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not usable, nor is it intended to exclude other embodiments from the scope of the present application.

[0048] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values of the range, as well as every value between the minimum and maximum values. Further, when the range refers to integers, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0049] In the description of the present application, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0050] In the description of the present application, the description of the reference terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the exemplary description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.

[0051] In the description of the present application, the saturated calcium hydroxide is saturated calcium hydroxide aqueous solution.

[0052] In the description of the present application, unless otherwise specified, "about" means that the allowable error is within ±2%.

[0053] Unless otherwise specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0054] Example 1

[0055] The present embodiment provides a high-bionic bovine bile and a preparation method thereof. The preparation raw materials include, by weight fraction:

[0056]

[0057] The present embodiment uses a 3D printing method to prepare a high-bionic bovine bile, which specifically includes the following steps.

[0058] (1) Preparation of bio-ink

[0059] The bio-ink of the present embodiment is composed of a first component and a second component, wherein the preparation method of the first component (fermented bovine bile precipitate) includes:

[0060] According to the above weight fraction, 95 parts of fermented bovine bile is collected after centrifugation to obtain a precipitate, and the viscosity thereof is about 5000 centipoise.

[0061] The preparation method of the fermented bovine bile is as follows:

[0062] Fresh bovine bile is taken, and 3% by volume of E. coli (purchased from China Typical Culture Collection Center, preservation number CCTCC AB 2012885) bacterial solution is added. The final concentration of E. coli is 2×10 7 Individuals / mL, and fermented and cultured at 37℃±2℃ and a rotation speed of 200 rpm for 72 h to obtain fermented bovine bile.

[0063] The preparation method of the second component includes:

[0064] According to the above weight fraction, saturated calcium hydroxide and hydroxypropyl methyl cellulose (HPMC) are mixed, and then the remaining 5 parts of fermented bovine bile, bilirubin, cholic acid, deoxycholic acid, zinc sulfate, taurine, ferrous sulfate and magnesium sulfate are added and uniformly mixed to obtain the product, and the viscosity thereof is about 3000 centipoise.

[0065] (2) Preparation of high-bionic natural bovine bile by 3D printing

[0066] The specific method of the high-bionic natural bovine bile of the present embodiment includes the following steps:

[0067] S1, using CAD computer aided design software to establish a diameter of 8 mm spherical, the model is saved as STL format digital model. STL format model is imported into the slice software for calculation, at the same time, G code (for controlling the nozzle path) is generated for the next step into the printer.

[0068] S2, using industrial type independent double nozzle 3D printer, independent double nozzle 3D printer can be two different materials at the same time, help to realize more complex structure of forming. Among them:

[0069] The main nozzle printing raw material is high viscosity fermented bovine bile precipitate (i.e. the first component), and the secondary nozzle printing raw material is bovine bile and its mixture (i.e. the second component), the diameter of the nozzle is about 0.2 mm; the movement of the two nozzles is started, completely synchronized, and ended at the same time. The printing method of this embodiment adopts layer-by-layer printing, and the printing speed is 50 mm / s. The printing is paused for 1 min, so that the material can fully react and form, achieving the effect of multiple layers.

[0070] S3, the printed high bionic natural bovine bezoar pill is placed in 37℃ for cultivation, and the cultivation time is 2h. After cultivation, freeze-drying is carried out, and the high bionic bovine bezoar is obtained.

[0071] The high bionic bovine bezoar prepared by this embodiment is well formed, has moderate hardness, and presents similar concentric layer structure and color as natural bovine bezoar. Further, according to the national standard, the total bilirubin and cholic acid content of the high bionic bovine bezoar is detected by spectrophotometry. The total bilirubin content of the high bionic bovine bezoar prepared in this embodiment is 55.28%, and the cholic acid content is 18.84%.

[0072] Example 2

[0073] The present embodiment provides a kind of high bionic bovine bezoar and its preparation method.The preparation raw material of the high bionic bovine bezoar includes:

[0074]

[0075] The present embodiment adopts 3D printing method to prepare high bionic bovine bezoar, which specifically includes the following contents.

[0076] (1) biological ink preparation

[0077] The biological ink of the present embodiment is composed of first component and second component, wherein the preparation method of the first component (fermented bovine bile precipitate) includes:

[0078] According to the above weight fraction, 95 parts of fermented bovine bile is collected after centrifugation to obtain the precipitate, and the viscosity is about 4000 centipoise.

[0079] The preparation method of the fermented bovine bile is as follows:

[0080] Fresh bovine bile is taken, and 3% of E. coli (purchased from China Center for Type Culture Collection, with the preservation number of CCTCC AB 2013330) bacterial solution is added, so that the final concentration of E. coli is 1.5×10 7 The fermented bovine bile is obtained by fermentation culture at 37℃±2℃ and a rotation speed of 200 rpm for 72 h.

[0081] The preparation method of the second component includes:

[0082] According to the above weight parts, saturated calcium hydroxide and hydroxypropyl methyl cellulose (HPMC) are mixed, and then the remaining 5 parts of fermented bovine bile, bilirubin, cholic acid, deoxycholic acid, zinc sulfate, taurine, ferrous sulfate and magnesium sulfate are added and uniformly mixed, to obtain the second component, and the viscosity thereof is about 3500 centipoise.

[0083] (2) 3D printing of high-biomimetic natural bovine bezoar

[0084] The specific method of the high-biomimetic natural bovine bezoar of the present embodiment includes the following steps:

[0085] S1, a CAD computer-aided design software is used to establish a spherical shape with a diameter of 8 mm, and the model is saved as a digital model in STL format. The STL format model is imported into a slicing software for calculation, and G code (used for controlling the nozzle path) is generated for the next step of being imported into a printer.

[0086] S2, an industrial independent double-nozzle 3D printer is used, wherein:

[0087] The printing raw material of the main nozzle is the fermented bovine bile precipitate with high viscosity (i.e., the first component, with a viscosity of about 4000 centipoise), and the printing raw material of the auxiliary nozzle is bovine bile and its mixture (i.e., the second component, with a viscosity of about 3500 centipoise). The diameter of the nozzles is about 0.25 mm. The movement of the two nozzles is simultaneously started, completely synchronized, and simultaneously ended. The printing method of the present embodiment adopts layer-by-layer printing, and the printing speed is 50 mm / s. After printing a layer, it is paused for 1 min, so that the substances can fully react and form, achieving a multi-layer effect.

[0088] S3, the printed high-biomimetic natural bovine bezoar pill is placed in a culture at 37℃ for 3 h, and then freeze-dried to obtain the high-biomimetic natural bovine bezoar pill.

[0089] The high bionic calculus bovis prepared by the embodiment has good shape, moderate hardness, and similar concentric layer structure and color as natural calculus bovis. Further, the total bilirubin content and cholic acid content of the high bionic calculus bovis are detected by spectrophotometry according to the national standard. The total bilirubin content of the high bionic calculus bovis prepared by the embodiment is 53.89%, and the cholic acid content is 17.23%.

[0090] Example 3

[0091] The embodiment provides a high bionic calculus bovis and a preparation method thereof, which is different from the embodiment 2 in that a single nozzle is used for preparation. The high bionic calculus bovis prepared by the embodiment contains the following preparation raw materials in terms of weight fraction:

[0092]

[0093] The embodiment adopts a 3D printing method to prepare a high bionic calculus bovis, and specifically includes the following contents.

[0094] (1) Preparation of biological ink

[0095] The biological ink of the embodiment is composed of the above preparation raw materials, and the preparation method includes the following steps.

[0096] S1, fresh bovine bile is taken, and 3% of E. coli (purchased from China Typical Culture Collection Center, preservation number CCTCC AB 2012885) bacterial solution is added, and the final concentration of E. coli is 2×10 7 / mL, and the fermentation culture is carried out at 37℃±2℃ and a rotation speed of 200 rpm for 72 h to obtain fermented bovine bile.

[0097] S2, according to the above weight fraction, 95 parts of fermented bovine bile is mixed with 100 parts of saturated calcium hydroxide solution, stirred for 25 min, heated and boiled for 20 min, then cooled and centrifuged to obtain a precipitate;

[0098] S3, the above weight fraction of bilirubin, cholic acid, deoxycholic acid, taurine, ferrous sulfate, zinc sulfate, magnesium sulfate and the remaining 5 parts of fermented bovine bile are added to the precipitate, then mixed with purified water, ultrasonically treated for 20 min, freeze-dried and crushed to obtain a composite bilirubin calcium powder;

[0099] S4, the composite bilirubin calcium powder, hydroxypropyl methyl cellulose and fermented bovine bile are mixed to obtain the biological ink, and water is added to adjust the viscosity to about 4000 centipoise for standby.

[0100] (2) Preparation of high bionic natural calculus bovis by 3D printing

[0101] The specific method of the high bionic natural calculus bovis of the embodiment includes the following steps:

[0102] S1. Use CAD software to create a sphere with a diameter of 8 mm and save the model as an STL digital model. Import the STL model into the slicing software for calculation and generate G-code (used to control the nozzle path) for the next step of importing it into the printer.

[0103] S2. An industrial independent single-nozzle 3D printer with a nozzle diameter of approximately 0.2 mm was used to print the bio-ink cartridge prepared above. The printing method adopted layer-by-layer printing at a printing speed of 50 mm / s. After printing a layer, a pause of 1 minute was made to allow the material to fully react and take shape, thereby achieving a multi-layered effect.

[0104] S3. The printed highly biomimetic natural bezoar pills are placed in a temperature of 32° C. for 2 hours, and then freeze-dried to obtain the obtained pills.

[0105] The total bilirubin and bile acid contents of the high bionic bezoar were detected by spectrophotometry according to national standards. The detection showed that the total bilirubin content of the high bionic bezoar prepared in this embodiment was 53.89%, and the bile acid content was 19.69%.

[0106] Comparative Example 1

[0107] This comparative example provides a natural bezoar, which is sourced from the China Food and Drug Inspection Institute.

[0108] The total bilirubin and bile acid contents of the natural bezoar were detected by spectrophotometry according to national standards. The results showed that the total bilirubin content of the natural bezoar was 64.23%, and the bile acid content was 10.38%.

[0109] Comparative Example 2

[0110] This comparative example provides an in vitro cultured bezoar, which was purchased from Wuhan Jianmin Dapeng Pharmaceutical Co., Ltd.

[0111] The total bilirubin and bile acid contents of the in vitro cultured bezoar were detected by spectrophotometry according to national standards. The results showed that the total bilirubin content of the in vitro cultured bezoar was 37.87%, and the bile acid content was 10.57%.

[0112] Comparative Example 3

[0113] This comparative example provides a bionic bezoar and a preparation method thereof. The high bionic bezoar is prepared from raw materials, calculated by weight, comprising:

[0114]

[0115] This embodiment adopts 3D printing method to prepare highly bionic bezoar, which specifically includes the following contents.

[0116] (1) Preparation of bio-ink

[0117] The bio-ink of the present comparative example is composed of the above preparation raw materials, and the preparation method comprises the following steps:

[0118] S1, fresh bovine bile is taken, and 3% of E. coli (purchased from China Center for Type Culture Collection, with the preservation number of CCTCC AB 2012885) bacterial solution is added, the final concentration of E. coli is 2×10 7 Individuals / mL, and fermentation culture is carried out at 37℃±2℃ and a rotation speed of 200 rpm for 72 h, and then 121℃ high-temperature sterilization is carried out for 30 min to obtain fermented bovine bile;

[0119] S2, 95 parts of the fermented bovine bile is mixed with a saturated calcium hydroxide solution according to the above weight fraction, stirred for 25 min, heated and boiled for 20 min, and then cooled, centrifuged to obtain a precipitate, and then the above weight fraction of bilirubin, cholic acid, 1 deoxycholic acid, taurine, ferrous sulfate, zinc sulfate and magnesium sulfate are added to the precipitate, and then mixed with purified water, ultrasonic treated for 20 min, frozen, vacuum dried and crushed to obtain a composite bilirubin calcium powder;

[0120] S3, the composite bilirubin calcium powder, hydroxypropyl methyl cellulose and the remaining 5% of the fermented bovine bile are mixed to obtain the bio-ink, and water is added to adjust the viscosity to about 3500 centipoises.

[0121] (2) 3D printing preparation of high-bionic natural bovine bezoar

[0122] The specific method of the high-bionic natural bovine bezoar of the present comparative example comprises the following steps:

[0123] S1, a CAD computer aided design software is used to establish a spherical shape with a diameter of 8 mm, and the model is saved as a digital model in STL format. The STL format model is imported into a slicing software for calculation, and G code (used for controlling the nozzle path) is generated for the next step of being imported into a printer.

[0124] S2, an industrial type independent single nozzle 3D printer is used, the diameter of the nozzle is about 0.2 mm, the above prepared bio-ink cartridge is used as the raw material for printing, the printing method adopts layer-by-layer printing, the printing speed is 50 mm / s, and the printing of one layer is paused for 1 min to make the substances fully react and form, so as to achieve the effect of multiple layers.

[0125] S3, the printed high-bionic natural bovine bezoar pill is placed in 32℃ for cultivation, the cultivation time is 2 h, and then freeze-drying is carried out after the cultivation is completed, and the high-bionic natural bovine bezoar pill is obtained.

[0126] The content of total bilirubin and cholic acid in the high-bionic bovine bile is detected according to the national standard by spectrophotometry. The total bilirubin content of the high-bionic bovine bile prepared in the pair of examples is 49.64%, and the cholic acid content is 17.56%.

[0127] Example 4

[0128] The pair of examples provides a bionic bovine bile and a preparation method thereof. The preparation raw materials include, in terms of weight fractions:

[0129]

[0130] The pair of examples adopts a 3D printing method to prepare a high-bionic bovine bile, which specifically includes the following contents.

[0131] (1) Preparation of biological ink

[0132] The biological ink of the example is composed of a first component and a second component. The preparation method of the first component (fermented bovine bile) includes:

[0133] Fresh bovine bile is taken, and 3% of E. coli (purchased from China Typical Culture Collection Center, with a preservation number of CCTCC AB 2012885) bacterial solution is added. The final concentration of E. coli is 2×10 7 Individuals / mL, and fermented and cultured at 37℃±2℃ and a rotation speed of 200 rpm for 72 h to obtain fermented bovine bile. Then, 95 parts of the fermented bovine bile are centrifuged according to the above weight fractions, and the precipitate is collected to obtain the fermented bovine bile, which has a viscosity of about 5000 centipoises.

[0134] The preparation method of the second component includes:

[0135] According to the above weight fractions, saturated calcium hydroxide and hydroxypropyl methyl cellulose (HPMC) are mixed, and then the remaining 5 parts of fermented bovine bile, bilirubin, cholic acid, deoxycholic acid, zinc sulfate, taurine, ferrous sulfate and magnesium sulfate are added and uniformly mixed to obtain the second component.

[0136] (2) Preparation of high-bionic bovine bile by 3D printing

[0137] The specific method of the high-bionic bovine bile of the pair of examples includes the following steps:

[0138] S1, a CAD computer-aided design software is used to establish a spherical shape with a diameter of 8 mm, and the model is saved as a digital model in STL format. The STL format model is imported into a slicing software for calculation, and G code (used for controlling the nozzle path) is generated for the next step of being imported into the printer.

[0139] S2, an industrial type independent double nozzle 3D printer is adopted, wherein the main nozzle prints the fermented bovine bile precipitate (i.e. the first component) as the printing raw material, and the auxiliary nozzle prints the bovine bile and its mixture (i.e. the second component) as the printing raw material, and the diameter of the nozzle is about 0.2 mm; the movement of the two nozzles is simultaneously started, completely synchronized and simultaneously ended. The printing mode of the pair of examples is layer-by-layer printing, and the printing speed is 50 mm / s, and the printing of one layer is paused for 1-2 min, and the result shows that it is difficult to form, and the expected multi-layer effect cannot be achieved.

[0140] Therefore, it can be seen that the raw material for the 3D printing of bovine bile is crucial for the final printing effect, and when the HPMC content is too high, the flow performance will be poor, the printing effect will be affected, and it is difficult to prepare ideal structure high-bionic natural bovine bile.

[0141] In summary, the present application provides a method for preparing high-bionic bovine bile by using 3D printing technology, and the high-bionic bovine bile prepared by the method of the present application can be comparable to natural bovine bile in composition, efficacy and appearance, and the main active ingredients can be adjusted according to actual needs, which can greatly reduce the production cost of bovine bile and meet the demand of people for bovine bile.

[0142] The above has made a detailed description of the embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary skilled persons in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A 3D printing method of high-bionic cow-bezoar, characterized in that, It comprises the following steps: S1, constructing a 3D printing digital model; S2, using the preparation raw material as a biological ink for 3D printing, wherein the printing method is layer-by-layer printing, the printing speed is 40-60 mm / s, and the layer-by-layer printing interval time is 30-90 s; S3, cultivating the printing sample obtained in step S2, and the temperature of the cultivation is 25-38℃, and the cultivation time is 2-3 h; According to the weight fraction, the preparation raw material of the high bionic bovine calculus comprises the following components: fermented bovine bile 80-200 parts; saturated calcium hydroxide 80-200 parts; hydroxypropyl methyl cellulose 0.5-2 parts; bilirubin 30-80 parts; cholic acid 1-10 parts; deoxycholic acid 1-10 parts; taurine 10-15 parts; zinc sulfate 0.1-1 part; ferrous sulfate 0.1-1 part; magnesium sulfate 0.1-1 part; The fermented bovine bile is obtained by fermenting bovine bile with Escherichia coli; The 3D printing is selected from double-nozzle printing or single-nozzle printing: When the 3D printing is double-nozzle printing, the main nozzle prints the precipitate of 90-95% by weight of the fermented bovine bile, and the auxiliary nozzle prints the mixture of the remaining preparation raw material; The viscosity of the precipitate of the fermented bovine bile is 3000-10000 centipoise, and the viscosity of the auxiliary nozzle printing raw material is 1300-4000 centipoise; When the 3D printing is single-nozzle printing, the preparation method of the biological ink comprises: S11, mixing 90-95% by weight of the fermented bovine bile with the saturated calcium hydroxide, heating, centrifuging, taking the precipitate, and then adding the bilirubin, cholic acid, deoxycholic acid, taurine, ferrous sulfate, zinc sulfate and magnesium sulfate to obtain a composite bilirubin calcium; S12, mixing the composite bilirubin calcium, the hydroxypropyl methyl cellulose and the remaining 5-10% by weight of the fermented bovine bile, and the viscosity of the biological ink is 2000-5000 centipoise. The Escherichia coli is selected from at least one of the Escherichia coli with the preservation numbers CCTCC AB 2012885, CCTCC AB 2013330 and CCTCC AB 2013342.

2. The 3D printing method according to claim 1, characterized in that, The construction of the 3D printing digital model specifically comprises: using a drawing software to establish a pre-printing model, generating a digital model, then using a slicing software to calculate, obtaining G code and importing it into a printer.

3. The 3D printing method of claim 1, wherein, The heating temperature is 90-105℃.

4. The 3D printing method of claim 1, wherein, The heating time is 15-20 min.

5. The 3D printing method of claim 1, wherein, 6. The 3D printing method according to any one of claims 1-5 for preparing a heat-clearing and detoxifying drug. ​

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