Calculus bovis with combination of in-vitro cultivation and in-vivo cultivation and preparation method of calculus bovis

Abstract

The invention discloses calculus bovis combining in-vitro cultivation and in-vivo cultivation and a preparation method. The preparation method specifically comprises the following steps: (1) preparing an escherichia coli mixed bacteria solution; (2) preparing composite mucoprotein paste; and (3) carrying out in-vivo cultivation of the calculus bovis. Bacteria are preserved in the composite mucoprotein paste, and the composite mucoprotein paste has viscosity and is used as condensation nuclei for inducing calculus bovis condensation. Compared with the traditional crystal nucleus, the scheme can better protect the activity of the strain, the effect of protecting and slowly releasing the strain is achieved by using an embedding method, and strain inactivation caused by direct contact of the strain with ox bile is avoided. The ox bile simulation liquid with the best escherichia coli amplification effect is used as a bacterial liquid amplification culture medium injected into the ox gall bladder; after the mixed solution is injected, the 10% calcium chloride solution is injected again, so that a sticky bezoar bed is formed; the bilirubin content of the prepared bezoar is greater than or equal to 30%, and the cholic acid content is greater than or Each cattle cattle yellow is greater than 8g of calculus bovis; the success rate of calculus bovis preparation is greater than 90%.

Description

Technical Field

[0001] The invention relates to a method for preparing bezoar, and in particular to a method for preparing bezoar combining in vitro cultivation and in vivo cultivation. Background Art

[0002] Bezoar is a gallstone of cattle. It is oval in shape, light in weight, with a golden to yellow-brown surface, delicate and shiny. It can be used to relieve fever, detoxify, and calm shock. Modern bezoars are divided into four types according to their quality: artificial bezoar, in vitro cultured bezoar, natural bezoar, and cultured bezoar. Cultivated bezoar is a gallstone produced in cattle by artificial means, similar to artificially cultivated pearls. High-quality cultured bezoars are completely consistent with the intrinsic quality of natural bezoars.

[0003] The existing traditional cultivation technology is to make an incision on the cow's gallbladder, implant the crystal nucleus with the strain, and then suture it. The operation will cause bleeding, and the success rate is 75%. The quality of cultivated bezoar varies, it is difficult to control, and the growth cycle is long. Since cultivated bezoar still needs to be inoculated in the cow's body, it usually takes half a year to a year to form stones. A stone is only 3 to 5 grams. The success rate of obtaining high-quality cultivated bezoar is low, so it is difficult to form large-scale production. In addition, due to the easy inactivation of strains and other reasons, the traditional technology has a 25% probability of failure.

[0004] In the prior art, CN118020715A discloses a method for cultivating bezoar in vivo. Although it discloses that after the bezoar bed is produced by injection, the cattle can eat after one day, and the influence on the normal growth and fattening of the cattle is very small, CN115067275B discloses a method for cultivating three-dimensional composite bezoar in vivo, which has the characteristics of better adhesion and is easier to gather free bile salt particles in bile. However, the above prior arts all have the technical problem of low success rate of traditional cultivation.

[0005] The prior art CN1686175A natural bezoar in vitro cultivation method discloses that ox bile is mixed with composite microbial preparation liquid for fermentation to produce saturated calcium oxide solution and composite calcium bilirubin; however, it is pure in vitro culture of bezoar and its quality is inferior to that of in vivo culture. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing bezoar by combining in vitro cultivation and in vivo cultivation in view of the shortcomings of the prior art. Specifically, the method comprises:

[0007] (1) preparing a mixed bacterial solution of Escherichia coli;

[0008] (2) preparing a composite mucin paste;

[0009] (3) Cultivate bezoar in vivo.

[0010] Furthermore, step (1) includes culturing Escherichia coli using a cow bile simulation solution or cow bile solution to obtain a mixed Escherichia coli solution.

[0011] Further, step (2) comprises,

[0012] The mixed bacterial liquid of cow bile and Escherichia coli is mixed and cultured; a calcium hydroxide solution is added to react, centrifuged, a precipitate is taken, an adhesive is added, and the mixture is mixed to obtain a composite mucin paste.

[0013] Further, step (3) comprises,

[0014] After the cattle are anesthetized, they are injected with a mixed solution of composite mucin paste and E. coli and cultured to turn them into yellow.

[0015] Furthermore, in step (1), the ox bile simulation liquid is prepared by using ox bile powder and water; for example, it is prepared into 1-20% ox bile simulation liquid; for example, 5% ox bile simulation liquid.

[0016] Furthermore, the Escherichia coli in step (2) is a mixed strain of three growth rate strains, namely, fast, medium and slow, separated from cow bile, to avoid the strain amplification rate being too fast, resulting in the inability to stably produce fermentation products, a decrease in bacterial productivity, and changes in the flow properties of the fermentation liquid.

[0017] Further, the three strains were shake-cultured for 6 hours (initial OD = 0.1) in 200 ml of culture medium. After 6 hours, the OD values ​​of each strain were monitored. The OD value of the fast strain was greater than 2, such as 2.5 and 2.1; the OD value of the medium strain was 1-2, such as 1.5, 1.8, and 1.2; and the OD value of the slow strain was less than 1, such as 0.8 and 0.7.

[0018] Furthermore, in step (2), the temperature of the mixed culture of the ox bile and the E. coli is 25-42°C, for example 37°C.

[0019] Furthermore, the calcium hydroxide solution in step (2) is a saturated solution.

[0020] Furthermore, in step (2), the binder is hydroxymethylpropyl cellulose and its derivatives and salts, or is one or a combination of two or more of polyvidone (PVP), hydroxypropyl cellulose, methyl cellulose (MC), and ethyl cellulose (EC); for example, 1.5% hydroxymethylpropyl cellulose.

[0021] Furthermore, in step (3), before injecting the composite mucin paste and the mixed bacterial solution of E. coli, the gallbladder is first punctured to extract bile, and then the bile is mixed with the composite mucin paste and the mixed bacterial solution of E. coli and injected into the cattle gallbladder again.

[0022] Furthermore, in step (3), the ratio of the composite mucin paste to the Escherichia coli mixed bacterial solution is (0.5-1.5): (1-5), for example, 1: 3. The viscosity is appropriate at this ratio, and the composite mucin paste is a paste with high viscosity. At this ratio, the injection solution will not be too viscous to be injected using a small-bore syringe, but still has a viscosity that is conducive to the formation of a bezoar bed.

[0023] Furthermore, in step (3), after the mixed solution is injected, 10% calcium chloride solution is injected again to form a viscous bezoar bed.

[0024] Furthermore, after the injection, the wound site was rinsed with saline to avoid infection.

[0025] Furthermore, the cattle were raised normally after the operation. After 12 months, the cattle were slaughtered, the gallbladders were removed, the bile was filtered, and the bezoar precipitate was taken.

[0026] The invention also provides a bezoar prepared by combining in vitro cultivation and in vivo cultivation.

[0027] Furthermore, the bilirubin content of bezoar is ≥30%, and the bile acid content is ≥5%.

[0028] Furthermore, each bezoar is older than 5 hours, and furthermore, is 8-13 g of bezoar.

[0029] Furthermore, the success rate of bezoar preparation is greater than 90%, and further, greater than 95%.

[0030] The excellent effects of the present invention are as follows: (1) the bacteria are preserved in the composite mucin paste, which is sticky and serves as a condensation nucleus for inducing the coagulation of bezoar. Compared with the traditional crystal nucleus, the present solution can better protect the activity of the strain, and the embedding method is used to protect and release the strain, so as to avoid the strain from directly contacting the cow bile and causing the strain to be inactivated; (2) the cow bile simulation liquid with the best effect of amplifying Escherichia coli is used as the bacterial liquid amplification medium injected into the cow gallbladder; (3) after the mixed solution is injected, 10% calcium chloride solution is injected again to form a sticky bezoar bed; (4) the bilirubin content of the bezoar is ≥30%, and the bile acid content is ≥5%; each cow bezoar is more than 5 hours, and further 8-13g of bezoar; the success rate of bezoar preparation is more than 90%, and further, more than 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 For plate expansion compound mucin paste bacterial test;

[0032] Figure 2 This is a photo of the composite mucin paste. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0034] Example 1

[0035] A preparation method of a composite mucin paste comprises the following steps:

[0036] Take 50ml of fresh ox bile, add 2.5ml of Escherichia coli mixed bacterial solution (screen out three fast, medium and slow growth strains isolated from ox bile to avoid the strain amplification speed being too fast, resulting in the inability to stably produce fermentation products, the decrease in bacterial productivity, and the change of the fluid properties of the fermentation liquid. The three strains were shaken for 6h (initial OD = 0.1) in 200ml culture medium. After 6h, the OD values ​​of each strain were monitored. Strain 1 OD = 2.03, strain 2 OD = 1.47, and strain 3 OD = 0.86. The growth rates of the three strains are quite different. The fastest growing strain will reach the plateau earlier, which is not conducive to the production of β-glucuronidase required for ox bile fermentation and cannot fully achieve the purpose of fermentation. Strain with slower growth rate is needed to inhibit growth and reduce the amplification rate), mix well and shake at 37°C for 72 hours. Add saturated calcium hydroxide solution and stir for 30 minutes. After the mucin complex and calcium hydroxide have fully reacted, centrifuge and take the precipitate. Add 1.5% hydroxymethylpropyl cellulose (hydroxypropyl methylcellulose is a partial methyl and partial polyhydroxypropyl ether of cellulose, which can be dissolved in cold water to form a solution with a certain viscosity, and its properties are similar to those of mucin in tears) and mix well to obtain a composite mucin paste.

[0037] The paste contains bacteria and is sticky, which serves as a condensation nucleus to induce bezoar coagulation. Compared with the traditional crystal nucleus, this scheme can better protect the activity of the strain. The embedding method is used to protect and release the strain, and prevent the strain from directly contacting the cow bile and causing the strain to be inactivated. The gun tip picks up the composite mucin paste and spreads it on the LB agar plate, incubating at 37℃ for 16h. A large number of bacteria were observed on the plate. Figure 1 .

[0038] Example 2

[0039] An Escherichia coli culture medium suitable for bovine gallbladder reaction, comprising the following steps:

[0040] Use ox bile powder and sterile water to prepare 5% ox bile simulation liquid, and compare the effect of amplifying E. coli with LB broth medium, EC broth medium and tryptic soy protein medium. Inoculate E. coli in 10ml LB broth medium, shake for 4h, and measure the OD600 value (~1.5). Dilute to 0.1OD in 10ml 5% ox bile simulation liquid, LB broth medium, EC broth medium and tryptic soy protein medium, shake for 6h, and then measure the OD600 value of E. coli amplified using each culture medium again (1OD contains 10 8 strains) to determine the best inoculation medium. The test data are as follows:

[0041] Table 1 Cultivation conditions of different culture media

[0042]

[0043]

[0044] Therefore, we selected 5% ox bile simulated solution which is closest in composition to ox bile and has the best effect in amplifying E. coli as the bacterial liquid amplification medium for injection into the ox gallbladder.

[0045] Example 3

[0046] A method for cultivating bezoar in vivo, comprising the following steps:

[0047] ① Restraint: A 1.5-year-old bull was selected for modeling. After fasting for 24 hours and depriving of water for 6-12 hours, the bull was restrained in a standing position in a pen with its head and neck raised to reduce the occurrence of anesthesia complications. Standing restraint can better stabilize the position of the gallbladder and prevent it from being deformed due to squeezing.

[0048] ② Anesthesia: Use traditional alcohol anesthesia. Fasting for 12-24 hours before surgery is required. Administer 40% alcohol, 250-300 ml per 100 kg body weight. Surgery can begin 30 minutes after anesthesia. The anesthesia effect can last for 1-2 hours.

[0049] ③Injection: Confirm that the cattle gallbladder is located at the intersection of the line connecting the right shoulder and hip joint of the cattle and the third rib from the bottom as the midpoint of the incision, and use a syringe to puncture the gallbladder to extract 50ml of bile. Mix 10ml of compound mucin paste and Escherichia coli mixed bacterial solution (1:3), and use a small-bore syringe to inject into the cattle gallbladder. Then immediately inject 10% calcium chloride solution to form a viscous bezoar bed. Rinse the wound site with saline to avoid infection.

[0050] ④ Become yellow: After the operation, the cattle are raised normally. After 12 months, the cattle are slaughtered, the gallbladders are removed, the bile is filtered, and the bezoar precipitates are taken. 8-13g of bezoar can be obtained from each cattle. The bilirubin and bile acid content of the obtained bezoar is measured by high performance liquid chromatography specified in the "Chinese Pharmacopoeia". Cultivated bezoar belongs to the category of natural bezoar. The pharmacopoeia stipulates that the bilirubin content must be ≥25% and the bile acid content must be ≥4%. The test results are as follows:

[0051] Table 2 Randomly select 3 bezoars for testing

[0052] sample Bilirubin cholic acid Bezoar① 31.5％ 6.16％ Bezoar② 32.7％ 5.47％ Bezoar③ 30.2％ 5.98％

[0053] The results showed that the quality of cultivated bezoar obtained through this scheme was much higher than the value required by the pharmacopoeia.

[0054] Parallel experiments were conducted on 20 cows, and all of them successfully produced more than 8g of bezoar.

[0055] Comparative Example 1

[0056] Only an equal volume of E. coli liquid was injected into 3 cows without composite mucin paste. Other means were operated and fed in the same manner as in the embodiment. After 12 months, no obvious coagulation of bezoar was observed in one cow. The bezoar precipitate obtained from two cows was less than 1g, and the yield was far lower than the technical solution of the present invention, and the success rate was also low.

[0057] Test conclusion: The present invention preserves bacteria in a composite mucin paste, which is sticky and serves as a condensation nucleus for inducing bezoar coagulation. Compared with the traditional crystal nucleus, this scheme can better protect the activity of the strain, and the use of the embedding method has the effect of protecting and slow-releasing the strain, avoiding the strain from directly contacting the cow bile and causing the strain to be inactivated; the cow bile simulation liquid with the best effect of amplifying Escherichia coli is used as the bacterial liquid amplification culture medium injected into the cow gallbladder; after the mixed solution is injected, 10% calcium chloride solution is injected again to form a sticky bezoar bed; and then the bezoar with a quality far higher than that of the pharmacopoeia is prepared, and the success rate is much higher than the traditional bezoar in vivo preparation method.

[0058] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless specifically defined as herein.

[0059] It should be understood that the detailed description of the technical solutions of the present invention by means of the preferred embodiments is illustrative rather than restrictive. A person skilled in the art may modify the technical solutions described in the embodiments, or replace some of the technical features by equivalents, based on reading the specification of the present invention; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing bezoar by combining in vitro cultivation and in vivo cultivation, characterized in that: include: (1) Preparing a mixed bacterial solution of Escherichia coli; (2) preparing a composite mucin paste; (3) Cultivate bezoar in vivo.

2. The method according to claim 1, characterized in that: Step (1) includes culturing Escherichia coli using a cow bile simulated liquid or cow bile liquid to obtain a mixed Escherichia coli liquid.

3. The method according to claim 1, characterized in that: Furthermore, step (2) includes, The mixed bacterial liquid of cow bile and Escherichia coli is mixed and cultured; a calcium hydroxide solution is added to react, centrifuged, a precipitate is taken, an adhesive is added, and the mixture is mixed to obtain a composite mucin paste.

4. The method according to claim 1, characterized in that: Step (3) includes, After the cattle are anesthetized, they are injected with a mixed solution of composite mucin paste and E. coli and cultured to turn them into yellow.

5. The method according to claim 1, characterized in that: The E. coli in step (2) is a mixed strain of three growth rate strains, namely, fast, medium and slow, which are screened and isolated from cow bile, to avoid the strain amplification speed being too fast, resulting in the inability to stably produce fermentation products, reducing the productivity of the bacteria, and changing the flow properties of the fermentation liquid.

6. The method according to claim 1, characterized in that: In step (2), the binder is hydroxymethylpropyl cellulose and its derivatives and salts, or one or a combination of two or more of polyvidone (PVP), hydroxypropyl cellulose, methyl cellulose (MC), and ethyl cellulose (EC); for example, 1.5% hydroxymethylpropyl cellulose.

7. The method according to claim 1, characterized in that: In step (3), before injecting the composite mucin paste and the mixed bacterial solution of E. coli, the gallbladder is first punctured to extract bile, and then the bile is mixed with the composite mucin paste and the mixed bacterial solution of E. coli and injected into the cattle gallbladder again.

8. The method according to claim 1, characterized in that: In step (3), the ratio of the composite mucin paste and the Escherichia coli mixed bacterial solution for injection is (0.5-1.5):(1-5), for example, 1:3; and / or After the mixed solution is injected, calcium chloride solution is injected again to form a viscous bezoar bed.

9. A bezoar prepared by combining in vitro cultivation and in vivo cultivation.

10. Bezoar according to claim 9, characterized in that: The bilirubin content of bezoar is ≥30%, and the bile acid content is ≥5%; Each bezoar is greater than 5h, and further 8-13g bezoar; The success rate of bezoar preparation is greater than 90%, and further, greater than 95%.

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