A microbial composition and its application

Abstract

The present invention belongs to the field of microorganisms, and in particular to a microbial composition and its application. The microbial composition of the present invention includes a microorganism with glucuronidase activity and Lactobacillus plantarum ZY-002, wherein the microorganism with glucuronidase activity includes Levilactobacillus brevis, preferably Lactobacillus brevis ZY-001, and the microbial composition of the present invention can realize the preparation of bezoar cultured in vitro, and can produce calcium bilirubin more efficiently than single strain fermentation.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a microbial composition and application thereof. Background Art

[0002] Bezoar has multiple pharmacological effects and is widely used in medicine and medical beauty products, with a huge market demand. However, the source of natural bezoar is limited, and exploring the use of artificial methods to make artificial bezoar and in vitro cultivation of bezoar has become a research hotspot. At present, the key raw materials bilirubin and bilirubin calcium for preparing artificial bezoar or in vitro cultivation of bezoar on the market are mainly extracted from various animal biles. The extraction methods adopt physical and chemical methods such as precipitation, solvent extraction and ion exchange resin adsorption. The Chinese invention patent CN101429152A discloses a method for reducing biliverdin in animal bile raw materials to increase the bilirubin yield, but due to the cumbersome process, the extraction yield is low and consumes a lot of acid, alkali and solvent, which is easy to cause environmental pollution. Patent documents (CN104774885A and CN102429926A) use Escherichia coli, Clostridium perfringens, Proteus and Enterococcus to ferment cattle bile. The above strains are all pathogenic bacteria or conditional pathogens, which makes it difficult to remove harmful bacterial toxins produced by pathogenic bacteria during product separation and purification. The application of the product in medical beauty or pharmaceutical products has certain safety risks and the application scale is limited. Patent document (CN115414434A) discloses a microbial combination for fermenting and preparing Arisaema ciliata slices. The microbial combination ferments cattle bile and only hydrolyzes the bound bile acid in the cattle bile into free bile acid. Under optimal conditions, the conversion rate is about 15%, and the fermentation takes 10 days. The non-patent literature "Study on the Changes of Effective Ingredients in Fermented Ox Bile and Its Mechanism of Action for Treating Ischemic Stroke" (Pei Yuying, Beijing University of Chinese Medicine, 2022) reported that the fermentation of ox bile with Shenqu as the strain only converted the bound bile acid into free bile acid, with a conversion rate of about 50%, which took 30 days. The strain composition used for ox bile fermentation was complex and difficult to control. The method disclosed in the Chinese patent (CN106265742A) uses copolymers, chelating agents and other methods to artificially add many chelating agents, enzyme preparations, hot ethanol and other organic solvents, which is not conducive to safe and environmentally friendly production. In addition, there are many additives, and many substances themselves are not the natural components that natural bezoar should have, which makes bezoar lack pharmacological safety demonstration. Summary of the invention

[0003] The present application combines microorganisms with glucuronidase activity (especially Levilactobacillus brevis) and Lactobacillus plantarum ZY-002 to achieve the preparation of in vitro cultured bezoar, and can produce calcium bilirubin more efficiently than fermentation of a single strain. Moreover, the microorganisms described in the present application are all safe edible microorganisms and / or probiotic microbial strains. In the production process, the production process has the advantages of mild and simple production process, low production cost, reduced use of flammable and explosive solvents, reduced environmental pollution, safe production process, reduced emissions and consumption, and environmental protection. The products are used in medicines and medical beauty products, and have quality and safety, and have good economic and social benefits. Specifically:

[0004] The first aspect of the present invention provides a microbial composition, comprising:

[0005] 1) a microorganism having glucuronidase activity; and,

[0006] 2) Lactobacillus plantarum ZY-002.

[0007] The plant lactobacillus ZY-002 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023. The storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC No: 63911.

[0008] Preferably, in the microbial composition, the ratio of the microorganism having glucuronidase activity to Lactobacillus plantarum ZY-002 is any value or interval in (1-9): (1-9); further preferably, in the microbial composition, the ratio of Lactobacillus breve ZY-001 to Lactobacillus plantarum ZY-002 is any value or interval in (1-5): (1-5), for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0009] Preferably, the microorganism having glucuronidase activity includes one or more of the genus Escherichia, Clostridium, Proteus or Lactobacillus.

[0010] Preferably, the genus Escherichia includes but is not limited to Escherichia coli.

[0011] Preferably, the genus Clostridium includes but is not limited to Clostridium perfringens.

[0012] Preferably, the Lactobacillus includes Levilactobacillus brevis.

[0013] In a specific embodiment of the present invention, the Levilactobacillus brevis includes Levilactobacillus brevis ZY-001, and the Levilactobacillus brevis ZY-001 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with a collection number of GDMCC No: 63910.

[0014] In a specific embodiment of the present invention, the microbial composition comprises 1) Lactobacillus breve ZY-001, and 2) Lactobacillus plantarum ZY-002.

[0015] Preferably, the Lactobacillus breve ZY-001 and Lactobacillus plantarum ZY-002 are strains tolerant to ox bile. Further preferably, the Lactobacillus breve ZY-001 and Lactobacillus plantarum ZY-002 can survive in pure ox bile for at least 5 days.

[0016] Preferably, the Lactobacillus breve ZY-001 also has bile salt hydrolase activity.

[0017] Preferably, the Lactobacillus plantarum ZY-002 has bile salt hydrolase activity.

[0018] Preferably, the 16S rDNA of the Lactobacillus breve ZY-001 is as shown in SEQ ID NO: 1.

[0019] SEQ ID NO: 1:

[0020]

[0021] Preferably, the 16S rDNA of the Lactobacillus plantarum ZY-002 is as shown in SEQ ID NO:2.

[0022] SEQ ID NO: 2:

[0023]

[0024] Preferably, the microbial composition may further comprise other microorganisms, and more preferably, the other microorganisms are edible microorganisms and / or probiotic microbial strains. More preferably, the microorganisms include various Lactobacillus and / or Bifidobacterium microorganisms, such as Limosilactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, Bifidobacterium longum, etc.

[0025] The second aspect of the present invention provides a use of the microbial composition described in the first aspect in the preparation of in vitro cultured bezoar.

[0026] The third aspect of the present invention provides a method for preparing in vitro cultured bezoar, wherein the method comprises using a microorganism, wherein the microorganism comprises one or more of the following groups:

[0027] 1) The microbial composition described in the first aspect above;

[0028] 2) Lactobacillus plantarum ZY-001; or,

[0029] 3) Lactobacillus breve ZY-002.

[0030] Preferably, the method comprises:

[0031] Step 1) preparing bezoar core: fermenting cattle bile using microorganisms, collecting brown-red precipitates, and obtaining bezoar core;

[0032] Step 2) preparing coated fine powder: taking part of the bezoar core obtained in step 1), adding at least one or two or more of bile acid, deoxycholic acid, taurine, cholesterol, bilirubin, ferrous sulfate, calcium ion donor, zinc sulfate, magnesium sulfate, ascorbic acid or B vitamins, mixing well and grinding into powder to obtain coated fine powder;

[0033] Step 3) preparing a coating concentrate: fermenting the cow bile using microorganisms, and concentrating the fermentation liquid (preferably low-temperature vacuum concentration) to obtain a coating concentrate;

[0034] Step 4) wetting the remaining part of the bezoar core obtained in step 1) with the coating concentrate obtained in step 3), and then coating it with the coating fine powder obtained in step 2);

[0035] Step 5) preparing crude bezoar: repeat the above step 4) at least 3 times, preferably any value or range between 3 and 10 times (for example, 3, 4, 5, 6, 7, 8, 9 or 10 times), to obtain crude bezoar cultured in vitro. Preferably, the step 5) further comprises a drying step.

[0036] Preferably, the preparation process of the crude bezoar includes a detection process until the encapsulated particles are continuously increased in value to a diameter of any value or interval of 1-5 cm, preferably any value or interval of 2-3 cm, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 cm, and the outer surface is not wet and no particles or dust fall off, and the spherical or quasi-spherical shape is obtained, that is, the crude bezoar cultured in vitro.

[0037] Preferably, the calcium ion donor includes one or more of calcium chloride, calcium sulfate, calcium nitrate, calcium lactate or calcium hydroxide, and more preferably calcium chloride;

[0038] Preferably, the B vitamins include one or more of B1, B2, B3, B5, B6, B7, B9 or B12.

[0039] Preferably, the method further comprises further processing the crude bezoar cultured in vitro, and the further processing comprises the following steps:

[0040] Step 6) preparing the in vitro cultured bezoar semi-finished product: rotating and vibrating the in vitro cultured bezoar crude product obtained in step 5) in a container to obtain the bezoar semi-finished product;

[0041] Step 7) wetting the in vitro cultured bezoar semi-finished product obtained in step 6) with the coating concentrate obtained in step 3) and then drying;

[0042] Step 8) preparing in vitro cultured bezoar: repeating the above step 7) at least 3 times, preferably any value or interval between 3 and 10 times (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 times), to obtain in vitro cultured bezoar;

[0043] Preferably, the method for preparing the bezoar core in step 1) comprises:

[0044] S1) preparing sterilized bovine bile; filtering and sterilizing the bovine bile;

[0045] S2) preparing microbial seed solution: inoculating the microorganism into a seed culture medium and culturing it to the late logarithmic growth stage;

[0046] S3) inoculating the microbial seed solution into sterilized ox bile to form a fermentation system;

[0047] Preferably, the preparation method further comprises step S4):

[0048] S4) adjusting the pH of the fermentation system to alkaline (preferably any value or range between 10 and 14 (e.g., 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5 or 14)), adding a calcium ion donor and stirring, filtering out the brown-red precipitate, and washing with purified water until the pH is neutral to obtain the bezoar core.

[0049] Preferably, in step S3), the ratio of sterilized ox bile to microbial seed solution is (3-7):1, such as 3:1, 4:1, 5:1, 6:1 or 7:1.

[0050] Preferably, the microbial seed solution in step S3) may be a microbial seed solution of a single microorganism, or may be a mixed seed solution of microbial seed solutions of two or more microorganisms.

[0051] In a specific embodiment of the present invention, the microbial seed solution in step S3) includes ZY-001 seed solution and ZY-002 seed solution. Preferably, the ratio of ZY-001 seed solution to ZY-002 seed solution is any value or interval in (1-9): (1-9); more preferably, it is any value or interval in (1-5): (1-5), for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0052] Preferably, in the microbial seed solution described in step S3), the content of ZY-001 seed solution can be the same as the content of ZY-002 seed solution, and of course can also be greater than or less than the content of ZY-002 seed solution.

[0053] Preferably, the seed culture medium can be any culture medium suitable for microbial proliferation in the prior art. It is further preferred that one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts be added to the seed culture medium. More preferably, in the seed culture medium, the volume fraction of one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts is at least 40%, preferably any value between 40% and 60%, for example 40%, 45%, 50%, 55% or 60%.

[0054] Preferably, the seed culture medium may be MRS culture medium (eg, MRS broth culture medium or MRS agar culture medium).

[0055] Preferably, the initial pH of the fermentation system is any value or range of 4.5-8.0, more preferably any value or range of 5.5-8.0, and even more preferably any value or range of 6.0-8.0, for example 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0.

[0056] Preferably, the temperature of the fermentation system is any value or range between 20°C and 45°C, further preferably any value or range between 25°C and 40°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C.

[0057] Preferably, the fermentation time is at least 36 hours, more preferably at least 48 hours, for example 36 hours to 150 hours or more, for example 36, 40, 45, 48, 50, 55, 60, 65, 70, 72, 75, 80, 84, 85, 90, 95, 96, 100, 105, 108, 110, 115, 120, 125, 130, 135, 140, 144 or 150 hours or more.

[0058] Preferably, adjusting the pH of the fermentation system and adding the calcium ion donor in step S4) can be performed step by step or simultaneously.

[0059] For example, the pH of the fermentation system is adjusted by adding calcium hydroxide, which is hydrolyzed into calcium ions and hydroxide ions, while the pH is adjusted to 10-14 and a calcium ion donor is added.

[0060] For another example, the pH of the fermentation system is adjusted by adding alkaline substances such as sodium hydroxide, adjusting the pH to alkaline (preferably 10-14), and then adding a calcium ion donor. Preferably, the calcium ion donor can be added all at once, in batches, or continuously.

[0061] Preferably, the method for preparing the coating concentrate described in step 3) comprises:

[0062] Fermenting sterilized bovine bile using the microbial composition described in the first aspect, and concentrating the fermentation liquid (preferably low-temperature vacuum concentration) to obtain a coating concentrate;

[0063] or,

[0064] The preparation method of the coating concentrate described in step 3) comprises: fermenting sterilized bovine bile using Lactobacillus breve ZY-001 and Lactobacillus plantarum ZY-002 respectively to obtain ZY-001 bovine bile fermentation liquid and ZY-002 bovine bile fermentation liquid, mixing the ZY-001 bovine bile fermentation liquid and the ZY-002 bovine bile fermentation liquid and concentrating (preferably low-temperature vacuum concentration) to obtain a coating concentrate.

[0065] Preferably, the contents of the components of the coated fine powder obtained in step 2) include: 12% of bile acid, 10% of deoxycholic acid, 6% of taurine, 2.5% of cholesterol, 45% of bilirubin, 0.5% of ferrous sulfate, 0.5% of calcium ions or calcium ion donors, 0.5% of zinc sulfate, 1% of magnesium sulfate, 1% of ascorbic acid, 0.5% of B vitamins (preferably B1:B2:B3:B9=2:1:1:1) and 20.5% of bezoar cores, accounting for 12% of the final total weight.

[0066] Preferably, the grinding into powder in step 2) includes grinding into powder in a sterile, light-proof environment, and sieving, preferably sieving with 200 mesh.

[0067] Preferably, the conditions for the rotational vibration in step 6) are: the temperature is set to any value or interval of 35-40°C (for example, 35, 36, 37, 38, 39 or 40°C), the relative humidity is set to any value or interval of 40-50% (for example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50%), the rotation and vibration are performed once every 10 minutes, each time for at least 10 seconds, preferably any value or interval of 10-20 seconds (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 seconds), the next rotation direction is opposite, and the culture is at least 10 days, preferably any value or interval of 10-20 days (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days).

[0068] Preferably, the container comprises a solid fermentation culture device for in vitro cultivation of bezoar.

[0069] Preferably, the measured water content of the in vitro cultured bezoar is less than 10%, preferably less than 6%;

[0070] Preferably, the drying method includes dehumidification drying, vacuum drying, freeze drying or hot air drying, etc.

[0071] Preferably, the drying comprises drying in a dryer for at least 1 hour, preferably any value or interval between 1 and 10 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours).

[0072] In a specific embodiment of the present invention, the morphology or detection indexes of bezoar cultured in vitro at various stages are as follows:

[0073] Bezoar core: brown-red precipitate (i.e., calcium bilirubin precipitate);

[0074] In vitro cultured bezoar crude product: 1-5cm spherical or quasi-spherical particles, with non-wet outer surface and no particle dust falling off;

[0075] In vitro cultured bezoar semi-finished product: smooth surface, with fine cracks on the exterior;

[0076] In vitro cultured bezoar: The surface is smooth and there are no obvious visible cracks.

[0077] The fourth aspect of the present invention provides an in vitro cultured bezoar, which is prepared by the method described in the third aspect.

[0078] The terms “include” or “comprising” described in the present invention are open-ended expressions, which include the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0079] 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". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0080] The benefits of the present invention are:

[0081] 1. Compared with single strain fermentation, the use of the microbial composition of this issue, or the combined use of Levilactobacillus brevis ZY-001 and Lactiplantibacillus plantarum ZY-002, can obtain high-yield bilirubin calcium precipitation in a shorter time, which can greatly shorten the preparation time of in vitro cultured bezoar.

[0082] 2. The microorganisms contained in the microbial composition described in the present application are all edible microorganisms and / or probiotic microorganisms. The products produced by the fermentation and transformation of the strains do not contain any substances harmful to human health, which is conducive to ensuring the safety of the medical beauty products or pharmaceutical products produced by the product.

[0083] 3. The fermentation metabolism process of the microbial composition produces bile salt hydrolase, which has the ability to decompose bound bile salts in bile. At the same time, the microbial combination can utilize the ability of protein and carbohydrate macromolecules in bile, resulting in a decrease in the pH of bile; the produced bile salt hydrolase can decompose bound bile acid into free bile acid, and under acidic conditions, free bile acid and cholesterol co-precipitate; the bile salt hydrolase hydrolyzes bound bile salts, promotes the combination of bilirubin and calcium ions in bile to form bilirubin calcium; the concentration of bile salts in bile is reduced under the action of bile salt hydrolase, which is beneficial to the survival of some microorganisms sensitive to bile salts and is beneficial to the addition of other functional microorganisms for mixed fermentation during the fermentation process.

[0084] 4. The fermentation metabolism process of the microbial composition produces glucuronidase, which can promote the enzymatic hydrolysis of a large amount of conjugated bilirubin in cattle bile into free bilirubin, making it easier for bilirubin in bile to combine with calcium ions to form bilirubin calcium.

[0085] 5. The microbial composition can survive at a relatively low pH, and the bile salt hydrolase and β-glucuronidase produced by it have good catalytic activity in a slightly acidic environment, which is beneficial to the conversion of conjugated bilirubin in bile into free bilirubin.

[0086] 6. The microbial composition produces a large amount of mucopolysaccharides, glycoproteins and other substances during the fermentation and metabolism process, which are beneficial to the combination of bile acid, bilirubin calcium, etc. to form bezoar and / or bezoar core, and are beneficial to the formation of concentric layers of bezoar cultured in vitro, which is beneficial to the spherical and quasi-spherical shape of the outer surface of bezoar cultured in vitro and the formation of a densely wrapped protective layer with antioxidant and anti-attenuation properties, which is more similar to the outer layer of natural bezoar, and is beneficial to the stability of the quality of in vitro cultured bezoar products and long-term preservation.

[0087] 7. The microbial composition produces a large amount of digestive enzymes during metabolism, which promotes the body's nutrient metabolism, and produces a large amount of organic acids, which increases the activity of digestive enzymes, degrades nutrients that are difficult for the body to digest and absorb, and effectively improves the body's immune regulation function and anti-inflammatory ability; it has good anti-infection effects, effectively inhibits the growth of drug-resistant pathogens, and improves the body's immune performance. When the microbial combination is used in the production of medical beauty or pharmaceutical products, there will be no negative effects caused by pathogenic bacteria or conditional pathogens such as Escherichia coli and Clostridium perfringens.

[0088] Strain deposit information:

[0089] Lactobacillus breve ZY-001:

[0090] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0091] Address of the preservation unit: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province;

[0092] Deposit number: GDMCC No.63910;

[0093] Deposit date: October 23, 2023;

[0094] Classification name: Levilactobacillus brevis.

[0095] Lactobacillus plantarum ZY-002:

[0096] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0097] Address of the preservation unit: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province;

[0098] Deposit number: GDMCC No:63911;

[0099] Deposit date: October 23, 2023;

[0100] Classification name: Lactiplantibacillus plantarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 : An in vitro cultured bezoar product prepared by the method described in this application;

[0102] Figure 2 : An in vitro cultured bezoar product prepared by the method in the prior art. DETAILED DESCRIPTION

[0103] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0104] The formula of MRS broth medium is as follows: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.1 g / L, manganese sulfate (MnSO4·H2O) 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1 mL / L, pH value 5.7±0.2.

[0105] MRS agar medium formula ingredients: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.1 g / L, manganese sulfate (MnSO4·H2O) 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1 mL / L, agar 15 g / L, pH 6.2±0.2.

[0106] The short growth-promoting lactobacillus is (Levilactobacillus brevis) ZY-001 (hereinafter referred to as ZY-001), which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023. The storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the storage number is GDMCC No: 63910.

[0107] The plant lactobacillus is (Lactiplantibacillus plantarum) ZY-002 (hereinafter referred to as ZY-002), which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023. The storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the storage number is GDMCC No: 63911.

[0108] Example 1: Microbial composition screening

[0109] Lactic acid bacteria (GUS bacteria, including GUS1: ATCC 15820, GUS2: ATCC 14869, ZY-001) with β-glucuronidase activity were screened and inoculated into 50% ox bile-MRS medium, and cultured at 37° C. and 80 rpm for 48 h to obtain a culture.

[0110] Lactic acid strains (BSH bacteria, including BSH1: ATCC BAA-793, BSH2: ATCC 43121, ZY-002) with bile salt hydrolase were screened and inoculated into 50% ox bile-MRS medium, and cultured at 37° C. and 80 rpm for 48 hours to obtain a culture.

[0111] Take 5mL of the above-mentioned different GUS bacteria and BSH bacteria cultures respectively, mix and inoculate into 40mL of sterilized ox bile, continue to culture at 80rpm at 37°C for 48 hours, take samples after the fermentation is completed, and detect the free bilirubin content in the fermented ox bile according to the quality detection method specified in the in vitro culture of bezoar in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181) (proportional to the subsequent product yield, that is, the higher the free bilirubin content, the higher the amount of in vitro cultured bezoar core and final product prepared). Take different lactobacillus strains with β-glucuronidase activity and different lactobacillus strains with bile salt hydrolase activity for combined testing, and set a single bacterial fermentation control.

[0112] When ZY-002 strains were combined with GUS strains to ferment cattle bile, the free bilirubin content was significantly increased. Among all the tested combinations, the combination of ZY-001 and ZY-002 had the best fermentation effect, with the free bilirubin content increased by 81% to 69.28 mg / L (see Table 1 below).

[0113] Table 1

[0114] Free bilirubin content (mg / L) BSH1 BSH2 ZY-002 / GUS1 23.62 9.38 32.66 10.53 GUS2 30.35 8.83 52.57 12.66 ZY-001 41.67 20.18 69.28 38.27 / 8.19 7.25 6.88 6.29

[0115] Example 2 Comparison of single fermentation of ZY-001 and mixed fermentation of ZY-001 and ZY-002

[0116] Comparison of single fermentation of Lactobacillus breve ZY-001 with β-glucuronidase activity and co-fermentation of Lactobacillus breve ZY-001 and Lactobacillus plantarum ZY-002:

[0117] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0118] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile into the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C to the late logarithmic growth stage to obtain ZY-001 seed solution and ZY-002 seed solution.

[0119] 1. Single-strain fermentation of ox bile using Lactobacillus breve ZY-001 with β-glucuronidase activity:

[0120] The seed liquid of the expanded Lactobacillus breve ZY-001 strain was inoculated into the sterile ox bile culture medium at a ratio of 200 mL of seed liquid per 800 mL of sterile ox bile, and cultured at 37°C and 80 rpm for 60 h, 72 h, 84 h, 96 h, and 120 h, respectively. 2000 mL of saturated calcium hydroxide solution was added to each fermented ox bile, stirred thoroughly, boiled at normal pressure for 30 minutes, cooled, and the brown-red precipitate was filtered out and dried and weighed.

[0121] 2. First, Lactobacillus plantarum ZY-002 was fermented and cultured with ox bile for 20 hours, and then the seed liquid of Lactobacillus breve ZY-001 with β-glucuronidase activity was added for co-cultivation:

[0122] The seed solution of ZY-002 strain was inoculated into sterile ox bile culture solution at a ratio of 100 mL of seed solution per 800 mL of sterile ox bile, and cultured at 37°C and 80 rpm for 20 h to obtain the ox bile culture of ZY-002;

[0123] Then, 100 mL of the seed solution of the ZY-001 strain was inoculated into the ZY-002 ox bile culture, and the culture was continued at 37°C and 80 rpm for 24 h, 36 h, 60 h, and 84 h, respectively, to obtain ZY-001 and ZY-002 mixed fermented ox bile. 2000 mL of saturated calcium hydroxide solution was added to the fermented ox bile, stirred thoroughly, boiled at normal pressure for 30 minutes, cooled, and the brown-red precipitate was filtered and dried and weighed.

[0124] 3. First, ferment and culture the cow bile with Lactobacillus breve ZY-001 for 20 hours, then add the seed solution of Lactobacillus plantarum ZY-002 for co-cultivation:

[0125] The seed solution of ZY-001 strain was inoculated into sterile ox bile culture solution at a ratio of 100 mL of seed solution per 800 mL of sterile ox bile, and cultured at 37°C and 80 rpm for 20 h to obtain the ox bile culture of ZY-001;

[0126] Then, 100 mL of the seed solution of the ZY-002 strain was inoculated into the ZY-001 ox bile culture, and the culture was continued at 37°C and 80 rpm for 24 h, 36 h, 60 h, and 84 h, respectively, to obtain mixed fermented ox bile of ZY-001 and ZY-002. 2000 mL of saturated calcium hydroxide solution was added to the fermented ox bile, stirred thoroughly, boiled at normal pressure for 30 minutes, cooled, and the brown-red precipitate was filtered and dried and weighed.

[0127] 4. Lactobacillus breve ZY-001 and Lactobacillus plantarum ZY-002 with β-glucuronidase activity were inoculated simultaneously to ferment cattle bile:

[0128] 100 mL of ZY-001 and 100 mL of ZY-002 seed solution were inoculated into every 800 mL of sterile ox bile, and the culture was continued at 37°C and 80 rpm for 36 h, 48 h, 60 h, and 72 h to obtain ZY-001 and ZY-002 mixed fermented ox bile. 2000 mL of saturated calcium hydroxide solution was added to the fermented ox bile, stirred thoroughly, boiled at normal pressure for 30 minutes, cooled, filtered, and the brown-red precipitate was dried and weighed.

[0129] Table 2 Quality of brown-red precipitate in bovine bile fermented by ZY-001 single strain and ZY-001 / ZY-002 combination

[0130]

[0131]

[0132] The fermentation results are shown in Table 2.

[0133] The results showed that when the cow bile was fermented with a single strain of Lactobacillus breve ZY-001 with β-glucuronidase activity, the brown-red precipitate produced after 72 hours of fermentation had the highest mass (8.6 g), indicating that the fermentation was nearly complete after 72 hours of fermentation.

[0134] After the cow bile was pre-fermented with ZY-002, it was added to ZY-001 and continued to ferment. The mass of the brown-red precipitate produced after 56 hours of fermentation could reach 8.5g, which was similar to the mass of the brown-red precipitate produced after a single fermentation of ZY-001 for 72 hours.

[0135] After the cow bile was pre-fermented with ZY-001 for 20 hours and then added to ZY-002 for continued fermentation, the mass of the brown-red precipitate produced after 44 hours of cumulative fermentation could reach 6.2g, which was similar to the mass of the brown-red precipitate produced after a single fermentation of ZY-001 for 60 hours, but lower than the mass of the brown-red precipitate produced after a pre-fermentation of ZY-002 for 20 hours and then added to ZY-001 for continued fermentation for 24 hours. This shows that the fermentation method of pre-fermenting cow bile with ZY-002 and then adding to ZY-001 can speed up the overall fermentation process compared with the fermentation method of pre-fermenting cow bile with ZY-001 and then adding to ZY-002. The mass of the brown-red precipitate produced after 56 hours of fermentation could reach 8.4g, which was similar to the mass of the brown-red precipitate produced after a single fermentation of ZY-001 for 72 hours.

[0136] When ZY-001 and ZY-002 were inoculated with cow bile and mixed fermented for 48 hours, the mass of the brown-red precipitate produced could reach 8.6g, which is similar to the mass of the brown-red precipitate produced by ZY-001 alone fermented for 72 hours.

[0137] The results show that, by fermenting cattle bile with a mixture of ZY-002 and ZY-001, the present application unexpectedly found that the mixed fermentation greatly shortened the time to obtain calcium bilirubin precipitation compared to fermentation with ZY-001 alone.

[0138] Example 3 Preparation of ZY-001 Fermented Ox Bile

[0139] 1. Activation of strains: freeze-dried ZY-001 strains were activated in MRS medium at 37°C for two generations;

[0140] 2. Pretreatment of cattle bile: Obtain fresh cattle bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0141] 3. Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve it with an appropriate amount of distilled water, slowly add 50 mL of the above-mentioned pretreated ox bile to the solution, dilute to 100 mL with distilled water, prepare 50% ox bile-MRS medium, inoculate the activated ZY-001 at a 2% inoculum into the 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage to obtain ZY-001 seed solution;

[0142] 4. Ox bile fermentation: inoculate the above ZY-001 seed solution into sterile ox bile culture medium at a ratio of 20 mL of seed solution per 80 mL of sterile ox bile, and ferment at 37°C and 80 rpm for 72 hours to obtain ZY-001 fermented ox bile.

[0143] Example 4 Preparation of ZY-002 Fermented Ox Bile

[0144] Activation of strains: The freeze-dried ZY-002 strain was activated in MRS medium at 37°C for two generations;

[0145] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0146] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with an appropriate amount of distilled water, slowly add 50 mL of the above-mentioned pretreated ox bile to the solution, dilute to 100 mL with distilled water, prepare 50% ox bile-MRS medium, inoculate the activated ZY-002 at a 2% inoculum into the 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage to obtain ZY-002 seed solution;

[0147] Ox bile fermentation: inoculate the above ZY-002 seed solution into sterile ox bile culture medium at a ratio of 20 mL of seed solution per 80 mL of sterile ox bile, and ferment at 37°C and 80 rpm for 72 h to obtain ZY-002 fermented ox bile.

[0148] Example 5: Preparation of in vitro cultured bezoar core by mixing ZY-001 and ZY-002 in deep fermented bovine bile

[0149] 1. Seed solution preparation:

[0150] The activated ZY-001 was inoculated with 2% of the inoculum into 500 mL of 50% ox bile-MRS medium, and cultured at 37°C until the late logarithmic growth phase to obtain the ZY-001 seed solution; the activated ZY-002 was inoculated with 2% of the inoculum into 500 mL of 50% ox bile-MRS medium, and cultured at 37°C until the late logarithmic growth phase to obtain the ZY-002 seed solution;

[0151] 2. Mixed fermentation: Place 3.2L of filtered bovine bile in a fermentation container with an automatic pH detection system and steam sterilize. Then take 400mL of ZY-001 and 400mL of ZY-002 seed liquid and transfer them into the above sterilized fermentation container. Under clean and aseptic conditions, mix and inoculate the sterile filtered bovine bile fermentation liquid of ZY-001 and ZY-002 and continue fermentation. Control the culture temperature at 37°C, the ventilation volume at 1:0.2V / VM, 50-80rpm, and the total fermentation time for 60h. After inoculation, when the pH of the fermentation liquid drops to 4.5±0.1, add saturated calcium hydroxide solution by aseptic operation to adjust and control the pH to maintain at 5.0-5.5 for 48 hours, then add saturated calcium hydroxide solution to adjust and control the pH to 5.5 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 5.75 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 6.0 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 6.25 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 6.5 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 6.75 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 7.0 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 7.25 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to 7.5 for 30 minutes, then add saturated calcium hydroxide solution to adjust and control the pH to The reaction mixture was run at 7.75 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 8.0, and then saturated calcium hydroxide solution was added to adjust and control the pH value to 8.25 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 8.5 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 8.75 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 9.0 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 9.5 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 10.0 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 10.5 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 11.0 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 11.5 for 30 min, then saturated calcium hydroxide solution was added to adjust and control the pH value to 12.0 and maintained.The pH is adjusted with saturated calcium hydroxide so that the bile salt hydrolase, glucuronidase, carotenoid, hydrogen peroxide reductase, superoxide dismutase and other biologically active enzymes (bioreductase) in the fermentation broth are catalyzed in their respective optimal pH ranges, and various amino acids, short peptides, polypeptides, proteins, bile acid, cholesterol, bilirubin, bile acid sodium salt, bile pigments, mucin bodies and a small amount of fat, cholesterol, lecithin, choline, urea, as well as sodium chloride, calcium phosphate, iron phosphate and the like produced by the mixed fermentation metabolism are flocculated, covalently bound and combined in the optimal pH range, and the operation of adding saturated calcium hydroxide solution at the same time can provide enough free calcium ions to achieve pairing and combination to form bilirubin calcium.

[0152] 3. Obtaining in vitro cultured bezoar core:

[0153] The mixed bovine bile fermentation broth with a final pH of 12.0, which was adjusted to different pH values ​​with a saturated calcium hydroxide solution and cultured for a certain period of time, was stirred in a single direction at a stirring speed of 120 rpm for 30 min, filtered or centrifuged to obtain a covalently bound flocculated spherical brown-red precipitate, washed with purified water until the purified water was pH neutral, and dried to obtain 35 g (dry weight) of in vitro cultured bezoar core.

[0154] Example 6: Preparation of in vitro cultured bezoar core by mixing ZY-001 and ZY-002 with deep fermented bovine bile:

[0155] 1. Seed solution preparation:

[0156] The activated ZY-001 was inoculated with 2% of the inoculum into 500 mL of 50% ox bile-MRS medium, and cultured at 37°C until the late logarithmic growth phase to obtain the ZY-001 seed solution; the activated ZY-002 was inoculated with 2% of the inoculum into 500 mL of 50% ox bile-MRS medium, and cultured at 37°C until the late logarithmic growth phase to obtain the ZY-002 seed solution;

[0157] 2. Mixed fermentation: 3.2L filtered cattle bile is placed in a fermentation container with an automatic pH detection system and steam sterilized. Then 400mL of ZY-001 and 400mL of ZY-002 seed solution are transferred to the sterilized fermentation container. Under clean and aseptic conditions, the sterile filtered cattle bile fermentation broth of ZY-001 and ZY-002 is mixed and inoculated and continued to ferment. The culture temperature is controlled at 37°C, the ventilation volume is 1:0.2V / VM, 50-80rpm, and the total fermentation time is 60h. After inoculation, when the pH of the fermentation broth drops to 4.5±0.1, sodium hydroxide solution is added to control the pH at 5.0-5.5 by aseptic operation for 48h, and then sterile calcium chloride solution is added to a final concentration of 0.25mol / L. At the same time, sodium hydroxide solution is added to control the pH at 5.5 for 30min, and then sodium hydroxide solution is added to control the pH at 5.75 for 30min, and then sodium hydroxide solution is added to control the pH. 6.0 run for 30min, then add sodium hydroxide solution to adjust and control pH 6.25 run for 30min, then add sodium hydroxide solution to adjust and control pH 6.5 run for 30min, then add sodium hydroxide solution to adjust and control pH 6.75 run for 30min, then add sodium hydroxide solution to adjust and control pH 7.0 run for 30min, then add sodium hydroxide solution to adjust and control pH 7.25 run for 30min, then add sodium hydroxide solution to adjust and control pH 7.5 run for 30min, then add sodium hydroxide solution to adjust and control pH 7.75 run for 30min, then add sodium hydroxide solution to adjust and control pH 8.0 run for 30min, then add sodium hydroxide solution to adjust and control pH 8.25 run for 30min, then add sodium hydroxide solution to adjust and control pH 8.5 run for 30min, then add sodium hydroxide solution to adjust and control pH 8.75 run for 30min, then add sodium hydroxide solution to adjust and control pH 9.0 run for 30min, then add sodium hydroxide solution to adjust and control pH The pH value was adjusted to 9.5 for 30 min, and then sodium hydroxide solution was added to adjust and control the pH value to 10.0 for 30 min. The pH value was adjusted to 10.5 for 30 min, and then sodium hydroxide solution was added to adjust and control the pH value to 11.0 for 30 min and maintained.The operation of adjusting pH makes the bile salt hydrolase, glucuronidase, carotenoid, hydrogen peroxide reductase, superoxide dismutase and other biologically active enzymes (bioreductase) in the fermentation broth catalyze in their respective optimal pH ranges, and various amino acids, short peptides, polypeptides, proteins, bile acid, cholesterol, bilirubin, bile acid sodium salt, bile pigments, mucin bodies and a small amount of fat, cholesterol, lecithin, choline, urea, as well as sodium chloride, calcium phosphate, iron phosphate and the like produced by the mixed fermentation metabolism are flocculated, covalently bound in their respective optimal pH ranges; the operation of adding calcium chloride solution can provide enough free calcium ions to achieve pairing and binding to form calcium bilirubin.

[0158] 3. Obtaining in vitro cultured bezoar core:

[0159] The mixed bovine bile fermentation broth with a final pH of 11.0, which had been adjusted to different pH values ​​with sodium hydroxide solution and cultured for a certain period of time, was stirred in a single direction at a stirring speed of 120 rpm for 30 min, filtered or centrifuged to obtain a covalently bound flocculated spherical brown-red precipitate, washed with purified water until the purified water was pH neutral, and dried to obtain 32 g (dry weight) of in vitro cultured bezoar core.

[0160] Example 7 In vitro cultivation of bezoar core proliferation and fermentation

[0161] 1. Prepare bezoar core, and obtain in vitro cultured bezoar core according to the method described in Example 5 or 6.

[0162] 2. Preparation of coated fine powder: according to pharmaceutical grade or food grade standards, add 12% of bile acid, 10% of deoxycholic acid, 6% of taurine, 2.5% of cholesterol, 45% of bilirubin, 0.5% of ferrous sulfate, 0.5% of calcium chloride, 0.5% of zinc sulfate, 1% of magnesium sulfate, 1% of ascorbic acid, and 0.5% of B vitamins (B1:B2:B3:B9=2:1:1:1) to the final total weight, and add the in vitro cultured bezoar core prepared in Example 5 or 6 to make up to 100% of the final total weight, grind into fine powder in a clean and sterile dark room or red light dark room and mix well.

[0163] 3. Preparation of coating concentrate: The ZY-001 fermented bovine bile prepared in Example 3 and the ZY-002 fermented bovine bile prepared in Example 4 were mixed in a ratio of 2:1, the pH of the mixed fermentation liquid was adjusted to 6.8±0.2, and vacuum concentrated under clean and sterile conditions, with the temperature controlled at 28°C±2°C and the vacuum degree being above -0.085 MPa; the mixed fermentation liquid was concentrated until the appearance of the mixed fermentation liquid was obviously sticky to the wall of a glass vacuum rotator and obvious stringing was obtained by picking with a glass rod, thereby obtaining a coating concentrate rich in bile, calcium bilirubin, various biologically active enzymes, mucopolysaccharides, glycoproteins and other active substances.

[0164] 4. Wrapping the bezoar core: under clean, sterile, and dark conditions, the in vitro cultured bezoar core obtained by mixing ZY-001 and ZY-002 with deep fermented ox bile or the in vitro cultured bezoar core obtained in the above step 1 is placed in an in vitro cultured bezoar solid fermentation solid culture device, first fully moistened with the above coating concentrate, then fully spherically wrapped with the above fully mixed ground fine powder, then fully moistened with the above coating concentrate, then fully spherically wrapped with the above fully mixed ground fine powder, after being wetted and wrapped 5 times (and the last time is wrapped with the concentrated solution), move it into a clean silica gel dryer and dry it for more than 6 hours, take it out and observe that the outer surface is not wet and no particles and dust fall off; the dried wrapped particles are fully moistened with the coating concentrate again, and the above process is repeated until the wrapped particles are continuously proliferated and grown into a spherical or quasi-spherical in vitro cultured bezoar crude product with a diameter of between 2cm and 3cm, an outer surface that is not wet and no particles and dust fall off.

[0165] 5. The obtained in vitro cultivated bezoar crude product is placed in an in vitro cultivated bezoar solid fermentation solid culture device in a clean and sterile dark room, the temperature of the solid culture device is controlled to be 38°C ± 1°C, the relative humidity is 40%-50%, and the solid culture device is rotated and vibrated once every 10 minutes for 15 seconds each time, and the next rotation direction is opposite, so that the in vitro cultivated bezoar has a slight rotation or rolling movement in the solid culture device. After 15 days of solid culture, the outer surface is smooth and fine cracks appear in some parts, thereby obtaining the in vitro cultivated bezoar semi-finished product.

[0166] 6. Fully moisten the above-mentioned in vitro cultivated bezoar semi-finished product with the prepared coating concentrate, place it in a dryer to dry for 6 hours, then fully moisten it with the coating concentrate, and then place it in a dryer to dry for 6 hours. Repeat the wetting and drying process for more than 5 times. The in vitro cultivated bezoar product is considered to have no obvious cracks on the appearance.

[0167] The in vitro cultured bezoar product obtained by the above method is tested under the item of cultured bezoar outside the carrier in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181):

[0168] Appearance: spherical or quasi-spherical, with a diameter of 2cm-3cm. The surface is smooth, yellow-red to brown-yellow. It is light, crispy, and has concentric layers on the cross section; it has a fragrant smell, tastes bitter and then sweet, and has a cool feeling; the measured moisture content is ≤6%; the bilirubin content of the in vitro cultivated bezoar obtained by the above method is 37%, the bile acid content is 9%, and the absorbance of free bilirubin at OD 453 is 0.13. The above determination indicators are in accordance with the provisions of the in vitro cultivated bezoar item of the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181).

[0169] In addition, the present application avoids using adhesives or other additives such as dextrin in the prior art, and chooses to use the concentrated liquid of the fermentation liquid as the coating concentrated liquid, and mixes part of the in vitro cultivated bezoar core with other ingredients to form a fine powder, which not only makes the internal and external components of the prepared in vitro cultivated bezoar uniform, but also because the coating concentrated liquid is a low-temperature vacuum concentration of the fermentation liquid, the microorganisms in the coating concentrated liquid can still remain alive. During the wetting and wrapping process, the microorganisms are still constantly simulating the in vivo bezoar cultivation process, so that the obtained in vitro cultivated bezoar product forms a layer-by-layer wheel pattern cross-section, which is more compact and uniform in composition. For the in vitro cultivated bezoar product obtained using the preparation method of the present application, see Figure 1 , and the in vitro cultured bezoar obtained by conventional means of the prior art is shown in Figure 2 .

[0170] Example 8: Production of bilirubin calcium precipitate by mixed fermentation of ZY-001 and ZY-002

[0171] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0172] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile to the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C until the late logarithmic growth stage;

[0173] Ox bile fermentation: inoculate the seed liquid of ZY-002 strain into sterile ox bile culture medium at a ratio of 100 mL of seed liquid per 800 mL of sterile ox bile, and culture at 37°C and 80 rpm for 20 h to obtain the ox bile culture of ZY-002; then inoculate 100 mL of the seed liquid of ZY-001 strain into the ox bile culture of ZY-002, and continue to culture at 37°C and 80 rpm for 72 h to obtain the mixed fermentation ox bile of ZY-001 and ZY-002.

[0174] Add 2000 mL of saturated calcium hydroxide solution to the fermented ox bile, stir thoroughly, boil at normal pressure for 30 minutes, cool, and filter out 8.7 g (dry weight) of brown-red precipitate.

[0175] Example 9: Production of Calcium Bilirubin Precipitate by Mixed Fermentation of ZY-001 and ZY-002

[0176] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0177] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile to the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C until the late logarithmic growth stage;

[0178] Ox bile fermentation: inoculate the seed liquid of ZY-002 strain into sterile ox bile culture medium at a ratio of 50 mL of seed liquid per 800 mL of sterile ox bile, and culture at 37°C and 80 rpm for 20 h to obtain the ox bile culture of ZY-002; then inoculate 150 mL of the seed liquid of ZY-001 strain into the ox bile culture of ZY-002, and continue to culture at 37°C and 80 rpm for 72 h to obtain the mixed fermentation ox bile of ZY-001 and ZY-002.

[0179] The pH of the fermented ox bile was adjusted to 12 with a saturated calcium hydroxide solution, and 100 mL of a 10 mmol / L calcium chloride solution was added, stirred thoroughly, and 8.9 g (dry weight) of a brown-red precipitate was filtered out.

[0180] Example 10: Production of bilirubin calcium precipitate by mixed fermentation of ZY-001 and ZY-002

[0181] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0182] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile to the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C until the late logarithmic growth stage;

[0183] Ox bile fermentation: 50 mL of ZY-002 and 150 mL of ZY-001 seed solution were inoculated into every 800 mL of sterile ox bile and cultured at 37°C and 80 rpm for 84 h to obtain ZY-001 and ZY-002 mixed fermented ox bile.

[0184] The pH of the fermented ox bile was adjusted to 12 with a saturated calcium hydroxide solution, and 150 mL of a 10 mmol / L calcium chloride solution was added. The mixture was stirred thoroughly, and 8.4 g (dry weight) of a brown-red precipitate was filtered out.

[0185] Example 11: Production of bilirubin calcium precipitate by mixed fermentation of ZY-001 and ZY-002

[0186] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0187] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile to the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C until the late logarithmic growth stage;

[0188] Ox bile fermentation: 150 mL of ZY-002 and 50 mL of ZY-001 seed solution were inoculated into every 800 mL of sterile ox bile and cultured at 37°C and 80 rpm for 96 h to obtain ZY-001 and ZY-002 mixed fermented ox bile.

[0189] The pH of the fermented ox bile was adjusted to 12 with a saturated calcium hydroxide solution, and 150 mL of a 10 mmol / L calcium chloride solution was added. The mixture was stirred thoroughly, and 8.5 g (dry weight) of a brown-red precipitate was filtered out.

[0190] Example 12: Production of calcium bilirubin precipitate by mixed fermentation of ZY-001 and ZY-002

[0191] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0192] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile to the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C until the late logarithmic growth stage;

[0193] Ox bile fermentation: 40 mL of ZY-002 and 160 mL of ZY-001 seed solution were inoculated into every 800 mL of sterile ox bile and cultured at 37°C and 80 rpm for 84 h to obtain ZY-001 and ZY-002 mixed fermented ox bile.

[0194] The pH of the fermented ox bile was adjusted to 12 with a saturated calcium hydroxide solution, and 150 mL of a 10 mmol / L calcium chloride solution was added. The mixture was stirred thoroughly and 8.2 g (dry weight) of a brown-red precipitate was filtered out.

[0195] Example 13: Production of bilirubin calcium precipitate by mixed fermentation of ZY-001 and ZY-002

[0196] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0197] Preparation of seed solution: weigh each component according to the MRS formula, fully dissolve it with appropriate amount of distilled water, slowly add appropriate amount of the above-mentioned pretreated ox bile to the solution to prepare 50% ox bile-MRS medium, inoculate ZY-002 and ZY-001 into 50% ox bile-MRS medium respectively, and culture at 37°C until the late logarithmic growth stage;

[0198] Ox bile fermentation: 180 mL of ZY-002 and 20 mL of ZY-001 seed solution were inoculated into every 800 mL of sterile ox bile and cultured at 37°C and 80 rpm for 108 h to obtain ZY-001 and ZY-002 mixed fermented ox bile.

[0199] The pH of the fermented ox bile was adjusted to 12 with a saturated calcium hydroxide solution, and 150 mL of a 10 mmol / L calcium chloride solution was added, stirred thoroughly, and 8.6 g (dry weight) of a brown-red precipitate was filtered out.

[0200] It can be understood that, in this embodiment, although the content of ZY001 is extremely low, only 20 ml, when used in combination with ZY002, the bilirubin calcium yield can be equivalent to that of ZY001 with a concentration of 200 ml.

[0201] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0202] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A microbial composition, characterized in that The microbial composition comprises: 1) a microorganism having glucuronidase activity; and, 2) Lactobacillus plantarum Lactiplantibacillus plantarum )ZY-002; The plant lactobacillus ZY-002 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the deposit number GDMCC No: 63911.

2. The microbial composition according to claim 1, characterized in that In the microbial composition, the ratio of the microorganism having glucuronidase activity to Lactobacillus plantarum ZY-002 is (1-9): (1-9).

3. The microbial composition according to claim 2, characterized in that In the microbial composition, the ratio of the microorganism having glucuronidase activity to Lactobacillus plantarum ZY-002 is (1-5): (1-5).

4. The microbial composition according to any one of claims 1 to 3, characterized in that The microorganisms having glucuronidase activity include Escherichia coli ( Escherichia ), Clostridium ( Clostridium ), Proteus ( Proteus ) or Lactobacillus spp. ( Lactobacillus ) one or more of the following.

5. The microbial composition according to claim 4, characterized in that The genus Lactobacillus includes Levilactobacillus brevis.

6. The microbial composition according to claim 5, characterized in that The probiotic lactobacillus ( Levilactobacillus brevis ) is Lactobacillus breve ZY-001, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the deposit number GDMCC No: 63910.

7. Use of the microbial composition according to any one of claims 1 to 6 in the preparation of in vitro cultured bezoar.

8. A method for preparing in vitro cultured bezoar, characterized in that: The method comprises using a microorganism, wherein the microorganism comprises a microbial composition according to any one of claims 1 to 6; The method comprises the following steps: Step 1) preparing bezoar core: fermenting cattle bile using microorganisms, collecting brown-red precipitates, and obtaining bezoar core; Step 2) preparing coated fine powder: taking part of the bezoar core obtained in step 1), adding at least one or two or more of bile acid, deoxycholic acid, taurine, cholesterol, bilirubin, ferrous sulfate, calcium ion donor, zinc sulfate, magnesium sulfate, ascorbic acid or B vitamins, mixing well and grinding into powder to obtain coated fine powder; Step 3) preparing a coating concentrate: fermenting the cow bile using microorganisms, and concentrating the fermentation liquid to obtain a coating concentrate; Step 4) wetting the remaining part of the bezoar core obtained in step 1) with the coating concentrate obtained in step 3), and then coating it with the coating fine powder obtained in step 2); Step 5) preparing crude bezoar: repeat the above step 4) at least 3 times to obtain crude bezoar cultured in vitro.

9. The method according to claim 8, characterized in that The step 5) includes repeating the step 4) 3-10 times.

10. The method according to claim 8, characterized in that The step 5) further comprises a drying step.

11. The method according to claim 8, characterized in that The calcium ion donor includes one or more of calcium chloride, calcium sulfate, calcium nitrate, calcium lactate or calcium hydroxide.

12. The method according to claim 8, characterized in that The B vitamins include one or more of B1, B2, B3, B5, B6, B7, B9 or B12.

13. The method according to any one of claims 8 to 12, characterized in that: The method further comprises further processing the crude in vitro cultured bezoar, wherein the further processing comprises the following steps: Step 6) preparing the in vitro cultured bezoar semi-finished product: rotating and vibrating the in vitro cultured bezoar crude product obtained in step 5) in a container to obtain the bezoar semi-finished product; Step 7) wetting the in vitro cultured bezoar semi-finished product obtained in step 6) with the coating concentrate obtained in step 3) and then drying; Step 8) Preparing in vitro cultured bezoar: repeat the above step 7) at least 3 times to obtain in vitro cultured bezoar.

14. The method according to claim 13, characterized in that The step 8) includes repeating the above step 7) 3-10 times.

15. The method according to any one of claims 8 to 12, characterized in that: The preparation method of the bezoar core in step 1) comprises: S1) preparing sterilized bovine bile; filtering and sterilizing the bovine bile; S2) preparing microbial seed solution: inoculating the microorganism into a seed culture medium and culturing it to the late logarithmic growth stage; S3) The microbial seed solution is introduced into sterilized ox bile to form a fermentation system.

16. The method according to claim 15, characterized in that The preparation method further comprises step S4): S4) adjusting the pH of the fermentation system to 10-14, adding a calcium ion donor and stirring, filtering out the brown-red precipitate, and washing with purified water until the pH is neutral to obtain the bezoar core.

17. The method according to any one of claims 8 to 12, characterized in that: The method for preparing the coating concentrate in step 3) comprises: fermenting sterilized bovine bile using the microbial composition described in any one of claims 1 to 6, and concentrating the fermentation liquid to obtain the coating concentrate; or The preparation method of the coating concentrate described in step 3) comprises: fermenting sterilized bovine bile using Lactobacillus breve ZY-001 and Lactobacillus plantarum ZY-002 respectively to obtain ZY-001 bovine bile fermentation liquid and ZY-002 bovine bile fermentation liquid, mixing the ZY-001 bovine bile fermentation liquid and the ZY-002 bovine bile fermentation liquid and concentrating them to obtain a coating concentrate.

18. The method according to claim 17, characterized in that The concentration includes low-temperature vacuum concentration.

19. The method according to any one of claims 8 to 12, characterized in that: The rotation and vibration conditions described in step 6) are: the temperature is set to 35-40° C., the relative humidity is set to 40-50%, the rotation and vibration are performed once every 10 minutes, each time for at least 10 seconds, and the next rotation direction is opposite, and the cultivation is at least 10 days.

20. The method according to claim 19, characterized in that Rotate and vibrate once every 10 minutes, for 10-20 seconds each time, and the next rotation direction will be opposite.

21. The method according to claim 19, characterized in that Cultivate for 10-20 days.

22. The method according to any one of claims 8 to 12, characterized in that: The measured water content of the in vitro cultured bezoar is less than 10%.

23. The method according to claim 22, characterized in that The measured water content of the in vitro cultured bezoar is less than 6%.

24. The method according to claim 13, characterized in that The drying in step 7) comprises drying in a dryer for at least 1 hour.

25. The method according to claim 24, characterized in that The drying in step 7) comprises drying in a dryer for 1-10 hours.

26. An in vitro culture of bezoar, characterized in that: The in vitro cultured bezoar is prepared by the method described in any one of claims 8-25.

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