Method for culturing calculus bovis in vitro

Abstract

The invention belongs to the technical field of in-vitro cultivation of calculus bovis, and particularly relates to a method for in-vitro cultivation of calculus bovis. According to the invention, firstly, the escherichia coli is subjected to bile domestication and ray induced mutation, a high-activity oxgall-resistant bezoar production engineering bacterium E.coli-NH01 is obtained through screening, a method for in-vitro cultivation of bezoar by using the engineering bacterium E.coli-NH01 is provided, the prepared bezoar meets the pharmacopoeia standard, the production efficiency of in-vitro cultivation of bezoar is improved, and the production cost is reduced. And the production cost is reduced, the overall forming period is shortened, and the key that precious traditional Chinese medicinal materials are popular can be achieved.

Description

Technical Field

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

[0002] Bezoar, a bilirubin stone found in the gallbladder, bile duct, and hepatic duct of bovines, is widely used in Traditional Chinese Medicine (TCM) to treat a variety of ailments, including epilepsy, stroke, pediatric shock, and sore throat. Known as the "King of Hundred Medicines," it is widely used in Traditional Chinese Medicine (TCM) to treat a wide range of conditions, including epilepsy, stroke, pediatric shock, and sore throat. However, due to the scarcity, high cost, and inconsistent quality of natural bezoar, it has been unable to meet clinical needs. In recent years, Chinese researchers have employed chemical synthesis and in vitro cultivation methods to produce artificial bezoar, cultured bezoar, and in vitro cultured bezoar substitutes. While these substitutes are essentially identical to natural bezoar in morphology, structure, and composition, artificial bezoar is simply a mixture of various ingredients, lacking the biological fermentation process in ox bile. Consequently, their active ingredients are limited, significantly reducing their efficacy. Cultured bezoar, in contrast, is achieved by implanting a nucleating material into the ox's gallbladder and then removing it through surgery or at slaughter. This method produces a product that closely resembles natural bezoar. However, the long production process, low yield, and cumbersome operation make it difficult to alleviate the shortage of natural bezoar.

[0003] Since the in vitro cultivated bezoar was developed by Professor Cai Hongjiao (CN1022668C, CN1044402A, CN1041105A) in the 1990s, Chinese scholars have continuously researched and improved its process. By simulating the pathological conditions for the formation of bovine gallstones, they use microorganisms and bovine bile to cultivate in vitro cultivated bezoar with the same chemical composition and clinical efficacy as natural bezoar.

[0004] Bacterial strain is the key point of in vitro cultivation bezoar into yellow, and its possible mechanism is that bacteria releases beta-glucuronidase (β-GD), decomposes the D-glucuronic acid in conjunction with bilirubin, makes bilirubin calcium precipitation.But not all bacterium all possesses the ability of bezoar conversion, and ox bile has broad-spectrum antibacterial action, and multiple bacteria can not survive for a long time in bile, has a strong impact on in vitro cultivation bezoar into yellow speed.At present only have the patent (CN102429927B of Wuhan Jianmin Dapeng Pharmaceutical Co., Ltd., CN102512457B) add in ox bile by single bacterial species such as Escherichia coli, Proteus or Enterobacter or composite bacterial species and ferment for several days, promote mucin generation, to gather bilirubin, cholic acid etc. to form colloidal precipitation, afterwards regulating pH is acidic, changes colloidal structure, and off-axis rotation forms bezoar coagulant. This method requires the addition of a large amount of complex bilirubin calcium and bile acid, which results in high production costs. This reflects the low efficiency of the enzymatic reaction of the engineered strain, the low bioconversion rate of key components such as bilirubin and bile acid, and the slow rate of yellow formation, resulting in the price of in vitro cultivated bezoar products still being expensive. Summary of the Invention

[0005] To address the above technical issues, the present invention screens a highly active, bile-resistant bezoar production strain by bile acclimation and radiation-induced mutation of Escherichia coli. The present invention also provides a method for in vitro cultivation of bezoar, which improves the production efficiency of in vitro cultivation of bezoar, reduces production costs, and shortens the overall formation cycle, achieving the key to making precious Chinese medicinal materials widely available to the public. Specifically, the present invention includes the following contents:

[0006] In a first aspect, the present invention provides a method for cultivating bezoar in vitro, the method comprising the following steps:

[0007] (1)(1) Inoculating the engineered bacterium E. coli-NH01 into ox bile for fermentation to obtain fermented bile; the engineered bacterium E. coli-NH01 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 30, 2024, with the deposit number CGMCC No. 32430, and the classification name is Escherichia coli;

[0008] (2) adding saturated calcium oxide solution and sodium metabisulfite to the ox bile, heating to boiling, and filtering the reaction solution to obtain a precipitate;

[0009] (3) adding the fermented bile, bilirubin, bile acid, zinc sulfate, magnesium sulfate, deoxycholic acid, and taurine described in step (1) to the precipitate described in step (2), stirring and mixing to obtain a mixture;

[0010] (4) drying and molding the mixture in step (3) to obtain a finished product.

[0011] Preferably, in step (1), 1-4 mL of the engineered bacteria E. coli-NH01 liquid is inoculated per 100 mL of ox bile.

[0012] Preferably, in step (1), 2 mL of the engineered bacteria E. coli-NH01 liquid is inoculated into every 100 mL of ox bile.

[0013] Preferably, the fermentation parameters in step (1) are fermentation at 37° C. for 48-96 hours, and sterilization is performed after fermentation is completed.

[0014] Preferably, in step (2), 50-200 mL of saturated calcium oxide solution and 0.01-0.02 g of sodium metabisulfite are added to every 100 mL of ox bile.

[0015] Preferably, in step (2), 100 mL of saturated calcium oxide solution and 0.01-0.02 g of sodium metabisulfite are added to every 100 mL of ox bile.

[0016] Preferably, the solution is heated and stirred until boiling for 10-20 minutes, then the heating is stopped and the precipitate is collected by filtration when the solution temperature drops to 50-60°C.

[0017] Preferably, the saturated calcium oxide solution is prepared by adding 2-4 g of calcium oxide to every 100 mL of deionized water to prepare a saturated calcium oxide solution.

[0018] Preferably, the saturated calcium oxide solution is prepared by adding 2 g of calcium oxide to every 100 mL of deionized water to prepare a saturated calcium oxide solution.

[0019] Preferably, step (3) comprises: adding 100 mL of the fermented bile of step (1) to the precipitate of step (2), adding 17-20% of bilirubin, 0.5-0.8% of bile acid, 0.03-0.1% of zinc sulfate, 0.03-0.1% of magnesium sulfate, 1-2% of deoxycholic acid, and 0.8-1.6% of taurine relative to the weight of the fermented bile, and stirring and mixing.

[0020] Preferably, 20% bilirubin, 0.5% bile acid, 0.06% zinc sulfate, 0.06% magnesium sulfate, 1.2% deoxycholic acid, and 1% taurine are added relative to the weight of the fermented bile.

[0021] Preferably, the molding method in step (4) is:

[0022] a) drying the mixture of step (3), selecting one piece as a stone, and grinding the rest into powder to obtain bezoar powder;

[0023] b) the stone is placed in a shaping machine and wrapped in a layer of body culture bezoar powder, after which the stone is taken out and sprayed with a layer of ox bile, and again placed in a shaping machine and wrapped in a layer of body culture bezoar powder;

[0024] c) Repeat step b) 10-14 times until the dough is formed into balls, and then dry to obtain the finished product.

[0025] Preferably, the drying method in step a) is low-temperature vacuum drying followed by freezing.

[0026] Preferably, the drying method in step c) is vacuum drying at 37° C. and 0.06-0.1 MPa.

[0027] In a second aspect, the present invention provides bezoar obtained by in vitro cultivation using the method described in the first aspect.

[0028] In a third aspect, the present invention provides the use of the bezoar described in the second aspect in pharmaceutical applications.

[0029] The beneficial effects of the present invention are as follows: the invention first screens a highly active ox bile-resistant bezoar production engineered bacterium E.coli-NH01 by bile acclimation and radiation-induced mutation of Escherichia coli, and provides a method for in vitro cultivation of bezoar using the engineered bacterium E.coli-NH01. The prepared bezoar meets the pharmacopoeia standards, improves the production efficiency of in vitro cultivation of bezoar, reduces production costs, shortens the overall forming cycle, and is the key to realizing the universal benefit of precious Chinese medicinal materials, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The gross appearance and microscopic structure of bezoar cultured in vitro; A is the gross appearance of bezoar, which is spherical or quasi-spherical, with a diameter between 5mm and 20mm, a smooth appearance, and a color ranging from yellow-red to brownish-yellow; B is a cross-sectional view of bezoar, showing concentric layers and a visible bezoar core; C is a microscopic photograph of bezoar (400x magnification), showing small yellowish-brown particles with irregular shapes and a typical golden color. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0032] (2) The engineered bacteria E. coli-NH01 described in the following examples was deposited in the General Microbiology Center of the China Culture Collection Administration on October 30, 2024, with the deposit number CGMCC No. 32430; the classification name is Escherichia coli; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the contact number is 010-64807355.

[0033] Example 1 Method for Cultivating Bezoar in Vitro

[0034] 1. Preparation Method

[0035] (1) The engineered bacteria E. coli-NH01 was inoculated into cattle bile, with 12 mL of bacterial solution inoculated into 1 L of cattle bile, and fermented at 37°C for 72 h. After fermentation, steam sterilization was performed.

[0036] (2) Add 100g of calcium oxide to 5L of deionized water to prepare a saturated calcium oxide solution. Add 1L of saturated calcium oxide solution and 0.1g of sodium metabisulfite to every 1L of fermented ox bile. Heat and stir until boiling for 15 minutes. Then let it stand at about 60°C and filter out the precipitate.

[0037] (3) Add 1 L of fresh ox bile to the precipitate, add 20% bilirubin, 0.5% bile acid, 0.06% zinc sulfate, 0.06% magnesium sulfate, 1.2% deoxycholic acid, and 1% taurine relative to the weight of the bile, and stir to mix.

[0038] (4) After freezing, vacuum dry at low temperature. Take the stone blocks of appropriate size as the core and grind the rest into powder. Place the stone blocks in a shaping machine and coat them with a layer of cultured bezoar powder. Then take them out and spray them with a layer of ox bile. Place them in the shaping machine again and coat them with a layer of cultured bezoar powder. Repeat this process 10-14 times. After forming into balls, place them in a vacuum drying oven at 37°C and 0.08MPa and dry them for 24 hours.

[0039] 2. Detection

[0040] High-performance liquid chromatography (HPLC) was used to determine the content of medicinal components in in vitro cultured bezoar, including free bilirubin, free bile acid, conjugated bile acids (taurocholic acid, taurodeoxycholic acid, glycocholic acid, and glycodeoxycholic acid), and bilirubin. The chromatographic column was packed with octadecylsilane-bonded silica (ACCHROM Xaqua C18 column, 25 cm length, 4.6 mm inner diameter, 5 μm particle size). Acetonitrile was used as mobile phase A, and 0.2% trifluoroacetic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1 at a flow rate of 1 mL / min. Detection was performed using an evaporative light scattering detector.

[0041] Table 1 Gradient elution parameters

[0042]

[0043] (1) Free bilirubin detection

[0044] Preparation of reference substance: Weigh an appropriate amount of bilirubin and add dichloromethane to prepare a 6.87 μg / mL solution.

[0045] Preparation of sample solution: Take about 10 mg of sample powder (passed through No. 6 sieve), place it in a brown conical flask, add 50 mL of dichloromethane, plug the flask tightly, weigh and mix, extract in an ice bath with ultrasound for 40 minutes, weigh and make up the weight loss with dichloromethane, centrifuge at 4000 rpm for 10 minutes, and take the supernatant for analysis.

[0046] (2) Determination of bile acid content

[0047] Preparation of reference substances: Weigh the free bile acid reference substance (cholic acid 0.6 mg / ml, deoxycholic acid 0.15 mg / ml), taurocholic acid reference substance (taurocholic acid 0.2 mg / ml), taurodeoxycholic acid reference substance (taurodeoxycholic acid 0.08 mg / ml), glycocholic acid reference substance (glycocholic acid 0.2 mg / ml), and glycodeoxycholic acid reference substance (glycodeoxycholic acid 0.08 mg / ml) respectively, and add the corresponding amount of methanol to prepare the reference substance.

[0048] Preparation of sample solution: Take about 200 mg of sample powder (passed through No. 3 sieve), place it in a brown conical flask, add 25 mL of methanol, plug the flask tightly, weigh and mix, extract in an ice bath with ultrasound for 40 min, weigh and make up the weight loss with methanol, centrifuge at 4000 rpm for 10 min, and take the supernatant for analysis.

[0049] (3) Determination of bilirubin content

[0050] Preparation of reference substance: Weigh an appropriate amount of bilirubin and add dichloromethane to prepare a 40 μg / mL solution.

[0051] Preparation of sample solution: Take about 10 mg of sample powder (passed through No. 6 sieve), place it in a brown conical flask, add 10 mL of 10% oxalic acid solution, plug the flask tightly, mix well, add 100 mL of water-saturated dichloromethane, weigh and mix well, extract in an ice bath with ultrasound for 40 min, weigh and make up the weight loss with dichloromethane, centrifuge at 4000 rpm for 10 min, and take the supernatant for analysis.

[0052] 3. Test results

[0053] A total of 1327g of raw materials were put in, and a total of 305g of bezoar product was obtained. Compared with the patent of Wuhan Jianmin Dapeng Pharmaceutical with the current application number of 202010577790.5, this process has the advantages of streamlined steps, low cost and high yield. The bezoar yield has been increased from 13.4% to 22.9%. After in vitro cultivation, the bezoar is spherical or quasi-spherical, with a diameter between 5mm and 20mm. The bezoar lumps have a smooth appearance and are yellow-red to brownish-yellow in color. The texture is crisp and light, with a special aroma and bitterness, and then a sweet taste, with a certain cool feeling. In cross-section, the bezoar presents concentric layers, and the nucleation core of the bezoar can be seen. Microscopic observation: The small yellow-brown particles are irregular in shape. After standing for a while, they will quickly dissolve and show a typical golden yellow color, with microscopic characteristics that meet the requirements of the standards.

[0054] In the spectrum of the in vitro cultivated bezoar prepared by this process, no chromatographic peak of free bilirubin appears at the position corresponding to the retention time of the chromatographic peak of the reference substance, which complies with the provisions on the free bilirubin content of in vitro cultivated bezoar in the "Pharmacopoeia of the People's Republic of China" (2025 public edition).

[0055] The average bilirubin content of the bezoar samples produced by the new process was 28.77±2.66%, meeting the standard of the Pharmacopoeia of the People's Republic of China (should be no less than 25%). The average total content of the four conjugated bile acids (taurocholic acid, taurodeoxycholic acid, glycocholic acid, and glycodeoxycholic acid) was 19.77±5.77%, meeting the standard of the Pharmacopoeia of the People's Republic of China (should be no less than 4%). The free bile acid content was 7.16±1.03%, meeting the standard of the Pharmacopoeia of the People's Republic of China (should be 6% to 13%). Through component analysis, the quality standards of in vitro cultivated bezoar met the official national drug standards, and the bilirubin and conjugated bile acid contents were significantly higher than the standard.

[0056] Table 2 Determination of the content of medicinal ingredients in bezoar cultured in vitro

[0057]

[0058] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for cultivating bezoar in vitro, characterized in that, The method comprises the following steps: (1) inoculating the engineered bacterium E. coli-NH01 into ox bile for fermentation to obtain fermented bile; the engineered bacterium E. coli-NH01 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 30, 2024, with a deposit number of CGMCC No. 32430 and a classification name of Escherichia coli; (2) adding saturated calcium oxide solution and sodium metabisulfite to the ox bile, heating to boiling, and filtering the reaction solution to obtain a precipitate; (3) adding the fermented bile, bilirubin, bile acid, zinc sulfate, magnesium sulfate, deoxycholic acid, and taurine described in step (1) to the precipitate described in step (2), stirring and mixing to obtain a mixture; (4) drying and molding the mixture in step (3) to obtain a finished product.

2. The method according to claim 1, wherein In the step (1), 1-4 mL of the engineered bacteria E. coli-NH01 solution is inoculated into every 100 mL of ox bile.

3. The method according to claim 2, wherein The fermentation parameters in step (1) are fermentation at 37° C. for 48-96 hours, and sterilization is performed after fermentation is completed.

4. The method according to claim 1, wherein In step (2), 50-200 mL of saturated calcium oxide solution and 0.01-0.02 g of sodium metabisulfite are added to every 100 mL of ox bile.

5. The method according to claim 4, wherein Heat and stir until it boils for 10-20 minutes, then stop heating and filter to obtain the precipitate when the solution temperature drops to 50-60°C.

6. The method according to claim 1, wherein The step (3) comprises: adding 100 mL of the fermented bile of step (1) to the precipitate of step (2), adding 17-20% of bilirubin, 0.5-0.8% of bile acid, 0.03-0.1% of zinc sulfate, 0.03-0.1% of magnesium sulfate, 1-2% of deoxycholic acid, and 0.8-1.6% of taurine relative to the weight of the fermented bile, and stirring and mixing.

7. The method according to claim 1, wherein The molding method in step (4) is: a) drying the mixture of step (3), selecting one piece as a stone, and grinding the rest into powder to obtain bezoar powder; b) the stone is placed in a shaping machine and wrapped in a layer of body culture bezoar powder, after which the stone is taken out and sprayed with a layer of ox bile, and again placed in a shaping machine and wrapped in a layer of body culture bezoar powder; c) Repeat step b) 10-14 times until the dough is formed into balls, and then dry to obtain the finished product.

8. The method according to claim 7, wherein The drying method in step a) is low-temperature vacuum drying followed by freezing.

9. The method according to claim 7, wherein: The drying method in step c) is vacuum drying at 37° C. and 0.06-0.1 MPa.

10. Bezoar obtained by in vitro cultivation according to any one of claims 1 to 9.

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