Bile-salt-tolerant yellow-causing bacterium and application thereof

Abstract

The invention provides cholate-tolerant yellow bacteria and application thereof, and belongs to the field of calculus bovis cultivation. According to the invention, a cholate-tolerant yellow-causing bacterium is obtained through screening, the cholate-tolerant yellow-causing bacterium is classified and named as Escherichia coli NCD04, the bacterial strain is preserved in China Center for Type Culture Collection on August 23, 2024, and the preservation number of the bacterial strain is CCTCC NO: M 20241834. The xanthogenic bacterium provided by the invention is derived from healthy oxgall, has excellent oxgall resistance, is high in activity of produced beta-glucuronidase and high in direct bilirubin conversion rate, can accelerate conversion of bile to calculus bovis, and is used for production of in-vivo or in-vitro artificial calculus bovis.

Description

Technical Field

[0001] The invention relates to the technical field of bezoar cultivation, and in particular to a bile-tolerant yellow-causing bacterium and 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] Prior art CN117965358A discloses a bile-tolerant flavonoid bacteria strain and its application, which is derived from anal swabs of healthy calves, has excellent tolerance to bovine bile, produces high β-glucuronidase activity, has high bezoar conversion rate and high safety.

[0005] Therefore, screening for efficient bezoar transformation strains with better bile tolerance is the key and difficulty in the in vivo cultivation of bezoar and in vitro bezoar production process. Summary of the invention

[0006] In view of the above prior art, the present invention screens out a bile-tolerant flavonoid fungus and applies it to in vitro bezoar cultivation. The flavonoid fungus of the present invention is derived from the bile of healthy cattle in cattle farms, has excellent bile tolerance, high bezoar conversion rate and high safety, and can be used for the production of live bezoar cultivation.

[0007] Specifically, the present invention relates to the following technical solutions: According to a first aspect of the present invention, a bile-tolerant flavonoid bacteria is provided, wherein the flavonoid bacteria is Escherichia coli; the strain has been deposited in the China Center for Type Culture Collection (CCTCC) on August 23, 2024, and its deposit number is: CCTCC NO: M 20241834.

[0008] The second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the above-mentioned yellow fungus.

[0009] Preferably, in the bacterial agent, the yellow fungi may be present in the form of cultured live bacteria or fermentation liquid of live bacteria.

[0010] Preferably, in the bacterial agent, the number of viable bacteria of yellow fungi is greater than or equal to (1, 2, 3, 4, 5) × 10 9 CFU / mL.

[0011] The third aspect of the present invention provides the use of the bile-tolerant flavonoids or the bacterial agent containing flavonoids in the production of bezoars, preferably including in vitro bezoar production.

[0012] Specifically, it includes the use of the above-mentioned bile-tolerant flavonoids or bacterial agents containing flavonoids in the production of bezoars in vitro. More specifically, the flavonoids or bacterial agents are added to the bile of cattle to produce artificial bezoars. The above-mentioned flavonoids need to be cultured to a concentration of (1-5)×10 9 CFU / ml or more, preferably 2, 3, 4, 5×10 9 CFU / ml. The concentration of yellow fungi can also be 1×10 6 CFU / ml~1×10 11 CFU / ml range, optional is 10 7 , 10 8 , 10 10 , 10 11 , 10 12 CFU / ml.

[0013] The fourth aspect of the present invention provides a method for cultivating artificial natural bezoar, comprising the step of injecting the above-mentioned bezoar-causing fungus into the gallbladder of a cow.

[0014] Preferably, the number of live bacteria of the injected yellow fungus is greater than or equal to (1-5)×109 CFU / ml.

[0015] Beneficial effects of the present invention: (1) The present invention provides a yellow fungus with a tolerance of up to 93.82%, which can tolerate high concentrations of bile salts.

[0016] (2) The flavonoids of the present invention have excellent resistance to bovine bile, high β-glucuronidase activity, high direct bilirubin conversion rate, and can accelerate the conversion of bile into bezoar. It is safe to use and can be used for efficient in vitro bezoar production and in vivo cultivation of bezoar.

[0017] Collection information: Bacteria species: Escherichia coli Latin: Escherichia coli Strain ID: Escherichia coli NCD04 Taxonomic nomenclature: Escherichia coli NCD04 Collection institution: China Center for Type Culture Collection Abbreviation of depository institution: CCTCC Address of depository institution: Wuhan University, Wuhan, Hubei Province Deposit number: CCTCC NO: M 20241834 Date of deposit: August 23, 2024 BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 To screen the strains, β-glucuronidase activity was detected.

[0019] Figure 2 Total protein consumption was detected for screening strains.

[0020] Figure 3 Direct bilirubin consumption assay for screening strains.

[0021] Figure 4 This is an experiment on the in vitro crystallization of NCD04 bovine bile to form bezoar. DETAILED DESCRIPTION

[0022] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0023] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0024] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

[0025] Example 1: Isolation and identification of bacterial species Bacterial isolation and culture: Bile was collected from healthy cattle in large-scale cattle farms, and the strains in the bile were isolated by coating and streaking technology; the genomic DNA of the isolated and purified strains was used as a template, 27 F (5'-CGGTGAATACGTTCYCGG-3') and 1492R (5'-AAGGAGGTGATCCRGCCGCA-3') 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 initially determined to be the same species). The 16S rRNA gene sequences of the four bacterial strains obtained were 98% to 100% similar to the model species. The results are shown in Table 1, which shows the similarity analysis of the 16S rRNA gene of the isolated Escherichia coli.

[0026] Table 1 Strain identification results

[0027] Note: The NCD 03 strain was not used in subsequent experiments because it was not a single colony during the purification step.

[0028] Example 2: Determination of the ability of strains to tolerate bile salts The bacterial solution (10 8 CFU / mL) was inoculated into MRS liquid culture medium at 2% inoculation volume, cultured at 37℃ for 18 h, centrifuged at 8000r / 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 it into 9 mL of MRS culture medium (adjust pH to 3 with 1 mol / L hydrochloric acid, continue to add ox bile salt to adjust to a final concentration of 5g / L, culture at 37℃ for 3 h, and determine the number of viable bacteria by plate count method at 0 h and 3 h, respectively, and set 3 replicates. The survival rate is calculated as shown in the following formula:

[0029] Where: N0 and N t Represent the colony counts before (0 h) and after (3 h) treatment of the strains, respectively.

[0030] Table 2 Comparison of bile salt tolerance of strains

[0031] The results are shown in Table 2. The tested E. coli showed high bile salt tolerance and grew normally in 5 g / L ox bile salt medium, especially the NCD04 strain, whose tolerance survival rate reached 93.82%.

[0032] Example 3: Biochemical identification of strains The bile-tolerant E. coli (NCD01, NCD02, NCD04, and NCD05) screened above were inoculated on MacConkey medium and placed in an incubator for 24 h at 37°C. Single colonies of the same size were strictly selected and inoculated into 5 mL of ox bile to ensure that the initial bacterial concentration was equal. The culture was shaken at 37°C and 180 r / min for 12 h, and the supernatant was separated by centrifugation. 100% (pure) bile without yellow bacteria inoculation was used as a control.

[0033] MacConkey medium (per liter): 20g peptone, 5g sodium chloride, 10g lactose, 13g agar, 1.5g bile salts, 0.03g neutral red, 0.001g crystal violet, 1000ml water.

[0034] The β-glucuronidase assay kit (catalog number: G0579W, microplate method), direct bilirubin (DBIL) content assay kit (G1223W, microplate method), and total protein content test kit (G0418W, microplate method) were used to determine the β-glucuronidase activity, direct bilirubin content, and total protein content in the supernatant, respectively; the above kits were purchased from Suzhou Grace Biotechnology Co., Ltd.

[0035] Table 3 Results of β-glucuronidase activity, direct bilirubin content and total protein content of strains

[0036] like Figure 1 , 2, 3 and Table 3, the β-glucuronidase activity, direct bilirubin (DBIL) content and total protein content of the isolated strain were determined using a kit. The β-glucuronidase activity of NCD04 reached 177.6 U / L, the total protein content was 1.51 g / L (consumption), and the direct bilirubin was 47.8 umol / L (consumption). It showed good β-glucuronidase activity and direct bilirubin utilization, which was beneficial to the conversion of bezoar bilirubin and the formation of bezoar.

[0037] The above results prove that the β-glucuronidase activity of E. coli NCD04 strain is the highest, and the ability to convert direct bilirubin into indirect bilirubin is the strongest. Therefore, the present invention selects E. coli NCD04 strain as the yellow fungus and deposits it biologically. The deposit information is as follows: NCD04 is Escherichia coli, taxonomic nomenclature Escherichia coli , deposited in China Center for Type Culture Collection on August 23, 2024, with the deposit number CCTCC NO: M 20241834.

[0038] Example 4: Application of E. coli NCD04 in a reactor Take a sterilized reactor, pour 250-300 mL of ox bile into it, and add 5 mL of 2×10 9 CFU / mL of E. coli NCD04 bacterial solution (Bacterial solution: cultured in MRS liquid medium for 3 days, centrifuged to remove the supernatant before use, and diluted with saline), turned on the peristaltic pump to vacuum and replenish the liquid after every 2 days of reaction. After 15 days of reaction, small crystals can be seen at the bottom of the reactor. It proves that E. coli NCD04 can crystallize ox bile in the reactor to form artificial bezoar. The resulting crystalline particles are irregular in shape and dark brown (such as Figure 4 ).

[0039] The flavonoids of the invention have excellent cattle bile resistance, high beta-glucuronidase activity, high direct bilirubin conversion rate, can accelerate the conversion of bile into bezoar, and are used for the production of artificial bezoar.

[0040] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bile-tolerant yellow-causing bacteria, characterized in that It can be used for bezoar cultivation. The yellow-causing bacteria is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241834 and a deposit date of August 23, 2024.

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

3. The bacterial agent according to claim 2, characterized in that In the bacterial agent, the number of viable bacteria of yellow fungi is 1~5×10 9 CFU / ml.

4. A composition, characterized in that: The composition comprises the yellow fungus and / or its fermentation product as claimed in claim 1.

5. Use of the bile-tolerant flavonoids according to claim 1 or the bacterial agent according to claim 2 in producing bezoar.

6. The use according to claim 5, characterized in that The application is the application in producing bezoar in vitro.

7. The use according to claim 5 or 6, characterized in that The yellow-causing bacteria or bacterial agent is added to the cattle bile to produce artificial bezoar.

8. The use according to claim 7, characterized in that The application is application in converting direct bilirubin into indirect bilirubin.

9. A method for cultivating bezoar in vitro, characterized in that: The method comprises introducing the yellow-causing fungus described in claim 1 or the bacterial agent described in claim 2 into cattle bile, and cultivating the bezoar product.

10. The method according to claim 9, characterized in that The number of viable bacteria of yellow fungi or bacterial agents is greater than or equal to 2×10 9 CFU / mL.

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