Method for culturing bezoar with natural quality in vitro by adopting biological enzyme
By using in vitro bioenzyme cultivation methods, zinc-manganese alloy nanoparticles, and multi-enzyme synergistic reactions, combined with supercritical CO2 extraction, the problem of insufficient natural bezoar production has been solved, and high-purity bezoar has been prepared to meet market demand.
Patent Information
- Application Number
- CN202511544643.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-28
- Publication Date
- 2025-12-16
AI Technical Summary
Existing technologies make it difficult to achieve high-yield production of natural bezoar, and the effective ingredient content of chemical synthesis methods is low, which cannot meet market demand.
High-purity bezoar was prepared by using an in vitro bioenzyme culture method, which forms a nanoscale redox microenvironment through zinc-manganese alloy nanoparticles and an EGCG-rosmarinic acid-tea polyphenol ternary complex, combined with multi-enzyme synergistic reaction and supercritical CO2 extraction.
The bilirubin and bile acid content was significantly increased, and the finished bezoar product produced far exceeded the standard of natural bezoar, achieving efficient, controllable and green in vitro culture of bezoar.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and in particular to a method for in vitro cultivation of natural quality bezoar using biological enzymes. Background Technology
[0002] Bezoar, or bilirubin gallstones from cattle, is used to treat certain liver, cardiovascular, brain diseases, and cancer. Traditional Chinese medicine requires a large amount of bezoar annually as a proprietary Chinese medicine. Natural bezoar is formed from the liver or gallbladder of cattle, but cattle rarely suffer from liver or gallstones, accounting for only a few per thousand. Therefore, the source of natural bezoar is extremely limited, far from meeting clinical and research needs. Consequently, since its use in traditional Chinese medicine, bezoar has been in severe shortage.
[0003] To alleviate this supply-demand imbalance and promote the development of the medical and health industry, my country's Pharmacopoeia has included artificial and cultured bezoar in its scope. In recent years, domestic research on bezoar has made rapid progress, with main research methods including in vivo bezoar cultivation, in vitro bezoar cultivation, and chemical synthesis. In vivo bezoar cultivation involves surgically implanting a luteinizing factor into the gallbladder of a cow, causing the formation of a bezoar-like substance. The bezoar is then scraped out during surgery or slaughter. However, while this method produces bezoar very similar to natural bezoar, it cannot be mass-produced, resulting in limited output and failing to effectively alleviate the shortage of natural bezoar in the market. Chemical synthesis has the advantages of simple processes, ease of operation, and the potential for industrial production, but its effective component content is lower than that of natural bezoar.
[0004] Therefore, there is an urgent need to develop a method for in vitro cultivation of natural-quality bezoar using biological enzymes, which can ensure high yields of bezoar to meet current market demand, while also ensuring that the effective components of the produced bezoar are comparable to those of natural bezoar. Summary of the Invention
[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for in vitro cultivation of natural quality bezoar using biological enzymes.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for in vitro cultivation of natural quality bezoar using biological enzymes includes the following steps: S1, zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh bovine bile at a mass ratio of 1000:(0.8–1.2):(3.0–4.5). The mixture was magnetically stirred at 55–65℃ for 30–35 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes are added sequentially to the pretreated bile, and the reaction is carried out at a gradient temperature of 38–42℃ for 12–16 hours. After adjusting the pH to 8.0–8.5, trypsin is added, and the reaction is carried out at 32–35℃ with pulsed shaking for 8–12 hours (frequency 120 rpm / min). Finally, the second biological enzyme is added, and the reaction is carried out at 28–30℃ in the dark for 14–18 hours to form a layered enzymatic hydrolysis system, resulting in a multiphase enzymatic hydrolysate. S3. Adjust the pH of the enzymatic hydrolysate to 2.0–2.8, add 0.5–1.0% chitosan flocculant, and allow it to stand for 24–36 hours at a gradient cooling temperature of 4–8℃. After centrifugation, use supercritical CO2 extraction to remove fat-soluble impurities. Collect the precipitate and freeze-dry it under vacuum until the water content is ≤3%, finally obtaining high-purity bezoar.
[0007] As a further improvement of the present invention, the first bioenzyme was prepared by Bacillus subtilis through a staged induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, and 0.1% lithium chloride and 0.05% ferrous sulfate were added. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared from Kluyveromyces lactis through carbon and nitrogen starvation: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.05. 600 =18–20; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
[0008] As a further improvement of the present invention, the gradient heating reaction process in S2 is as follows: 38°C for 2 hours, 40°C for 3 hours, and 42°C for 7–11 hours, thereby breaking the intermolecular hydrogen bond network of bile components through the temperature gradient.
[0009] As a further improvement of the present invention, the parameters of the pulsed oscillation reaction in S2 are: oscillation for 10 minutes followed by a 5-minute pause, amplitude of 6 cm, and protein conformational changes are promoted through intermittent shear force.
[0010] As a further improvement of the present invention, the light-shielding reaction in S2 adopts an infrared shielding reactor to block light radiation with a wavelength of 400–500 nm and prevent photosensitive bilirubin isomerization.
[0011] As a further improvement of the present invention, the conditions for supercritical CO2 extraction in S3 are: pressure 25–30 MPa, temperature 45–50 °C, CO2 flow rate 20–25 L / h, extraction time 1.5–2 hours, selectively removing cholesterol and free fatty acids.
[0012] As a further improvement of the present invention, the vacuum freeze-drying process in S3 is sequentially maintained at -60°C for 12 hours, at -20°C for 8 hours, and at room temperature for 4 hours.
[0013] The beneficial effects of this invention are: Through the antioxidant effect of zinc-manganese nanoparticles and the synergistic protective mechanism of the ternary complex (EGCG-rosmarinic acid-tea polyphenols), the bilirubin retention rate is greatly improved, significantly increasing the bilirubin and bile acid content in the finished product, far exceeding the standard of natural bezoar.
[0014] Synergistic effects of multiple enzymes were achieved through staged gradient heating and pulsed oscillation reaction to realize the targeted cleavage of bile lipoproteins. The synergistic effect of trypsin and glycosidase significantly improved the taurine binding rate. After IPTG induction, Bacillus subtilis showed a multiple-fold increase in phospholipase A2 activity, accelerating bilirubin calcium crystallization. After carbon and nitrogen source starvation acclimatization, Kluyveromyces lactis showed a significant increase in β-glucuronidase secretion, enhancing the bile acid dissociation efficiency. Bacillus stearothermophilus induced heat shock proteins under 0.8 MPa high pressure, increasing the temperature tolerance to 65℃ and broadening the reaction condition window.
[0015] This invention utilizes a three-dimensional technological innovation—nanomaterial enhancement, multi-enzyme engineering coupling, and supercritical purification—to construct the world's first controllable, standardized, and green in vitro culture system for bovine gallstones. Detailed Implementation
[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.
[0017] Example 1 A method for in vitro cultivation of natural quality bezoar using biological enzymes includes the following steps: S1. Zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh ox bile at a mass ratio of 1000:0.8:3.0. The mixture was then magnetically stirred at 55°C for 30 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes were added sequentially to the pretreated bile, and the reactions were carried out at 38℃ for 2 hours, 40℃ for 3 hours, and 42℃ for 7 hours. The intermolecular hydrogen bond network of bile components was broken by the temperature gradient. After adjusting the pH to 8.0, trypsin was added, and the reaction was carried out at 32℃ with pulsed oscillation for 8 hours (frequency 120 rpm / min), with a 5-minute pause every 10 minutes of oscillation and an amplitude of 6 cm. The protein conformational change was promoted by intermittent shear force. Finally, the second biological enzyme was added, and the reaction was carried out at 28℃ in the dark for 14 hours. An infrared shielded reactor was used to block light radiation at a wavelength of 400 nm to prevent the isomerization of photosensitive bilirubin, forming a layered enzymatic hydrolysis system and obtaining a multiphase enzymatic hydrolysate. S3. The pH of the enzymatic hydrolysate was adjusted to 2.0, and 0.5% chitosan flocculant was added. The mixture was allowed to stand at a gradient temperature of 4℃ for 24 hours. After centrifugation, supercritical CO2 extraction was used to remove lipid-soluble impurities. The supercritical CO2 extraction pressure was 25 MPa, the temperature was 45℃, the CO2 flow rate was 20 L / h, and the extraction time was 1.5 hours. Cholesterol and free fatty acids were selectively removed. The precipitate was collected and freeze-dried under vacuum until the water content was ≤3%. The vacuum freeze-drying process was carried out at -60℃ for 12 hours, at -20℃ for 8 hours, and at room temperature for 4 hours in sequence, finally obtaining high-purity bezoar.
[0018] The first bioenzyme was prepared by Bacillus subtilis through a phased induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, with the addition of 0.1% lithium chloride and 0.05% ferrous sulfate. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared from Kluyveromyces lactis through carbon and nitrogen starvation: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.05. 600 =18; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
[0019] Example 2 A method for in vitro cultivation of natural quality bezoar using biological enzymes includes the following steps: S1, zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh ox bile at a mass ratio of 1000:0.9:3.2. The mixture was then magnetically stirred at 58°C for 33 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes were added sequentially to the pretreated bile, and the reactions were carried out at 38℃ for 2 hours, 40℃ for 3 hours, and 42℃ for 8 hours. The intermolecular hydrogen bond network of bile components was broken by the temperature gradient. After adjusting the pH to 8.2, trypsin was added, and the reaction was carried out at 33℃ with pulsed oscillation for 9 hours (frequency 120 rpm / min), with a 5-minute pause every 10 minutes of oscillation and an amplitude of 6 cm. The protein conformational change was promoted by intermittent shear force. Finally, the second biological enzyme was added, and the reaction was carried out at 29℃ in the dark for 15 hours. An infrared shielded reactor was used to block light radiation at a wavelength of 450 nm to prevent the isomerization of photosensitive bilirubin, forming a layered enzymatic hydrolysis system and obtaining a multiphase enzymatic hydrolysate. S3. The pH of the enzymatic hydrolysate was adjusted to 2.2, and 0.6% chitosan flocculant was added. The mixture was then allowed to stand at a gradient temperature of 5°C for 25 hours. After centrifugation, supercritical CO2 extraction was used to remove lipid-soluble impurities. The supercritical CO2 extraction pressure was 26 MPa, the temperature was 46°C, the CO2 flow rate was 22 L / h, and the extraction time was 1.6 hours. Cholesterol and free fatty acids were selectively removed. The precipitate was collected and freeze-dried under vacuum until the water content was ≤3%. The vacuum freeze-drying process was carried out at -60°C for 12 hours, -20°C for 8 hours, and room temperature for 4 hours in sequence, finally obtaining high-purity bezoar.
[0020] The first bioenzyme was prepared by Bacillus subtilis through a phased induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, with the addition of 0.1% lithium chloride and 0.05% ferrous sulfate. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared from Kluyveromyces lactis through carbon and nitrogen starvation: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.05. 600 =19; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
[0021] Example 3 A method for in vitro cultivation of natural quality bezoar using biological enzymes includes the following steps: S1, zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh ox bile at a mass ratio of 1000:1.0:3.5. The mixture was then magnetically stirred at 58°C for 33 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes were added sequentially to the pretreated bile, and the reactions were carried out at 38℃ for 2 hours, 40℃ for 3 hours, and 42℃ for 9 hours. The intermolecular hydrogen bond network of bile components was broken by the temperature gradient. After adjusting the pH to 8.3, trypsin was added, and the reaction was carried out at 33℃ with pulsed oscillation for 10 hours (frequency 120 rpm / min), with a 5-minute pause every 10 minutes of oscillation and an amplitude of 6 cm. The protein conformational change was promoted by intermittent shear force. Finally, the second biological enzyme was added, and the reaction was carried out at 29℃ in the dark for 16 hours. An infrared shielded reactor was used to block light radiation of wavelength 480 nm to prevent the isomerization of photosensitive bilirubin, forming a layered enzymatic hydrolysis system and obtaining a multiphase enzymatic hydrolysate. S3. The pH of the enzymatic hydrolysate was adjusted to 2.5, and 0.7% chitosan flocculant was added. The mixture was then allowed to stand at a gradient temperature of 6°C for 28 hours. After centrifugation, supercritical CO2 extraction was used to remove lipid-soluble impurities. The supercritical CO2 extraction pressure was 28 MPa, the temperature was 47°C, the CO2 flow rate was 23 L / h, and the extraction time was 1.7 hours. Cholesterol and free fatty acids were selectively removed. The precipitate was collected and freeze-dried under vacuum until the water content was ≤3%. The vacuum freeze-drying process was carried out at -60°C for 12 hours, -20°C for 8 hours, and room temperature for 4 hours in sequence, finally obtaining high-purity bezoar.
[0022] The first bioenzyme was prepared by Bacillus subtilis through a phased induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, with the addition of 0.1% lithium chloride and 0.05% ferrous sulfate. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared from Kluyveromyces lactis through carbon and nitrogen starvation: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.05. 600 =19; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
[0023] Example 4 A method for in vitro cultivation of natural quality bezoar using biological enzymes includes the following steps: S1, zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh ox bile at a mass ratio of 1000:1.1:4.4. The mixture was then magnetically stirred at 64°C for 34 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes were added sequentially to the pretreated bile, and the reactions were carried out at 38℃ for 2 hours, 40℃ for 3 hours, and 42℃ for 10 hours. The intermolecular hydrogen bond network of bile components was broken by the temperature gradient. After adjusting the pH to 8.4, trypsin was added, and the reaction was carried out at 34℃ with pulsed oscillation for 11 hours (frequency 120 rpm / min), with a 5-minute pause every 10 minutes of oscillation and an amplitude of 6 cm. The protein conformational change was promoted by intermittent shear force. Finally, the second biological enzyme was added, and the reaction was carried out at 29℃ in the dark for 17 hours. An infrared shielded reactor was used to block light radiation of wavelength 490 nm to prevent photosensitive bilirubin isomerization, forming a layered enzymatic hydrolysis system and obtaining a multiphase enzymatic hydrolysate. S3. The pH of the enzymatic hydrolysate was adjusted to 2.7, and 0.9% chitosan flocculant was added. The mixture was then allowed to stand at a gradient temperature of 7°C for 35 hours. After centrifugation, supercritical CO2 extraction was used to remove lipid-soluble impurities. The supercritical CO2 extraction pressure was 29 MPa, the temperature was 49°C, the CO2 flow rate was 24 L / h, and the extraction time was 1.9 hours. Cholesterol and free fatty acids were selectively removed. The precipitate was collected and freeze-dried under vacuum until the water content was ≤3%. The vacuum freeze-drying process was carried out at -60°C for 12 hours, -20°C for 8 hours, and room temperature for 4 hours in sequence, finally obtaining high-purity bezoar.
[0024] The first bioenzyme was prepared by Bacillus subtilis through a phased induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, with the addition of 0.1% lithium chloride and 0.05% ferrous sulfate. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared from Kluyveromyces lactis through carbon and nitrogen starvation: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.05. 600 =19; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
[0025] Example 5 A method for in vitro cultivation of natural quality bezoar using biological enzymes includes the following steps: S1. Zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh ox bile at a mass ratio of 1000:1.2:4.5. The mixture was then magnetically stirred at 65°C for 35 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes were added sequentially to the pretreated bile, and the reactions were carried out at 38℃ for 2 hours, 40℃ for 3 hours, and 42℃ for 11 hours. The intermolecular hydrogen bond network of bile components was broken by the temperature gradient. After adjusting the pH to 8.5, trypsin was added, and the reaction was carried out at 35℃ with pulsed oscillation for 12 hours (frequency 120 rpm / min), with a 5-minute pause every 10 minutes of oscillation and an amplitude of 6 cm. The protein conformational change was promoted by intermittent shear force. Finally, the second biological enzyme was added, and the reaction was carried out at 30℃ in the dark for 18 hours. An infrared shielded reactor was used to block light radiation at a wavelength of 500 nm to prevent the isomerization of photosensitive bilirubin, forming a layered enzymatic hydrolysis system and obtaining a multiphase enzymatic hydrolysate. S3. The pH of the enzymatic hydrolysate was adjusted to 2.8, and 1.0% chitosan flocculant was added. The mixture was then allowed to stand at 8°C for 36 hours with a gradient cooling process. After centrifugation, supercritical CO2 extraction was used to remove lipid-soluble impurities. The supercritical CO2 extraction pressure was 30 MPa, the temperature was 50°C, the CO2 flow rate was 25 L / h, and the extraction time was 2 hours. Cholesterol and free fatty acids were selectively removed. The precipitate was collected and freeze-dried under vacuum until the water content was ≤3%. The vacuum freeze-drying process was carried out at -60°C for 12 hours, -20°C for 8 hours, and room temperature for 4 hours in sequence to finally obtain high-purity bezoar.
[0026] The first bioenzyme was prepared by Bacillus subtilis through a phased induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, with the addition of 0.1% lithium chloride and 0.05% ferrous sulfate. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared from Kluyveromyces lactis through carbon and nitrogen starvation: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.05. 600 =20; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
[0027] Table 1. Bilirubin content (%)
[0028] As can be seen from Table 1, the bilirubin content of the bezoar prepared in Examples 1-5 is much higher than the requirement in Part I of the 2025 edition of the Chinese Pharmacopoeia (bilirubin content shall not be less than 35.0%). This shows that the enzymatic reaction of bile in this application results in a bilirubin content in the bezoar prepared that is significantly higher than the standard in the pharmacopoeia.
[0029] Table 2. Bile acid content (%)
[0030] As shown in Table 2, the cholic acid content in the bezoar prepared in Examples 1-5 is much higher than the requirements for in vitro cultured bezoar stipulated in the 2025 edition of the Chinese Pharmacopoeia, Part I (cholic acid content shall not be less than 6.0%). This application uses enzymatic reactions of bile to prepare bezoar with a significantly higher cholic acid content than the standard in the pharmacopoeia.
[0031] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for in vitro cultivation of natural quality bezoar using biological enzymes, characterized in that, Includes the following steps: S1, zinc-manganese alloy nanoparticles (zinc / manganese atomic ratio 1:0.5) and EGCG-rosmarinic acid-tea polyphenol ternary complex (mass ratio 1:1.2:0.8) were added to fresh bovine bile at a mass ratio of 1000:(0.8–1.2):(3.0–4.5). The mixture was magnetically stirred at 55–65℃ for 30–35 minutes to form a nanoscale redox microenvironment, thus obtaining pretreated bile. S2, the first and third biological enzymes are added sequentially to the pretreated bile, and the reaction is carried out at a gradient temperature of 38–42℃ for 12–16 hours. After adjusting the pH to 8.0–8.5, trypsin is added, and the reaction is carried out at 32–35℃ with pulsed shaking for 8–12 hours (frequency 120 rpm / min). Finally, the second biological enzyme is added, and the reaction is carried out at 28–30℃ in the dark for 14–18 hours to form a layered enzymatic hydrolysis system, resulting in a multiphase enzymatic hydrolysate. S3. Adjust the pH of the enzymatic hydrolysate to 2.0–2.8, add 0.5–1.0% chitosan flocculant, and allow it to stand for 24–36 hours at a gradient cooling temperature of 4–8℃. After centrifugation, use supercritical CO2 extraction to remove fat-soluble impurities. Collect the precipitate and freeze-dry it under vacuum until the water content is ≤3%, finally obtaining high-purity bezoar.
2. The method for in vitro culture of natural quality bezoar using biological enzymes according to claim 1, characterized in that, The first bio-enzyme was prepared by Bacillus subtilis through a staged induction culture: the culture medium contained 8 g / L beef extract, 12 g / L tryptone, and 5 g / L sodium chloride, with the addition of 0.1% lithium chloride and 0.05% ferrous sulfate. After shaking culture at 37°C for 48 hours, IPTG at a final concentration of 0.5 mmol / L was added to induce expression for 6 hours. The second biological enzyme was prepared by starvation of Kluyveromyces lactis using carbon and nitrogen sources: the culture medium contained 25 g / L whey powder, 3 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. After three consecutive cycles of starvation-enrichment with added glucose (0.5% v / v each time) and methanol (0.2% v / v), the final cell density reached OD0.
05. 600 =18–20; The third bioenzyme was prepared by pressure-responsive domestication of Bacillus stearothermophilus: the culture medium contained 20 g / L maltose, 8 g / L peptone, and 1.0 g / L magnesium sulfate, and was cultured at 55°C with the pressure gradually increased to 0.8 MPa for 24 hours to induce the expression of heat shock protein.
3. The method for in vitro culture of natural quality bezoar using biological enzymes according to claim 1, characterized in that, The gradient heating reaction process in S2 is as follows: 38℃ for 2 hours, 40℃ for 3 hours, and 42℃ for 7–11 hours, breaking the intermolecular hydrogen bond network of bile components through the temperature gradient.
4. The method for in vitro culture of natural quality bezoar using biological enzymes according to claim 1, characterized in that, The parameters of the pulsed oscillation reaction in S2 are: oscillation for 10 minutes followed by a 5-minute pause, amplitude of 6 cm, and intermittent shear force to promote protein conformational changes.
5. The method for in vitro culture of natural quality bezoar using biological enzymes according to claim 1, characterized in that, The light-shielding reaction in S2 uses an infrared shielding reactor to block light radiation with a wavelength of 400–500 nm, preventing the isomerization of photosensitive bilirubin.
6. The method for in vitro culture of natural quality bezoar using biological enzymes according to claim 1, characterized in that, The conditions for supercritical CO2 extraction in S3 are: pressure 25–30 MPa, temperature 45–50 °C, CO2 flow rate 20–25 L / h, extraction time 1.5–2 hours, selectively removing cholesterol and free fatty acids.
7. The method for in vitro culture of natural quality bezoar using biological enzymes according to claim 1, characterized in that, The vacuum freeze-drying process in S3 is as follows: maintaining -60℃ for 12 hours, maintaining -20℃ for 8 hours, and maintaining room temperature for 4 hours.