A screening medium and screening method for β-glucuronidase-producing bacteria

Abstract

This invention belongs to the field of microbiology, specifically relating to a screening culture medium and screening method for β-glucuronidase-producing bacteria. The screening culture medium for β-glucuronidase-producing bacteria comprises the following raw materials in parts by weight: 3-10 parts crude bovine bile extract, 12-24 parts ammonium nitrate, 10-20 parts sodium chloride, 1-10 parts dipotassium hydrogen phosphate, 1-8 parts quercetin, and 0.1-0.5 parts 5-bromo-4-chloro-3-indolyl-β-D-glucuronide; wherein the crude bovine bile extract is an ethanol extract of bovine bile powder. This screening culture medium allows for the rapid isolation of β-glucuronidase-producing bacteria from bovine bile, and these isolated bacteria, when used in the preparation of bezoar, can yield high-yield and high-quality bezoar, thus improving the production efficiency of bezoar.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a screening culture medium and screening method for β-glucuronidase-producing bacteria. Background Technology

[0002] Bezoar is a gallstone found in the gallbladder, bile duct, or hepatic duct of cattle (Bovine cattle or buffalo). It possesses medicinal properties such as clearing heat and detoxifying, calming the nerves, and relieving convulsions. As a precious traditional Chinese medicine, its medicinal value primarily relies on its various active ingredients, such as bile acids, bilirubin, and cholesterol, with bilirubin being a crucial component. The Chinese Pharmacopoeia stipulates that the bilirubin content in bezoar must not be less than 25.0%.

[0003] The formation of bilirubin mainly involves the deglucuronidation of water-soluble conjugated bilirubin in the bovine gallbladder by β-glucuronase produced by bacteria, generating free bilirubin that is insoluble in bile. The complex bilirubin calcium formed by the combination of these free bilirubin and calcium ions constitutes the core of bezoar and plays a crucial role in the formation of natural bezoar, cultured bezoar, and in vitro cultured bezoar.

[0004] Currently, the use of β-glucuronidase-producing bacteria mainly focuses on *Escherichia coli*. While research on other bacterial species has been reported in recent years, the screening of strains primarily involves measuring the β-glucuronidase activity of the obtained strains to verify their luteinizing properties, a process that is time-consuming. To improve the screening efficiency of β-glucuronidase-producing bacteria, and thus increase the yield and quality of bezoar, optimizing the screening medium composition to obtain bacteria with high β-glucuronidase activity is a major research direction. JP2012000076A discloses a culture medium for detecting β-glucuronidase-producing *Escherichia coli* containing food and beverages. This medium effectively screens for β-glucuronidase-producing bacteria by using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide or its salt as the enzyme matrix. However, the activity of β-glucuronidase produced by bacteria is not high, and it cannot effectively improve the yield and quality of bezoar. Therefore, it is necessary to find a culture medium that can not only efficiently screen out β-glucuronidase-producing bacteria, but also make the screened bacteria produce high β-glucuronidase activity, so as to improve the production efficiency of bezoar. Summary of the Invention

[0005] The purpose of this invention is to provide a screening culture medium for β-glucuronidase-producing bacteria, and a method for isolating and purifying β-glucuronidase-producing bacteria using the culture medium, so as to rapidly obtain bacteria with high β-glucuronidase activity.

[0006] This invention provides a screening medium for β-glucuronidase-producing bacteria, the screening medium comprising a β-glucuronidase indicator, a carbon source, a nitrogen source, and other auxiliary components;

[0007] The β-glucuronidase indicator is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide;

[0008] The carbon source is quercetin;

[0009] The nitrogen source is ammonium nitrate;

[0010] The other auxiliary components include inorganic salts and trace substances.

[0011] Furthermore, the inorganic salt is sodium chloride and dipotassium hydrogen phosphate;

[0012] The trace substance is crude bovine bile extract; the crude bovine bile extract is an ethanol extract of bovine bile powder.

[0013] Furthermore, the screening culture medium comprises the following components in parts by weight:

[0014] 3-10 parts crude bovine bile extract, 12-24 parts ammonium nitrate, 10-20 parts sodium chloride, 1-10 parts dipotassium hydrogen phosphate, 1-8 parts quercetin, and 0.1-0.5 parts 5-bromo-4-chloro-3-indolyl-β-D-glucoside.

[0015] Preferably, the ingredients are: 10 parts crude bovine bile extract, 24 parts ammonium nitrate, 20 parts sodium chloride, 10 parts dipotassium hydrogen phosphate, 4 parts quercetin, and 0.5 parts 5-bromo-4-chloro-3-indolyl-β-D-glucoside.

[0016] Furthermore, the screening medium also includes 1-3 parts of a glucuronic acid compound; the glucuronic acid compound includes glycyrrhizic acid and / or bilirubin diglucuronide;

[0017] The glycyrrhizic acid is 0.5-1.5 parts; the bilirubin diglucuronide is 0.5-1.5 parts.

[0018] Furthermore, it also includes 5 to 15 parts of agar.

[0019] This invention also provides a method for screening β-glucuronidase-producing bacteria, which includes the following steps:

[0020] 1) Take cattle bile and dilute it with ultrapure water in a gradient to prepare bile solutions of various concentrations;

[0021] 2) Take bile solutions of various concentrations and spread them on solid culture medium for culture. Pick a single colony and streak it on solid culture medium for culture. Pick a single colony and inoculate it on liquid culture medium for culture. When an indigo blue insoluble precipitate appears in the liquid culture medium, take the bacterial solution to obtain the bacterial solution.

[0022] The solid culture medium is prepared by dissolving the aforementioned screening culture medium in water and then sterilizing it.

[0023] The liquid culture medium is prepared by dissolving the aforementioned screening culture medium in water and then sterilizing it.

[0024] Further, in step 2), the bile solution or single colony is cultured in a solid culture medium at a temperature of 35-38°C for 24-36 hours, using an inverted culture method.

[0025] Further, in step 2), the single colony is cultured in liquid culture medium at a temperature of 35-38°C, a rotation speed of 100-200 rpm, and a time of 24-36 h.

[0026] Furthermore, the preparation method of the solid culture medium or liquid culture medium is as follows:

[0027] Weigh the raw materials according to the formula, take the crude extract of bovine bile, dissolve it in water, sterilize it, then add the remaining raw materials, add water to make up the difference, stir, adjust the pH to 6.5, sterilize it, and the product is ready.

[0028] Further, the crude bovine bile extract is dissolved in water to form an aqueous solution with a concentration of 6-20%; water is added to make up to 1.5-3 times the volume of the aqueous solution; the stirring temperature is 40℃, the stirring speed is 500 rpm, and the stirring time is 60 min.

[0029] In the process of developing this invention, it was discovered that some bacteria producing β-glucuronidase can metabolize quercetin, and the β-glucuronidase produced by these bacteria has high enzyme activity. Preliminary analysis has determined the possible metabolic mechanism as follows: bacteria glucuronize quercetin, and the resulting β-glucuronidase hydrolyzes the glucuronized form of quercetin. The hydrolyzed glucuronic acid can be further metabolized by the bacteria, serving as a carbon and energy source. According to this metabolic mechanism, the stronger the bacteria's ability to metabolize quercetin, the higher the β-glucuronidase activity, the more available carbon sources, and the faster and better their growth.

[0030] This invention provides a screening culture medium for β-glucuronidase-producing bacteria. By using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide to indicate the production of β-glucuronidase by the bacteria, and quercetin as the carbon source to indicate the enzyme activity of β-glucuronidase, the medium, combined with ammonium nitrate as a nitrogen source and trace amounts of crude bovine bile extract, allows for the rapid isolation of high-activity β-glucuronidase-producing bacteria suitable for bezoar preparation from bovine bile. This solves the problem of the cumbersome steps currently required in artificial bezoar preparation, which involve first screening for β-glucuronidase-producing bacteria and then conducting biochemical experiments to detect the enzyme activity. This new medium allows for efficient screening of high-activity β-glucuronidase-producing bacteria in a single step. Furthermore, these isolated and purified β-glucuronidase-producing bacteria, when used in bezoar preparation, yield high-yield and high-quality bezoar, improving production efficiency. The screening culture medium of this invention has practical application value for the isolation and purification of β-glucuronidase-producing bacteria.

[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 Image of fermentation broth for strain WLK001;

[0034] Figure 2 Image of fermentation broth for strain WLK002;

[0035] Figure 3 Image of fermentation broth for strain WLK003;

[0036] Figure 4 Image of fermentation broth for strain WLK004;

[0037] Figure 5 Image of bacterial colonies on TSA solid medium. Detailed Implementation

[0038] The raw materials, reagents, and equipment used in the specific embodiments of this invention were all purchased commercially. The crude bovine bile extract was prepared according to the following method:

[0039] Take ox bile, dry it to constant weight, grind it into powder and pass it through a No. 6 sieve. Add ethanol at a ratio of 5g / 100mL, soak for 48h, centrifuge at 5500rpm for 30min, take the supernatant and filter it. Rotate the filtrate to a paste, take it out and dry it to obtain the product.

[0040] TSA culture medium was purchased from Qingdao Haibo Biotechnology.

[0041] Example 1: Preparation of liquid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0042] Formula: 3g crude bovine bile extract, 12g ammonium nitrate, 10g sodium chloride, 5g dipotassium hydrogen phosphate, 8g quercetin, 0.1g 5-bromo-4-chloro-3-indolyl-β-D-glucoside;

[0043] Preparation method:

[0044] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0045] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, quercetin, and 5-bromo-4-chloro-3-indolyl-β-D-glucoside, add them to the sterilized aqueous solution obtained in step 1), then add deionized water to make up the volume to 1000 ml, stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, and autoclave at 121℃ for 20 min to obtain the final product.

[0046] Example 2: Preparation of solid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0047] Formula: 3g crude bovine bile extract, 12g ammonium nitrate, 10g sodium chloride, 5g dipotassium hydrogen phosphate, 8g quercetin, 0.1g 5-bromo-4-chloro-3-indolyl-β-D-glucoside, 10g agar;

[0048] Preparation method:

[0049] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0050] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, quercetin, and 5-bromo-4-chloro-3-indolyl-β-D-glucoside, add them to the sterile aqueous solution obtained in step 1), add agar, and add deionized water to make up the volume to 1000 ml. Stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, autoclave at 121℃ for 20 min, and finally pour into disposable sterile empty culture dishes to obtain the final product.

[0051] Example 3: Preparation of liquid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0052] Formula: 5g crude bovine bile extract, 18g ammonium nitrate, 15g sodium chloride, 8g dipotassium hydrogen phosphate, 5g quercetin, 0.3g 5-bromo-4-chloro-3-indolyl-β-D-glucoside;

[0053] Preparation method:

[0054] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0055] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, quercetin, and 5-bromo-4-chloro-3-indolyl-β-D-glucoside, add them to the sterilized aqueous solution obtained in step 1), then add deionized water to make up the volume to 1000 ml, stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, and autoclave at 121℃ for 20 min to obtain the final product.

[0056] Example 4: Preparation of solid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0057] Formula: 5g crude bovine bile extract, 18g ammonium nitrate, 15g sodium chloride, 8g dipotassium hydrogen phosphate, 5g quercetin, 0.3g 5-bromo-4-chloro-3-indolyl-β-D-glucoside, 10g agar;

[0058] Preparation method:

[0059] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0060] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, quercetin, and 5-bromo-4-chloro-3-indolyl-β-D-glucoside, add them to the sterile aqueous solution obtained in step 1), add agar, and add deionized water to make up the volume to 1000 ml. Stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, autoclave at 121℃ for 20 min, and finally pour into disposable sterile empty culture dishes to obtain the final product.

[0061] Example 5: Preparation of liquid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0062] Formula: 10g crude bovine bile extract, 24g ammonium nitrate, 20g sodium chloride, 10g dipotassium hydrogen phosphate, 4g quercetin, 0.5g 5-bromo-4-chloro-3-indolyl-β-D-glucoside;

[0063] Preparation method:

[0064] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0065] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, quercetin, and 5-bromo-4-chloro-3-indolyl-β-D-glucoside, add them to the sterilized aqueous solution obtained in step 1), then add deionized water to make up the volume to 1000 ml, stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, and autoclave at 121℃ for 20 min to obtain the final product.

[0066] Example 6 Preparation of solid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0067] Formula: 10g crude bovine bile extract, 24g ammonium nitrate, 20g sodium chloride, 10g dipotassium hydrogen phosphate, 4g quercetin, 0.5g 5-bromo-4-chloro-3-indolyl-β-D-glucoside, and 10g agar;

[0068] Preparation method:

[0069] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0070] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, quercetin, and 5-bromo-4-chloro-3-indolyl-β-D-glucoside, add them to the sterile aqueous solution obtained in step 1), add agar, and add deionized water to make up the volume to 1000 ml. Stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, autoclave at 121℃ for 20 min, and finally pour into disposable sterile empty culture dishes to obtain the final product.

[0071] Example 7 Preparation of liquid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0072] Formula: 10g crude bovine bile extract, 20g ammonium nitrate, 10g sodium chloride, 1g dipotassium hydrogen phosphate, 1g glycyrrhizic acid, 1g quercetin, 1g bilirubin diglucuronide, 0.5g 5-bromo-4-chloro-3-indolyl-β-D-glucuronide.

[0073] Preparation method:

[0074] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0075] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, glycyrrhizic acid, quercetin, bilirubin diglucuronide, and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, add them to the sterilized aqueous solution obtained in step 1), then add deionized water to make up the volume to 1000 ml, stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, and autoclave at 121℃ for 20 min to obtain the final product.

[0076] Example 8: Preparation of solid culture medium for screening β-glucuronidase-producing bacteria according to the present invention

[0077] Formula: 10g crude bovine bile extract, 20g ammonium nitrate, 20g sodium chloride, 10g dipotassium hydrogen phosphate, 1g glycyrrhizic acid, 1g quercetin, 1g bilirubin diglucuronide, 0.5g 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 10g agar;

[0078] Preparation method:

[0079] 1) Weigh the raw materials according to the ratio, take the crude bovine bile extract and add deionized water to prepare a 10% g / mL aqueous solution, and autoclave at 121℃ for 40 min;

[0080] 2) Take ammonium nitrate, sodium chloride, dipotassium hydrogen phosphate, glycyrrhizic acid, quercetin, bilirubin diglucuronide, and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, add them to the sterile aqueous solution obtained in step 1), then add agar and deionized water to make up the volume to 1000 ml. Stir at 40℃ and 500 rpm for 60 min, adjust the pH to 6.5, autoclave at 121℃ for 20 min, and finally pour into disposable sterile empty culture dishes to obtain the final product.

[0081] Example 9: Screening method for β-glucuronidase-producing bacteria of the present invention

[0082] 1) Take cattle bile and dilute it with ultrapure water in a gradient to prepare bile solutions of various concentrations;

[0083] 2) Take bile solutions of various concentrations and spread them on the solid culture medium prepared in Example 6. Incubate at 37°C upside down for 24 hours. Pick a single colony and streak it on the solid culture medium prepared in Example 6. Incubate at 37°C upside down for 24 hours. Pick a single colony and inoculate it on the liquid culture medium prepared in Example 5. Incubate at 37°C and shake at 120 rpm for 24 hours. An indigo blue insoluble precipitate appears in the liquid culture medium. Take the bacterial solution to obtain the β-glucuronidase-producing bacterial strain.

[0084] The following experimental examples illustrate the beneficial effects of the present invention.

[0085] Experiment 1: Validation of the effectiveness of the screening medium for β-glucuronidase-producing bacteria

[0086] I. Test on the effectiveness of the screening culture medium for β-glucuronidase-producing bacteria of the present invention

[0087] 1. Culture medium preparation

[0088] 1.1 Culture medium formulation

[0089] The specific formulation of the screening culture medium for this invention is shown in Table 1.

[0090] Table 1. Selection medium formulations for β-glucuronidase-producing bacteria

[0091]

[0092] 1.2 Preparation of experimental culture medium

[0093] Prepare four types of solid culture media and corresponding liquid culture media according to the raw material ratios in Table 1, and label them as solid culture media ①~④ and liquid culture media ①~④ respectively.

[0094] 1.2.1 Preparation of solid culture medium

[0095] Weigh the raw materials according to the proportions in the formula; add the crude bovine bile extract to deionized water to prepare a 10% aqueous solution, and autoclave at 121℃ for 40 minutes to obtain a sterilized aqueous solution; weigh the remaining raw materials and add them to the sterilized aqueous solution, then add 10g of agar, and finally add deionized water to make up the volume to 1000ml. Stir at 40℃ and 500rpm for 60 minutes, adjust the pH to 6.5, and autoclave at 121℃ for 20 minutes to obtain the final product.

[0096] 1.2.2 Preparation of liquid culture medium

[0097] The preparation method is the same as that for solid culture media, except that agar is not added.

[0098] 2. Isolation and purification of β-glucuronidase-producing bacteria from bovine bile

[0099] Bovine bile was collected, and β-glucuronidase-producing bacteria with high enzyme activity were screened using prepared solid and appropriate liquid culture media. The specific method was as follows:

[0100] 1) Spreading and separation: Take fresh cattle bile, dilute it with ultrapure water in a gradient, spread it on a solid culture medium, invert the petri dish, and incubate at 37°C for 24 hours.

[0101] 2) Primary purification culture: Select a single colony with good separation and number it WLK002. Take a new solid culture medium (the same as in step 1) and streak it in sections. Invert the petri dish and incubate at 37°C for 24 hours.

[0102] 3) Secondary purification culture: Pick a single colony with good isolation from the primary purification culture, take a new solid culture medium (the same as in step 1), streak it in sections, invert the plate, and incubate at 37°C for 24 hours;

[0103] 4) Tertiary purification culture: Pick a single colony with good separation from the secondary purification culture, inoculate it into the corresponding liquid culture medium as in step 1), and incubate at 37°C and 120 rpm for 24 h with shaking.

[0104] 5) Preservation of bacterial strains: Mix the tertiary bacterial suspension with 40% sterile glycerol solution at a 1:1 ratio to achieve a final glycerol concentration of 20%. Dispense the mixture into sterilized cryovials and store them in an ultra-low temperature freezer at -80°C for later use.

[0105] Note: The solid culture medium ① used in the screening process corresponds to the liquid culture medium ①, the solid culture medium ② corresponds to the liquid culture medium ②, the solid culture medium ③ corresponds to the liquid culture medium ③, and the solid culture medium ④ corresponds to the liquid culture medium ④.

[0106] 3. Verification of β-glucuronidase-producing bacterial strains

[0107] 3.1 Confirmation of β-glucuronidase production during bacterial isolation and purification process

[0108] During the isolation and purification of β-glucuronidase-producing bacteria using culture medium formula number ① in Table 1, an indigo-blue insoluble precipitate was observed in the liquid culture medium of the tertiary purification culture. Figure 1 Preliminary identification indicates that the bacteria in the bacterial suspension are β-glucuronidase-producing bacteria, designated WLK001.

[0109] During the isolation and purification of β-glucuronidase-producing bacteria using culture medium formula number ② in Table 1, an indigo-blue insoluble precipitate was observed in the liquid culture medium of the tertiary purification culture. Figure 2 Preliminary identification indicates that the bacteria in the bacterial suspension are β-glucuronidase-producing bacteria, designated WLK002.

[0110] During the isolation and purification of β-glucuronidase-producing bacteria using culture medium formula number ③ in Table 1, an indigo-blue insoluble precipitate was observed in the liquid culture medium of the tertiary purification culture. Figure 3 Preliminary identification indicates that the bacteria in the bacterial suspension are β-glucuronidase-producing bacteria, designated WLK003.

[0111] During the isolation and purification of β-glucuronidase-producing bacteria using culture medium formula number ④ in Table 1, an indigo-blue insoluble precipitate was observed in the liquid culture medium of the tertiary purification culture. Figure 4 Preliminary identification indicates that the bacteria in the bacterial suspension are β-glucuronidase-producing bacteria, designated WLK004.

[0112] 3.2 Confirmation of the bioactivity of the purified and preserved strains

[0113] 3.2.1 Detection of β-glucuronidase activity in bacterial strains

[0114] ① Activation of microbial strains

[0115] Take 50 μL of the preserved bacterial strain and inoculate it into 50 mL of TSB liquid medium. Incubate at 37°C and 120 rpm for 24 h to obtain the activated bacterial strain.

[0116] ② Fermentation of microorganisms

[0117] 500 μL of the activated bacterial culture was inoculated into 50 mL of TSB liquid medium and cultured at 37°C and 120 rpm for 24 hours to obtain TSB fermentation broth.

[0118] ③ Detection of β-glucuronidase activity

[0119] The β-glucuronidase activity in TSB fermentation broth was detected using a β-glucuronidase activity assay kit.

[0120] Table 2 β-glucuronidase activity

[0121]

[0122] 3.2.2 Detection of the ability of bacterial strains to transform bezoar

[0123] ① Activation of microbial strains

[0124] Take 50 μL of the preserved bacterial strain, inoculate it into 50 mL of bovine bile, and incubate at 37°C and 120 rpm for 24 h to obtain the activated bacterial strain;

[0125] ② Fermentation of microorganisms

[0126] 500 μL of activated bacterial culture was inoculated into 50 mL of bovine bile and cultured at 37°C and 120 rpm for 24 hours to obtain bovine bile fermentation broth.

[0127] ③ Detection of bezoar conversion ability

[0128] According to the quality testing methods specified in the in vitro culture of bezoar section of the Chinese Pharmacopoeia (2025, Part I, Medicinal Materials and Processed Pieces, page 186), the bilirubin and conjugated bile acid content in fermented bovine bile was tested to assess the ability of the strain to transform bezoar.

[0129] Table 3. Results of bilirubin and conjugated bile acid content detection:...

[0130]

[0131] The above verification results show that using the culture medium formulations in Table 1, especially formulation ③, can isolate and purify bacteria with high β-glucuronidase activity and strong bezoar conversion ability from bovine bile. A further analysis of the culture medium formulations in Table 1 reveals that quercetin serves as the carbon source for the bacteria, ammonium nitrate as the nitrogen source, and sodium chloride and dipotassium hydrogen phosphate, being inorganic salts, maintain the osmotic and acid-base balance of the bacteria. The crude bovine bile extract provides the necessary trace elements for bacterial growth, enabling the efficient screening of high-activity β-glucuronidase from bovine bile. Without any of these components, especially the carbon and nitrogen sources, bacteria cannot grow and reproduce normally in the culture medium, and it is impossible to screen for bacteria with high β-glucuronidase activity and strong bezoar conversion ability.

[0132] II. Using commercially available culture media as a control, the effectiveness of the screening medium of this invention in screening β-glucuronidase-producing bacteria was tested.

[0133] 1. Culture medium preparation

[0134] Take TSA medium and prepare it into TSA solid medium according to the instructions.

[0135] 2. Isolation and purification of β-glucuronidase-producing bacteria from bovine bile

[0136] 1) Spreading and separation: Fresh bovine bile was serially diluted with ultrapure water and spread onto TSA solid medium. The plates were then inverted and incubated at 37°C for 24 hours. The number of bacterial strains growing on the TSA solid medium was greater than that on solid mediums ①~④ under the section "I. Test on the effect of screening medium for β-glucuronidase-producing bacteria of this invention". Figure 5 );

[0137] 2) Purification culture: Four single colonies with good separation characteristics that differed from strains WLK001, WLK002, WLK003 and WLK004 were selected from TSA solid medium, and four single colonies with good separation characteristics that were the same as strains WLK001, WLK002, WLK003 and WLK004 were selected, for a total of eight colonies. These eight colonies were inoculated into liquid culture media ① to ④ under the section "I. Test on the effect of screening culture medium for β-glucuronidase-producing bacteria of the present invention" and cultured at 37°C and 120 rpm for 24 h.

[0138] 3. Microbial enzyme activity test

[0139] 50 μL of bacterial culture was inoculated into 50 mL of TSB liquid medium and cultured at 37 °C and 120 rpm for 24 h. The β-glucuronidase activity in the TSB fermentation broth was detected using a β-glucuronidase activity assay kit. Four bacteria with colony characteristics different from strains WLK001, WLK002, WLK003, and WLK004 failed to show β-glucuronidase activity. Four bacteria with colony characteristics identical to strains WLK001, WLK002, WLK003, and WLK004 showed β-glucuronidase activity. The enzyme activity of bacteria with the same colony characteristics as WLK002 was greater than 800 u / mL, and the enzyme activity of bacteria with the same colony characteristics as WLK003 was greater than 1500 u / mL.

[0140] 4. In vivo culture capacity test for bezoar

[0141] The selected bacterial strains and bezoar beds were implanted into cattle through minimally invasive surgery. Four bacteria with colony characteristics different from strains WLK001, WLK002, WLK003, and WLK004 on TSA solid culture medium failed to yield bezoar one year after implantation. Four bacteria with colony characteristics identical to strains WLK001, WLK002, WLK003, and WLK004 were implanted into cattle one year after implantation. The bezoar was then removed from the cattle, dried, and weighed.

[0142] Table 4 Weight of bezoar

[0143]

[0144] The results above show that although many colonies grow on TSA medium, they do not necessarily possess β-glucuronidase activity. The bacteria on the screening medium of this invention all possess β-glucuronidase activity, indicating that TSA medium cannot directly screen for bacteria with β-glucuronidase activity, while the screening medium of this invention can specifically screen for β-glucuronidase-producing strains. Compared to the β-glucuronidase-producing bacteria screened on TSA medium, the β-glucuronidase-producing strains screened on the screening medium of this invention have higher enzyme activity. Using these strains as in vivo or in vitro cultures for bezoar production eliminates the need for further enzyme activity determination after isolating and purifying the strains using general culture media.

Claims

1. A screening medium for the production of β-glucuronidase-producing bacteria, characterized in that: It is composed of the following raw materials by weight: The crude bovine bile extract is an ethanol extract of bovine bile powder. It is composed of the following raw materials by weight:

2. The screening medium according to claim 1, characterized in that: The crude bovine bile extract is an ethanol extract of bovine bile powder. It is composed of the following raw materials by weight:

3. A screening medium for the production of β-glucuronidase bacteria, characterized in that: The crude bovine bile extract is an ethanol extract of bovine bile powder. It is composed of the following raw materials by weight: The crude bovine bile extract is an ethanol extract of bovine bile powder. The glucuronic acid compound includes glycyrrhizic acid and / or bilirubin diglucuronide; The glycyrrhizic acid is 0.5~1.5 parts; The bilirubin diglucuronide is 0.5~1.5 parts.

4. A screening medium for the production of β-glucuronidase bacteria, characterized in that: It is prepared by adding 5~15 parts of agar to the screening medium of any one of claims 1~3.

5. A method of screening for a β-glucuronidase producing bacterium, characterized by: It comprises the following steps: 1) Take bovine bile, and dilute it with ultrapure water to obtain a series of bile solutions with different concentrations; 2) Take the bile solutions with different concentrations respectively, and culture them on solid culture medium, pick single colonies, inoculate them on solid culture medium, pick single colonies, and inoculate them on liquid culture medium, wherein the liquid culture medium has indigo insoluble precipitate, and the bacteria solution is obtained; The solid culture medium is prepared by dissolving the screening medium of claim 4 in water and sterilizing it; The liquid culture medium is prepared by dissolving the screening medium of any one of claims 1~3 in water and sterilizing it.

6. The screening method according to claim 5, characterized in that: In step 2), the temperature for culturing the bile solution or single colony on solid culture medium is 35~38℃, the time is 24~36h, and the culture mode is inverted culture.

7. The screening method according to claim 5, characterized in that: In step 2), the temperature for culturing the single colony on liquid culture medium is 35~38℃, the rotation speed is 100~200rpm, and the time is 24~36h.

8. The screening method according to claim 5, characterized in that: The preparation method of the solid culture medium or liquid culture medium is as follows: According to the proportion, weigh the raw materials, dissolve the crude bovine bile extract in water, sterilize it, then add the remaining raw materials, make up the water, stir, adjust the pH to 6.5, and sterilize it to obtain the medium.

9. The screening method according to claim 8, characterized in that: Dissolve the crude bovine bile extract in water to obtain a water solution with a concentration of 6~20%, make up the water to 1.5~3 times the volume of the water solution, stir at a temperature of 40℃ and a rotation speed of 500rpm for 60min.

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