A composite yellow-causing bacteria and its application

Abstract

The present invention discloses a composite yellow-causing bacteria and its application, wherein the composite yellow-causing bacteria comprises Bacillus brevis ( Brevibacillus sp.) NCD08, Bacillus ( Bacillus sp.) MCD04 and Escherichia coli ( Escherichia coli )NCD04. The present invention uses composite flavonoids to ferment cattle bile to obtain in vitro cultured bezoar, which provides a new method for the production of artificial bezoar and can alleviate the problem of shortage of bezoar in the pharmaceutical market.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a composite yellow-causing bacteria and an application thereof. Background Art

[0002] Bezoar is a stone formed in the gallbladder, bile duct and hepatic duct of bovine animals, and is a precious Chinese medicinal material. The natural occurrence rate of bovine gallstones is very low, and the monomer yield is usually a few grams, which is far from meeting the needs of medicine. In order to solve the problem of scarce resources and high prices of bezoar, Chinese medical workers developed a variety of substitutes for bezoar in the 20th century, such as cultivated bezoar, in vitro cultivated bezoar and artificial bezoar. Natural bezoar is a pathological gallstone in cattle, and microbial infection of the gallbladder is one of the pathological conditions for the formation of bezoar stones in gallbladder bile. However, bovine bile itself has a broad-spectrum antibacterial effect, and most intestinal bacteria cannot survive in bovine bile, let alone the ability to transform bezoar. It has been reported in China that Escherichia coli and the β-glucuronidase (β-G enzyme) it produces also play an important role in the formation of bezoar. β-G enzyme can decompose conjugated bilirubin (CB) into free bilirubin (UCB), which is less soluble in water and easily combines with metal ions such as calcium and magnesium to form bilirubin calcium salt precipitates, which can further polymerize with protein-polysaccharide complexes to form small bezoar particles.

[0003] Studies have shown that high concentrations of live bacteria produce a large amount of β-G enzymes, under which free bilirubin is generated, and the latter undergoes a series of changes with related components in bile. There are two main changes: one is that when free bilirubin is in a low concentration supersaturated state, it promotes cholesterol crystallization and precipitates together or successively with a large amount of cholesterol and other components, thereby forming stones with cholesterol as the main component; the other is that when free bilirubin is in a high concentration supersaturated state, it self-aggregates as the main form, and precipitates together with a small amount of cholesterol and other components, forming stones with bile pigments as the main component. There are also reports that bilirubin reduces bile lipids disproportionately without affecting the secretion of bile salts. As the types of bile phospholipids change, the crystallization of cholesterol is accelerated, thereby promoting the formation of stones.

[0004] Therefore, screening microorganisms that produce highly active β-G enzymes and using them for the production of bezoar in vitro is of great significance for meeting the demand for bezoar in the pharmaceutical market. Summary of the invention

[0005] In order to alleviate the contradiction between supply and demand of bezoar in the pharmaceutical market and provide a method for cultivating bezoar in vitro and its yield, the present invention provides the following technical solutions.

[0006] In the first aspect, the present invention provides some microorganisms with strong bile salt tolerance, capable of long-term stable reproduction in the gallbladder environment, and producing highly active β-glucuronidase, specifically including the following strains:

[0007] (1) Bacillus brevis Brevibacillus sp.) NCD08, deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: M 20242134, and the deposit date is September 30, 2024.

[0008] (2) Bacillus Bacillus sp.) MCD04, deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20242133, and the deposit date being September 30, 2024.

[0009] (3) Escherichia coli ( Escherichia coli )NCD04, deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: M 20241834, and the deposit date is August 23, 2024.

[0010] In a second aspect, the present invention provides a composite yellow fungus comprising the above-mentioned Bacillus brevis NCD08, Bacillus MCD04 and Escherichia coli NCD04.

[0011] In a third aspect, the present invention provides a bacterial agent, comprising any one strain or a combination of two or more strains of the above-mentioned Bacillus brevis NCD08, Bacillus MCD04 or Escherichia coli NCD04.

[0012] Preferably, the bacterial agent comprises one or both strains of Bacillus brevis NCD08 or Bacillus MCD04.

[0013] Furthermore, the bacterial agent contains Bacillus brevis NCD08.

[0014] Furthermore, the bacterial agent contains Bacillus MCD04.

[0015] Furthermore, the bacterial agent contains Bacillus brevis NCD08 and Escherichia coli NCD04.

[0016] Furthermore, the bacterial agent comprises Bacillus MCD04 and Escherichia coli NCD04.

[0017] Furthermore, the bacterial agent comprises Bacillus brevis NCD08, Bacillus MCD04 and Escherichia coli NCD04.

[0018] In a fourth aspect, the present invention provides a composition comprising the composite yellow fungus of the second aspect or the bacterial agent of the third aspect.

[0019] In a fifth aspect, the present invention provides an application of the composite yellow fungus of the second aspect, the bacterial agent of the third aspect, or the composition of the fourth aspect, wherein the application comprises at least one of the following:

[0020] (1) Application in fermentation of ox bile;

[0021] (2) Application in the preparation of in vitro cultured bezoar.

[0022] In a sixth aspect, the present invention provides a method for fermenting bovine bile, the method comprising introducing the composite flavonoids of the second aspect into a system containing bovine bile.

[0023] Preferably, the method comprises filtering and sterilizing the system containing ox bile to obtain a sterilized system.

[0024] Preferably, the method comprises inoculating the composite yellow fungus of the second aspect into a seed culture medium to obtain a microbial seed solution.

[0025] Preferably, one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts are added to the seed culture medium. More preferably, the concentration of one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts in the seed culture medium is at least 5 g / L, for example: 5 g / L, 10 g / L, 15 g / L, 20 g / L.

[0026] Preferably, the seed culture medium may be MRS culture medium (eg, MRS broth culture medium or MRS agar culture medium).

[0027] Preferably, the temperature of the seed culture medium is adjustable according to the physiological characteristics of the microorganism, preferably any value or range between 30-40°C, for example: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40°C.

[0028] Preferably, the method comprises connecting the above-mentioned microbial seed liquid to the above-mentioned sterilization system to obtain a fermentation system.

[0029] Preferably, the access ratio of the microbial seed liquid accounts for any value or range of 1%-15% of the volume fraction of the fermentation system, preferably any value or range of 2%-10%, for example: 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0030] Preferably, the initial pH of the fermentation system is any value or range between 3 and 5, for example, 3, 3.2, 3.5, 3.7, 4, 4.2, 4.5, 4.7, or 5.

[0031] In a specific embodiment of the present invention, the initial pH of the fermentation system is controlled to be 3.

[0032] Preferably, the temperature of the fermentation system is any value or range between 25-40°C, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40°C.

[0033] In a specific embodiment of the present invention, the temperature of the fermentation system is controlled at 37°C.

[0034] Preferably, the fermentation time is at least 30 h, preferably any value or interval between 30 and 96 h, for example, 30, 35, 36, 40, 45, 48, 50, 55, 60, 65, 70, 72, 75, 80, 84, 85, 90, 95 or 96 h;

[0035] In a seventh aspect, the present invention provides a method for culturing bezoar in vitro, the method comprising introducing the composite flavonoids of the second aspect into cattle bile, and fermenting to obtain the in vitro cultured bezoar.

[0036] In an eighth aspect, the present invention provides an in vitro cultured bezoar, characterized in that the in vitro cultured bezoar is prepared according to the method described in the seventh aspect.

[0037] The term “comprising” or “including” used in the present invention is an open-ended writing method, which includes the specified components or steps described, as well as other specified components or steps that will not substantially affect the described components or steps.

[0038] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example: "A and / or B" includes "A", "A and B" and "B". "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0039] Beneficial effects of the present invention:

[0040] 1. The present invention screened out Bacillus brevis NCD08, Bacillus MCD04 and Escherichia coli NCD04, which are acid-resistant (such as pH=3), bile-resistant, and produce highly active β-glucuronidase, enriching the resources of yellow fungi.

[0041] 2. The present invention uses a composite strain combination of Bacillus brevis NCD08, Bacillus MCD04 and Escherichia coli NCD04 to ferment cattle bile to obtain in vitro cultivated bezoar, which provides a new method for the production of artificial bezoar and can alleviate the problem of bezoar shortage in the pharmaceutical market. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1Shown are the results of determination of β-glucuronidase activity, direct bilirubin content and total protein content of different strains in Example 3, A is β-glucuronidase activity, B is direct bilirubin content, and C is total protein content;

[0043] Figure 2 Shown is the appearance of bezoar samples produced by fermentation of different composite bacteria in Example 4;

[0044] Figure 3 Shown is the bilirubin content in the bezoar sample of Example 4.

[0045] Culture deposits for patent procedures:

[0046] Bacillus brevis Brevibacillus sp.) NCD08, the deposit number is CCTCC NO: M 20242134, and the deposit date is September 30, 2024.

[0047] Bacillus ( Bacillus sp.) MCD04, the deposit number is CCTCC NO: M 20242133, and the deposit date is September 30, 2024.

[0048] Escherichia coli ( Escherichia coli )NCD04, the deposit number is CCTCC NO: M 20241834, and the deposit date is August 23, 2024.

[0049] Depository: China Center for Type Culture Collection (CCTCC);

[0050] Address of the depository: Wuhan University, Wuhan, China, Postal Code: 430072. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.

[0052] The culture medium formula in the embodiment of the present invention is as follows:

[0053] MRS medium: beef extract 10.0 g / L, tryptone 10.0 g / L, yeast extract powder 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium citrate 5.0 g / L, anhydrous sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.05 g / L, anhydrous manganese sulfate 0.05 g / L, glucose 20.0 g / L, agar 15 g / L, Tween 80 1.0 mL, autoclave at 121°C for 20 min.

[0054] NB medium: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, 15 g / L agar, autoclaved at 121°C for 15 min.

[0055] Example 1: Isolation and identification of bacterial species

[0056] Feces collected from healthy cattle in large-scale cattle farms were used as samples, and the strains in bile were separated by coating and streaking techniques on MRS and NB solid culture medium plates. The genomic DNA of the isolated and purified strains was used as a template, 27F and 1492R were used as primers, and the 16S rRNA gene of the strain was amplified by PCR. The amplified products were sequenced and the obtained sequences were classified by online EzTaxon (16S rRNA gene sequence similarity greater than 97% was preliminarily determined to be the same species). The specific sequences of 27F and 1492R are as follows:

[0057] 27F: 5'-CGGTGAATACGTTCYCGG-3' (SEQ ID NO: 1)

[0058] 1492R: 5'-AAGGAGGTGATCCRGCCGCA-3' (SEQ ID NO: 2)

[0059] As shown in Table 1, 8 bacterial strains were isolated whose 16SrRNA gene sequences had a similarity of 98% to 100% with the model species.

[0060] Table 1 Strain identification results

[0061]

[0062] Example 2: Determination of the ability of strains to tolerate bile salts

[0063] The 8 bacterial strains isolated in the example were cultured and the effective viable count was 10 8 CFU / mL of cultured bacteria, then inoculated into MRS liquid medium at 2% inoculation volume, cultured at 37℃ for 18 h, centrifuged at 8000 r / min for 5 min, collected the bacteria, washed twice with sterilized saline (pH 7.0), and resuspended in saline. Take 1 mL of bacterial suspension and inoculate into 9 mL of MRS medium (pH=3, final concentration of ox bile salt=5 g / L), cultured at 37℃ for 3 h, and determined the number of viable bacteria by plate count method at 0 h and 3 h, respectively, with 3 replicates. Calculate the survival rate of the strain, the calculation formula is as follows:

[0064] Survival rate (%) = logN t / logN0×100%

[0065] Where: N0 and N tRepresent the number of viable bacteria before (0 h) and after (3 h) treatment, respectively.

[0066] As shown in Table 2 , the survival rates of strains NCD01, NCD02, NCD04, NCD05, NCD08, and MCD04 were 80.75%, 41.91%, 93.82%, 55.22%, 167.9%, and 151.26%, respectively, showing high bile salt tolerance.

[0067] Table 2 Comparison of bile salt tolerance of strains

[0068]

[0069] Note: Since strains NCD 03, NCD 06, NCD 07 and MCD 03 were not single colonies during the purification step, no subsequent experiments were performed.

[0070] Example 3: Biochemical identification of strains

[0071] NCD01, NCD02, NCD04, NCD05, NCD08, and MCD04 were inoculated on solid MacConkey medium and cultured at 37°C for 24 h. Single colonies of the same size were strictly selected and inoculated into 5 mL of sterilized ox bile, cultured at 37°C and 180 r / min with shaking for 12 h, centrifuged, and the supernatant was separated. 100% (pure) ox bile without inoculation of yellow bacteria was used as a control.

[0072] The β-glucuronidase assay kit (catalog number: G0579W, Suzhou Grace Biotechnology Co., Ltd., microplate method), direct bilirubin (DBIL) content assay kit (G1223W, Suzhou Grace Biotechnology Co., Ltd., microplate method) and total protein content test kit (G0418W, Suzhou Grace Biotechnology Co., Ltd., microplate method) were used to determine the β-glucuronidase activity, direct bilirubin content and total protein content in the supernatant of the six bacterial strains.

[0073] like Figure 1 As shown, compared with the control (CK), the NCD04, NCD08 and MCD04 strains had higher β-glucuronidase activity, stronger ability to reduce direct bilirubin in bovine bile, and lower total protein content, indicating that the NCD04, NCD08 and MCD04 strains had strong growth activity in bile.

[0074] Example 4: Application of composite bacteria fermentation in the preparation of bezoar

[0075] (1) Preparation of composite bacterial solution: strains NCD01, NCD02, NCD04, NCD05, NCD08 and MCD04 were inoculated into MRS liquid medium and cultured for 3 days. The supernatant was removed by centrifugation and the bacterial precipitate was diluted with physiological saline until the number of viable bacteria was 2×10 9 CFU / mL, compound bacteria SC01 and SC02 were obtained according to the volume ratio of 1:1:1 (see Table 3)

[0076] Table 3 Composition of composite bacteria

[0077]

[0078] (2) Compound bacteria fermentation of cow bile: Take a sterilized reactor, add 250-300 mL of 2-fold concentrated cow bile, and add 2×10 9 CFU / mL of the composite bacterial solution was 5 mL, and the peristaltic pump was turned on to vacuum and replenish the solution every 2 days. Compared with the control (pure bile), a large number of fine crystals appeared at the bottom of the reactor after 30 days of reaction (see Figure 2 ). The crystal particles are irregular in shape, dark brown, crispy, and light in weight, and are bezoar particles.

[0079] Example 5: Determination of bilirubin in bezoar samples

[0080] The bilirubin content in the bezoar sample prepared in Example 4 was determined according to the diazotization reagent method.

[0081] Preparation of diazo reagent: Solution A: 1 g of p-aminobenzenesulfonic acid, 15 mL of concentrated hydrochloric acid, add distilled water to 1000 mL; Solution B: 0.5 g of sodium nitrite, add distilled water to 100 mL, store in a brown bottle, and can be used for up to 2 months; before use, take 50 mL of Solution A and add 1.5 mL of Solution B to make the diazo reagent, which is effective within 3 hours.

[0082] Accurately weigh 10 mg of bilirubin reagent, dissolve it in a 10 mL brown volumetric flask with chloroform and dilute it, shake it well, and use it as the reference solution. Take 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of the reference solution in a 10 mL volumetric flask, and then add ethanol solution to make the total volume 9.0 mL. Then add 1.0 mL of diazotization reagent to each bottle, mix and shake well. After standing for 1 hour, use the corresponding reagent (without bilirubin) as the blank, measure the absorbance at a wavelength of 533 nm, and draw a standard curve with the ordinate as the ordinate and the mg of bilirubin contained as the abscissa. Draw the standard curve: Y=1.933X+0.036, R=0.99.

[0083] Weigh 10 mg of the bezoar powder after the reaction of the composite bacteria SC01 and SC02, add 30 mL of a mixed solution of chloroform and ethanol reagents (V chloroform: V ethanol = 7:3), add 3 drops of concentrated HCl and shake well, and place in a water bath at 85°C for 30 min. After cooling, place it in a 50 mL brown volumetric flask and dilute it at 95°C to obtain a bilirubin test solution. Place 5.0 mL of the test solution in a 10 mL brown volumetric flask, add 1.0 mL of the heavy nitrogen reagent, dilute it to 10 mL with ethanol, mix it, place it indoors away from light for 1 h, use the corresponding reagent (without bilirubin) as a blank, measure the absorbance at a wavelength of 533 nm, and calculate the bilirubin content using the standard curve.

[0084] like Figure 3 As shown in the figure, the bilirubin content in the bezoar sample treated with SC01 compound bacteria increased by 42.81%. The bilirubin content in the bezoar sample treated with SC02 compound bacteria increased by 12.92%. This shows that both compound bacteria SC01 and SC02 can increase the bilirubin content in bezoar samples and are used to cultivate bezoar in vitro, but the effect of compound bacteria SC01 is higher.

[0085] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A composite yellow fungus, characterized in that: The composite yellow-causing bacteria consists of Bacillus brevis, Bacillus and yellow-causing bacteria; The Brevibacillus is Brevibacillus ( Brevibacillus sp.) NCD08, deposited in China Center for Type Culture Collection, with the deposit number of CCTCC NO: M 20242134 and the deposit date of September 30, 2024; The Bacillus is Bacillus ( Bacillus sp.) MCD04, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242133 and the deposit date of September 30, 2024; The yellow-causing bacteria is NCD04, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20241834 and the deposit date is August 23, 2024.

2. A bacterial agent, characterized in that: The bacterial agent comprises the composite yellow fungus according to claim 1.

3. A composition, characterized in that: The composition comprises the complex yellow fungus according to claim 1 or the bacterial agent according to claim 2.

4. The use of the composite yellow-causing bacteria according to claim 1, the bacterial agent according to claim 2 or the composition according to claim 3, characterized in that: The application includes at least one of the following: (1) Application in fermentation of ox bile; (2) Application in the preparation of in vitro cultured bezoar.

5. A method for fermenting ox bile, characterized in that: The method comprises introducing the composite flavonoids according to claim 1 into a system containing ox bile.

6. A method for in vitro cultivation of bezoars, characterized in that: The method comprises introducing the composite flavonoids of claim 1 into cattle bile, fermenting the mixture, and obtaining the in vitro cultured bezoar.

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