CN117562927A - Preparation process for in-vitro cultivation of bezoar - Google Patents
Preparation process for in-vitro cultivation of bezoar Download PDFInfo
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- CN117562927A CN117562927A CN202311702673.7A CN202311702673A CN117562927A CN 117562927 A CN117562927 A CN 117562927A CN 202311702673 A CN202311702673 A CN 202311702673A CN 117562927 A CN117562927 A CN 117562927A
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- 206010004542 Bezoar Diseases 0.000 title claims abstract description 108
- 238000000338 in vitro Methods 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims abstract description 72
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 27
- 238000012258 culturing Methods 0.000 claims abstract description 24
- 230000001954 sterilising effect Effects 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000006052 feed supplement Substances 0.000 claims abstract description 17
- 102000053187 Glucuronidase Human genes 0.000 claims abstract description 15
- 108010060309 Glucuronidase Proteins 0.000 claims abstract description 15
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 14
- 239000001110 calcium chloride Substances 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 14
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 13
- 239000002994 raw material Substances 0.000 claims abstract description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 8
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- 235000015278 beef Nutrition 0.000 claims abstract description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims description 17
- 238000001035 drying Methods 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims description 6
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 208000001130 gallstones Diseases 0.000 claims description 6
- 229940097043 glucuronic acid Drugs 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 238000003860 storage Methods 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 239000002699 waste material Substances 0.000 claims description 4
- 238000007599 discharging Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 6
- 239000004615 ingredient Substances 0.000 abstract description 5
- 230000000857 drug effect Effects 0.000 abstract description 4
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- 230000008569 process Effects 0.000 description 11
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- 239000004480 active ingredient Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 235000013619 trace mineral Nutrition 0.000 description 5
- 239000011573 trace mineral Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 239000004380 Cholic acid Substances 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 229940107161 cholesterol Drugs 0.000 description 2
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 2
- 229960002471 cholic acid Drugs 0.000 description 2
- 235000019416 cholic acid Nutrition 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 229960003964 deoxycholic acid Drugs 0.000 description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
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- 238000009776 industrial production Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 238000012840 feeding operation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/413—Gall bladder; Bile
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention provides a preparation process for in vitro culturing bezoar, which comprises the following steps: culturing the strain producing the beta-glucuronidase in high density, concentrating the ceramic membrane, homogenizing and crushing under high pressure to obtain the beta-glucuronidase; step two: adding bilirubin and glucose into a reaction kettle for reaction to obtain combined bilirubin; step three: pouring the cultivation raw materials into a feed tank for sterilization treatment to obtain sterilized oxgall; step four: the prepared strain is obtained through activation treatment and bacteria culture; step five: uniformly stirring calcium chloride and pure water, and then sterilizing to obtain a prepared calcium chloride solution; step six: uniformly stirring water, glucose and beef extract, and then sterilizing to obtain a prepared feed supplement liquid; step seven: culturing calculus bovis in an in vitro calculus bovis culturing tank by fermentation method; solves the problem of slight difference between the ingredients of the cultured bezoar and the natural bezoar in vitro, improves the drug effect and reduces the manufacturing cost.
Description
Technical Field
The invention belongs to the technical field of pharmacy, and particularly relates to a preparation process for culturing bezoar in vitro.
Background
Bezoar is a rare traditional Chinese medicine, and the natural bezoar has extremely low probability of forming in the ox gall, so that bezoar is extremely lacking, and clinical requirements are difficult to meet.
Three natural bezoar substitutes, namely cultivated bezoar, cultivated bezoar in vitro, artificial bezoar and the like, are approved by the national drug administration in 1972, the cultivated bezoar is equivalent to the natural bezoar, a large number of calves need to be cultivated for cultivating the bezoar, the yield is extremely low, and the market demand is difficult to meet. The efficacy of the artificial bezoar is poor compared with that of the natural bezoar by a large scale; the in vitro cultured bezoar has 90 percent of efficacy compared with natural bezoar, is suitable for industrial production, and is put into mass production in the current Wuhan Dapeng pharmaceutical industry.
However, in the patent of the medical industry of the Wuhan Jianmen Dapeng with the application number of 202010577790.5, the in-vitro cultured bezoar produced by the Wuhan Jianmen Dapeng is prepared by mixing various components according to a certain proportion and making into pills. The trace elements are definitely different from trace elements compared with natural bezoar.
In order to make the in vitro cultured bezoar similar to natural bezoar in each component, the invention takes oxgall as raw material, and cultures in an in vitro cultured bezoar culture tank simulating the environment formed by bezoar in the oxgall, can accurately control the pH, temperature, dissolved oxygen, bilirubin and Ca+ amount, and yellow in the tank body, thus obtaining the in vitro cultured bezoar.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation process of in-vitro cultured bezoar, which aims to solve the problem of small difference between in-vitro cultured bezoar and natural bezoar components in the prior art and improve the drug effect of in-vitro cultured bezoar.
The invention discloses a preparation process for in vitro culturing bezoar, which is characterized by comprising the following steps:
a preparation process of in vitro cultured bezoar comprises the following steps:
step one: preparation of beta-glucuronidase:
culturing the strain producing the beta-glucuronidase in high density, then concentrating the ceramic membrane, and then homogenizing and crushing the ceramic membrane under high pressure to finally obtain the prepared beta-glucuronidase for later use;
step two: preparation of bilirubin:
adding bilirubin and glucose into a reaction kettle, heating to 35-10 ℃, and regulating the PH value to maintain the PH value between 6 and 8;
step three: preparation of sterilized oxgall:
pouring the cultivation raw materials into a feed supplementing tank, heating to 60-70 ℃, maintaining the temperature for 1 hour, and sterilizing to finally obtain sterilized oxgall for later use;
step four: preparation of strains:
and taking out the strain from the strain storage cabinet, and performing activation treatment and bacterial culture to obtain the required strain.
Step five: preparing a calcium chloride solution: adding calcium chloride into a feed supplement tank, adding pure water according to the amount, uniformly stirring, heating to 121 ℃ and sterilizing to obtain a prepared calcium chloride solution;
step six: preparing a feed supplement liquid:
adding water, glucose and beef extract into a feed supplement tank, uniformly stirring, then sterilizing the mixed solution, and heating to 121 ℃ for sterilization to obtain a prepared feed supplement liquid for later use;
step seven: culturing in vitro bezoar:
introducing ox gall into an external ox gall cultivation tank, sealing, adopting a fermentation method to simulate the environment generated by ox gall, cultivating under constant temperature, constant PH and constant oxygen, fermenting for the next day, observing that tiny ox gallparticles are in the tank body through a sight glass, then the ox gallstone particles grow up in one day, cultivating for 8 days, observing that the ox gallstone diameter in the ox gallstone cultivation tank is 2.5-3cm, and stopping feeding; continuously culturing for 6-8 hours, raising the temperature of the bezoar culture tank to 60-70 ℃, maintaining for 1 hour, cooling, discharging liquid, opening a discharge hole to take out cultured bezoar in vitro, drying, packaging and warehousing after sampling and detection are qualified.
In the seventh step, the fermentation process includes raising the temperature of the bezoar culture tank to 60-70 deg.c, maintaining for 1 hr, cooling the tank material to 37 deg.c, regulating the pH value of the tank material to 7.2 via the microcomputer controlled feeding pump, opening the inoculation port and pouring the strain into the bezoar culture tank; opening ventilation, maintaining the tank pressure at 0.05MPa, maintaining the pH of the tank material at 7.0-7.2, and culturing at 37deg.C for 5 hr.
As a further aspect of the present invention, feeding the bilirubin and calcium chloride solution to the bezoar culture tank is started, feeding the bilirubin and calcium chloride solution is started, and simultaneously, a waste liquid is discharged; the liquid level of the material in the bezoar culture tank is reduced by one third, and the feed supplement is started to sterilize the oxgall.
In the third step, the cultivation material is lyophilized fel bovis seu Bubali powder, and water is added for reconstitution or fresh fel bovis seu Bubali.
As a further scheme of the invention, the sterilization treatment temperature of the cultivation raw materials is 50-80 ℃.
As a further scheme of the invention, in the step four, the strain is obtained by separating and screening from yellow oxgall.
As a further aspect of the present invention, in step seven, the bilirubin contained in the feed is conjugated bilirubin or a bilirubin salt, wherein the conjugated bilirubin is glucuronic acid bilirubin.
As a further scheme of the invention, the calcium chloride contained in the feed adopts a solution with the concentration of 0.05-2%.
As a further aspect of the present invention, the drying method of the drying treatment is freeze vacuum drying.
Compared with the prior art, the invention has the following beneficial effects:
1. compared with the prior art, the preparation process has the advantages that the yellow-forming is carried out in the cultivation tank, no additional pill preparation is needed, the cultivated in-vitro cultivated bezoar has golden cross section, fine concentric circle layers and white cores are arranged, the layers are clear, the bezoar is similar to the natural bezoar, the problem of slight difference between the ingredients of the in-vitro cultivated bezoar and the natural bezoar is solved, and the drug effect of the in-vitro cultivated bezoar is improved; the process ensures that the preparation of the in vitro cultured bezoar is simpler, more convenient and more efficient, saves the time and the cost for preparing the bezoar, maintains the natural characteristics of the bezoar and improves the quality and the stability of the bezoar.
2. The environment formed by the bezoar in the ox gall is simulated, and the feeding pump is controlled by the microcomputer to feed or control the temperature of the cultivation tank, so that the accurate control of parameters such as PH, dissolved oxygen, temperature and the like is realized; the preparation process not only ensures that the cultured in vitro cultured bezoar accords with various indexes of the first edition of Chinese pharmacopoeia 2020, but also does not need to additionally add cholic acid, deoxycholic acid, cholesterol, trace elements and the like, and has simple operation and low manufacturing cost, thereby being suitable for laboratory and industrial production and improving the economical efficiency of the product.
3. Through the arrangement of freeze vacuum drying, when the preparation process is used, the in vitro cultured bezoar can better keep the active ingredients and natural characteristics thereof in the drying treatment process, and the loss and denaturation of ingredients possibly caused in the heat treatment process are avoided; the active ingredients of the bezoar can be effectively reserved in the drying mode, the quality and the efficacy of the product are ensured, the stability and the storage life of the product are improved, and the method has the advantages in the aspects of maintaining the quality and the active ingredients of the product.
Drawings
FIG. 1 is a technical scheme of a preparation process for culturing bezoar in vitro according to the present invention.
Detailed Description
For a further understanding of the present invention, preferred embodiments of the invention are described below with reference to the drawings and examples, but it is to be understood that these descriptions are merely intended to illustrate further features and advantages of the invention and are not limiting of the invention as claimed.
Examples:
referring to fig. 1, the invention provides a preparation process for in vitro culturing bezoar, comprising the following steps:
step one: preparation of beta-glucuronidase:
firstly, selecting a proper strain for culture, wherein the strain can produce beta-glucuronidase, and the growth density of the strain is improved by a high-density culture technology, so that the yield of the beta-glucuronidase is increased; then concentrating the beta-glucuronidase in the culture solution by adopting a ceramic membrane concentration treatment method to remove irrelevant substances and improve the concentration of the beta-glucuronidase; then, crushing the concentrated thalli by utilizing a high-pressure homogenizing and crushing technology to release beta-glucuronidase, and finally, obtaining the prepared beta-glucuronidase for standby after proper purification and treatment;
step two: preparation of bilirubin:
firstly adding bilirubin and glucose into a reaction kettle, then raising the temperature in the reaction kettle to 35-10 ℃, and regulating the PH value to ensure that the PH value is maintained between 6 and 8; then, detecting by using a thin layer chromatography scanner until the detection result meets the requirement, and preparing the prepared combined bilirubin for later use;
step three: preparation of sterilized oxgall:
pouring the cultivation raw materials into a feed supplement tank, then raising the temperature of the feed supplement tank to 60-70 ℃, keeping the temperature for 1 hour, and then sterilizing to ensure that the oxgall is not polluted by bacteria and other microorganisms; after the sterilization treatment, taking the obtained sterilized oxgall as a subsequent standby;
step four: preparation of strains:
taking out the needed strain from the strain preservation cabinet, and performing activation treatment to activate the biological activity of the strain; then, culturing the activated strain, and rapidly propagating the strain through proper culture conditions and culture medium, thereby obtaining a large number of required strains, and reserving the strains for subsequent use;
step five: preparing a calcium chloride solution:
firstly, adding a proper amount of calcium chloride into a material supplementing tank, adding pure water according to a proportion, and then fully stirring to ensure that the calcium chloride is fully dissolved; then, heating the mixed solution to 121 ℃ for sterilization treatment to ensure that the solution is not polluted by bacteria and other microorganisms, and obtaining a prepared calcium chloride solution after the sterilization treatment for subsequent application;
step six: preparing a feed supplement liquid:
firstly, adding water into a feed supplementing tank, adding required glucose and beef extract, and then fully stirring to ensure that all components are fully and uniformly mixed; subsequently, the mixed solution is sterilized and the temperature is raised to 121 ℃ to ensure that the feed solution is not contaminated by bacteria and other microorganisms. After sterilization treatment, the prepared feed supplement liquid is obtained and is reserved for subsequent use;
step seven: culturing in vitro bezoar:
introducing fel bovis Seu Bubali into in vitro calculus bovis culture tank, and sealing; then, culturing under constant temperature, constant PH and constant oxygen by adopting a fermentation method, and simulating the natural environment generated by the bezoar in the ox gall; on the next day of fermentation, microscopic bezoar particles were observed in the tank through the sight glass, and then gradually increased until day 8 of incubation, the bezoar particles were observed to have a diameter of 2.5-3cm, at which time feeding was stopped; then, continuing to cultivate for 6-8 hours, raising the temperature of the bezoar cultivation tank to 60-70 ℃, keeping for 1 hour, and then cooling; then, opening a liquid outlet to discharge liquid, and then opening the liquid outlet to take out the cultured in-vitro cultured bezoar, and drying the bezoar; and finally, packaging and warehousing after the sampling and detection are qualified.
By simulating the environment formed by the bezoar in the ox gall, the fermentation cultivation is carried out, so that the cultivated in-vitro cultivated bezoar has golden section, fine concentric circle layers and white cores, has distinct layers, is similar to natural bezoar, solves the problem of tiny difference between the ingredients of the in-vitro cultivated bezoar and the natural bezoar, and improves the drug effect of the in-vitro cultivated bezoar; and no additional cholic acid, deoxycholic acid, cholesterol, trace elements and the like are needed, so that the production cost is reduced, and the economical efficiency of the product is improved.
In the seventh step, the fermentation process of bezoar comprises the following detailed steps: firstly, heating a bezoar cultivation tank to 60-70 ℃, keeping the temperature range for 1 hour, and cooling materials in the bezoar cultivation tank to 37 ℃ after the heat preservation time of 1 hour, wherein the temperature is suitable for microorganism growth and fermentation, and a suitable environment is created for microorganism propagation and fermentation through accurate temperature control; then, the pH value of the tank material is regulated to be 7.2 by controlling a feed pump through a microcomputer, and the regulation of the pH value is very important for the fermentation process, because the pH value directly influences the growth and metabolic activity of microorganisms, and the microorganisms can perform fermentation reaction better under the condition of proper pH value; opening an inoculation port, pouring the strain after screening and culturing into a bezoar culture tank, and promoting the fermentation process of bezoar by the strain after screening and culturing, and improving the quality and yield of the product; then, ventilation is started, the tank pressure is kept at 0.05MPa, oxygen can be fully dissolved in the tank material, and the needed oxygen supply is provided for microorganisms; the pH value of the tank material is maintained between 7.0 and 7.2 by a microcomputer, and the temperature is kept at 37 ℃ for 5 hours.
In the culturing process of the bezoar culture tank, the bezoar culture tank is subjected to feeding operation, specifically, bilirubin and calcium chloride solution are fed to the bezoar culture tank, so as to provide microelements required by bezoar growth; meanwhile, in order to keep the environment in the bezoar culture tank stable, a water outlet on the membrane filter water storage bin is opened to discharge waste liquid, so that metabolic waste and impurities generated in the culture process can be effectively removed, and the cleanness and sanitation in the tank are maintained; when the liquid level of the material in the bezoar culture tank is reduced by one third, the feed supplement sterilizing bezoar is started, the bezoar is an important nutrition source for bezoar growth, and the bezoar can be provided with sufficient nutrients by supplementing the sterilizing bezoar, so that the bezoar growth and development are promoted.
In the third step, the cultivation raw material is freeze-dried ox gall powder, and water is added for re-dissolution or fresh ox gall is used; the freeze-dried oxgall powder is a powdery substance obtained by freeze-drying oxgall, and after being re-dissolved by adding water, the oxgall powder is restored to the original liquid state, so that the nutritional ingredients and bioactive substances in the oxgall are reserved, and the oxgall powder is convenient to store and transport; the other raw material is fresh oxgall which is collected from the gall bladder of the cattle, contains rich nutrition and bioactive substances, and is an ideal basic material for the growth of bezoar; whether the freeze-dried ox gall powder is re-dissolved in water or fresh ox gall powder contains nutrient substances and bioactive components required by the growth of bezoar, and can be used as a base material for culturing bezoar.
Referring to fig. 1, the oxgall needs to be sterilized, so that potentially harmful microorganisms can be effectively killed, and the sanitation and safety of the cultivation process are ensured; to achieve this, the sterilizing temperature of the sterilized oxgall is usually 50-80 ℃ for sterilization; in the temperature range, microorganisms in the cultivation raw materials can be inhibited or killed, the higher temperature can accelerate the metabolic process of the microorganisms, so that the microorganisms cannot grow and reproduce normally, and meanwhile, the high temperature can damage the cell walls and cell membranes of the microorganisms, so that the proteins in the cells are denatured, and the microorganisms are effectively killed; the sterilization treatment is carried out on the cultivation raw materials at the temperature of 50-80 ℃, so that the interference of bacteria, fungi and other microorganisms on the bezoar cultivation process is reduced, and the quality and safety of the product are improved.
In the fourth step, the strain used needs to collect a sample from the yellow oxgall and then bring the sample back to a laboratory for separation and screening; in the separation process, an aseptic technique is required to be used to avoid the pollution of external mixed bacteria; a sample of oxgall is diluted appropriately and then spread evenly on a plate of a culture medium containing appropriate nutrients, after a period of incubation, a number of colonies of different forms and colors appear on the plate, and then these colonies are screened to select strains with specific characteristics; by this separation and screening process we can obtain pure species from yellow oxgall, which can be used for further applications.
In step seven, the bilirubin contained in the feed may be bound bilirubin, which is bilirubin bound to glucuronic acid, also known as glucuronic acid bilirubin, or a bilirubin salt; the conjugated bilirubin is formed by a series of biotransformation processes of bilirubin in the liver, wherein the bilirubin is conjugated with glucuronic acid to form stable glucuronic acid bilirubin; making bilirubin more soluble in water; the choice of the conjugated bilirubin or bilirubin salt contained in the feed will depend on the particular application and experimental requirements, and the choice of the appropriate bilirubin type is critical to the success of the experiment.
The calcium chloride contained in the feed is usually 0.05-2% solution to ensure that the calcium chloride can play the best effect in the feed process; calcium chloride is a yellow trace element supplement, and by using a calcium chloride solution with proper concentration, we can provide calcium ions required by cells; the concentration range of 0.05% to 2% was selected based on the requirements for cell culture and the solubility of calcium chloride; lower concentration calcium chloride solutions may provide the proper amount of calcium ions, while higher concentration calcium chloride solutions may be toxic to cells or cause osmotic imbalance; in practical applications, the specific calcium chloride concentration should be adjusted according to the culture system, cell type and experimental purpose.
The bezoar product is freeze-dried in vacuum, and the drying method combines the freezing and vacuum processes to achieve the purposes of removing water and keeping the original characteristics of the sample; firstly, freezing a sample to be dried to a low temperature, and then, reducing the boiling point of water by reducing the pressure in a vacuum environment, so that the drying time is reduced, and the production efficiency is improved; the original structure and active ingredients of the sample can be maintained through freeze-vacuum drying, the problems of thermal denaturation, oxidization and caking possibly occurring in the traditional drying method are avoided, the active ingredients of the bezoar can be effectively maintained through the drying mode, the quality and the efficacy of the product are ensured, the stability and the storage life of the product are improved, and the method has the advantages in the aspects of maintaining the quality and the active ingredients of the product.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
Claims (9)
1. A preparation process for culturing bezoar in vitro is characterized in that: the method comprises the following steps:
step one: preparation of beta-glucuronidase:
culturing the strain producing the beta-glucuronidase in high density, then concentrating the ceramic membrane, and then homogenizing and crushing the ceramic membrane under high pressure to finally obtain the prepared beta-glucuronidase for later use;
step two: preparation of bilirubin:
adding bilirubin and glucose into a reaction kettle, heating to 35-10 ℃, and regulating the PH value to maintain the PH value between 6 and 8;
step three: preparation of sterilized oxgall:
pouring the cultivation raw materials into a feed supplementing tank, heating to 60-70 ℃, maintaining the temperature for 1 hour, and sterilizing to finally obtain sterilized oxgall for later use;
step four: preparation of strains:
taking out the strain from the strain storage cabinet, and performing activation treatment and bacterial culture to obtain the required strain;
step five: preparing a calcium chloride solution: adding calcium chloride into a feed supplement tank, adding pure water according to the amount, uniformly stirring, heating to 121 ℃ and sterilizing to obtain a prepared calcium chloride solution;
step six: preparing a feed supplement liquid:
adding water, glucose and beef extract into a feed supplement tank, uniformly stirring, then sterilizing the mixed solution, and heating to 121 ℃ for sterilization to obtain a prepared feed supplement liquid for later use;
step seven: culturing in vitro bezoar:
introducing ox gall into an external ox gall cultivation tank, sealing, adopting a fermentation method to simulate the environment generated by ox gall, cultivating under constant temperature, constant PH and constant oxygen, fermenting for the next day, observing that tiny ox gallparticles are in the tank body through a sight glass, then the ox gallstone particles grow up in one day, cultivating for 8 days, observing that the ox gallstone diameter in the ox gallstone cultivation tank is 2.5-3cm, and stopping feeding; continuously culturing for 6-8 hours, raising the temperature of the bezoar culture tank to 60-70 ℃, maintaining for 1 hour, cooling, opening a liquid outlet, discharging liquid, opening the liquid outlet, taking out cultured bezoar in vitro, drying, packaging and warehousing after sampling and detection are qualified.
2. The process for preparing the in vitro cultured bezoar according to claim 1, wherein: in the seventh step, the fermentation method comprises heating the bezoar culture tank to 60-70deg.C, maintaining for 1 hr, cooling tank material to 37deg.C, adjusting the pH of the tank material to 7.2, opening an inoculation port, and pouring the strain into the bezoar culture tank; and (3) starting ventilation, maintaining the tank pressure at 0.05MPa, maintaining the pH of the tank material at 7.0-7.2 by the microcomputer, and culturing for 5 hours at 37 ℃.
3. The process for preparing the in vitro cultured bezoar according to claim 2, wherein: feeding the bilirubin and the calcium chloride solution to the bezoar culture tank, feeding the bilirubin and the calcium chloride solution, and discharging waste liquid; the liquid level of the material in the bezoar culture tank is reduced by one third, and the feed supplement is started to sterilize the oxgall.
4. The process for preparing the in vitro cultured bezoar according to claim 1, wherein: in the third step, the cultivation raw material is freeze-dried ox gall powder, and water is added for re-dissolving or fresh ox gall is used as the cultivation raw material.
5. The process for preparing in vitro cultured bezoar according to claim 4, wherein: the sterilization treatment temperature of the cultivation raw materials is 50-80 ℃.
6. The process for preparing the in vitro cultured bezoar according to claim 1, wherein: in the fourth step, the strain is obtained by separating and screening from yellow oxgall.
7. The process for preparing the in vitro cultured bezoar according to claim 1, wherein: in step seven, the bilirubin contained in the feed supplement is conjugated bilirubin or a bilirubin salt, wherein the conjugated bilirubin is glucuronic acid bilirubin.
8. The process for preparing in vitro cultured bezoar according to claim 7, wherein: the calcium chloride contained in the feed adopts a solution with the concentration of 0.05-2%.
9. The process for preparing in vitro cultured bezoar according to claim 8, wherein: the drying mode of the drying treatment is freeze vacuum drying.
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