A kind of growth-promoting lactobacillus breve and application thereof

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a short growth-promoting lactobacillus and its application. The short growth-promoting lactobacillus is preserved in the Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No: 63910. The short growth-promoting lactobacillus can be used for bovine bile fermentation. A large amount of glucuronidase is produced during the fermentation process to promote the conversion of bound bilirubin in bile into free bilirubin, so that the free bilirubin is combined with exogenously added calcium ions to form bilirubin calcium precipitation.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a growth-promoting lactobacillus breve and an application thereof. Background Art

[0002] Free bilirubin (indirect bilirubin) is the main raw material for the manufacture of in vitro cultured bezoar and artificial bezoar. It has multiple pharmacological effects and is widely used in pharmaceutical and medical beauty products. The market demand is huge. At present, the main method for obtaining free bilirubin and bilirubin calcium in the market is to extract them from the bile of various animals. The extraction methods adopt physical and chemical methods such as precipitation, solvent extraction and ion exchange resin adsorption:

[0003] Chinese patent CN101429152A "Method for extracting bilirubin from cattle bile" reduces 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 large amount of acid, alkali and solvent, resulting in high production costs.

[0004] Chinese patent CN104774885A "A method for extracting bilirubin from bovine bile by fermentation" uses a biological method combined with a solvent extraction method to obtain bilirubin from animal bile, which improves the yield and purity of bilirubin. However, the strains used in microbial fermentation are pathogenic bacteria or conditional pathogens such as Escherichia coli and Clostridium perfringens, which makes it difficult to separate and purify the product to remove harmful bacterial toxins produced by pathogenic bacteria. The application of the product in medical beauty or pharmaceutical products has certain safety risks, and the scale of application is limited.

[0005] Chinese invention patent application CN115851497A "A strain of cow bile-resistant bezoar transforming bacteria and its application" discloses a strain of Citrobacter rodinii CGMCC No.24893, which can tolerate cow bile and has the ability to ferment cow bile to produce free bilirubin and free bile acid. However, the delay period of this strain of Citrobacter rodinii in cow bile is 48 hours, and the fermentation cycle is as long as 16 days. In addition, Citrobacter rodinii is a Gram-negative bacterium, which will produce endotoxins and other toxic factors during the fermentation process. There are certain safety risks in the application of the product in medical beauty or pharmaceutical products. Summary of the invention

[0006] The present invention uses safe and effective probiotic strains to ferment cattle bile to produce free bilirubin and bilirubin calcium, realizes in vitro cultivation of bezoar, 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, environmental protection, product quality and safety, etc., and has good economic and social benefits. Specifically:

[0007] The first aspect of the present invention provides a brevis lactobacillus (Levilactobacillus brevis), which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the storage address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the storage number is GDMCC No: 63910.

[0008] Preferably, the sequence of 16s rDNA of the Lactobacillus breve is shown as SEQ ID NO: 1.

[0009] Preferably, the Lactobacillus breve has the ability to tolerate ox bile, and further preferably, the Lactobacillus breve can survive in pure ox bile for at least 6 days.

[0010] The Lactobacillus breve described in the present invention is derived from traditional fermented food and is a generally recognized as safe (GRAS) strain.

[0011] The second aspect of the present invention provides a method for culturing the breve lactobacillus described in the first aspect, the culturing method comprising using a culture medium, the culture medium comprising ox bile with a volume fraction of at least 10%, preferably any value or range between 10% and 50%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0012] The third aspect of the present invention provides an application of the Lactobacillus breve described in the first aspect in fermenting bovine bile, converting conjugated bilirubin into free bilirubin, preparing calcium bilirubin, and / or preparing in vitro cultured bezoar.

[0013] The fourth aspect of the present invention provides a method for converting conjugated bilirubin into free bilirubin, the method comprising introducing a microorganism into a system containing conjugated bilirubin, the microorganism comprising the Lactobacillus breve described in the first aspect above.

[0014] Preferably, the method comprises fermenting a system containing conjugated bilirubin using a microorganism.

[0015] Preferably, the method comprises filtering and sterilizing the system containing conjugated bilirubin to obtain a sterilized system.

[0016] Preferably, the method comprises inoculating the microorganism into a seed culture medium to obtain a microorganism seed solution.

[0017] Preferably, the seed culture medium can be any culture medium in the prior art that is suitable for the proliferation of microorganisms (especially lactobacilli). It is further preferred that one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts is 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%.

[0018] Preferably, the seed culture medium may be MRS culture medium (eg, MRS broth culture medium or MRS agar culture medium).

[0019] 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.

[0020] Preferably, the method comprises connecting the above-mentioned microbial seed liquid to the above-mentioned sterilization system to obtain a fermentation system.

[0021] Preferably, the access ratio of the microbial seed liquid accounts for any value or interval of 5%-90 of the volume fraction of the fermentation system, preferably any value or interval of 20%-50%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0022] Preferably, the initial pH of the fermentation system is any value or interval between 4 and 8, such as 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8. In one embodiment of the present invention, the initial pH of the fermentation system is controlled to be 6.5 or 7.

[0023] 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.

[0024] In a specific embodiment of the present invention, the temperature of the fermentation system is controlled at 37°C.

[0025] Preferably, the fermentation time is at least 30 hours, preferably any value or interval between 30 and 150 hours, for example, 30, 35, 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, 145 or 150 hours;

[0026] Preferably, the system comprising conjugated bilirubin comprises ox bile.

[0027] In a specific embodiment of the present invention, the method comprises fermenting bovine bile using microorganisms.

[0028] The fifth aspect of the present invention provides a fermentation product obtained by the method described in the fourth aspect.

[0029] Preferably, the fermentation product comprises a system with high free bilirubin.

[0030] The sixth aspect of the present invention provides a method for preparing calcium bilirubin, the method comprising introducing a microorganism into a system containing bound bilirubin, wherein the microorganism comprises the Lactobacillus breve described in the first aspect.

[0031] Preferably, the preparation method further comprises adding calcium ions.

[0032] Preferably, the preparation method comprises fermenting a system containing conjugated bilirubin using a microorganism;

[0033] Preferably, the preparation method comprises filtering and sterilizing the system containing conjugated bilirubin to obtain a sterilized system;

[0034] Preferably, the preparation method comprises inoculating the microorganism into a seed culture medium to obtain a microorganism seed solution.

[0035] Preferably, the seed culture medium can be any culture medium in the prior art that is suitable for the proliferation of microorganisms (especially lactobacilli). It is further preferred that one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts is 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%.

[0036] Preferably, the seed culture medium may be MRS culture medium (eg, MRS broth culture medium or MRS agar culture medium).

[0037] 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.

[0038] Preferably, the method comprises connecting the microbial seed liquid to the sterilization system to obtain the fermentation system.

[0039] Preferably, the access ratio of the microbial seed liquid accounts for any value or interval of 5%-90 of the volume fraction of the fermentation system, preferably any value or interval of 20%-50%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0040] Preferably, the initial pH of the fermentation system is any value or interval between 4 and 8, such as 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8. In one embodiment of the present invention, the initial pH of the fermentation system is controlled to be 6.5 or 7.

[0041] Preferably, the temperature of the fermentation system is any value or interval between 25-40°C, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40;

[0042] In a specific embodiment of the present invention, the temperature of the fermentation system is controlled at 37°C.

[0043] Preferably, the fermentation time is at least 30 hours, preferably any value or interval between 30 and 150 hours, for example 30, 35, 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, 145 or 150 hours.

[0044] Preferably, the system comprising conjugated bilirubin comprises ox bile.

[0045] Preferably, the adding of calcium ions includes adding in the middle or late stage of fermentation or after the end of fermentation.

[0046] Preferably, calcium ions are added before or after adjusting the pH of the fermentation system to alkaline (preferably adjusting the pH to 11-14), or calcium ions are added while adjusting the pH of the fermentation system to alkaline (preferably adjusting the pH to 11-14).

[0047] Preferably, adding calcium ions comprises adding a calcium ion donor;

[0048] Further preferably, the calcium ion donor includes one or more of calcium oxide, calcium sulfate, calcium chloride or calcium hydroxide.

[0049] Preferably, the calcium ions are added in a one-time manner, or in batches, or continuously.

[0050] Preferably, the step of adding calcium ions and the step of adjusting pH can be the same step. For example, the step of adjusting pH includes adding calcium hydroxide, which produces calcium ions and hydroxide ions after hydrolysis, thereby achieving the effects of adjusting pH and adding calcium ions at the same time.

[0051] Preferably, after adding calcium ions into the system, the process further comprises the steps of stirring, separating, filtering and washing, and more preferably, further comprises the step of drying;

[0052] More preferably, the stirring includes stirring at normal temperature and pressure, or stirring under heating;

[0053] More preferably, the drying includes dehumidification drying, vacuum drying, freeze drying or hot air drying.

[0054] In a specific embodiment of the present invention, the method comprises fermenting bovine bile using microorganisms.

[0055] The seventh aspect of the present invention provides a mixture containing calcium bilirubin, wherein the mixture containing calcium bilirubin is obtained by the preparation method described in the sixth aspect.

[0056] The eighth aspect of the present invention provides a method for cultivating bezoar in vitro, the method comprising subjecting the bilirubin calcium prepared by the preparation method described in the ninth aspect, or the mixture containing bilirubin calcium described in the tenth aspect, to the following steps:

[0057] Step 1) preparing bezoar core: washing with calcium bilirubin or a mixture containing calcium bilirubin and adjusting the pH to neutral to obtain bezoar core;

[0058] Step 2) preparing coated fine powder: taking part of the bezoar core obtained in step 1), adding one 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 fine powder to obtain coated fine powder;

[0059] Step 3) preparing a coating concentrate: fermenting bovine bile using a microorganism, and concentrating the fermentation liquid (preferably low-temperature vacuum concentration) to obtain a coating concentrate; preferably, the microorganism includes the Lactobacillus breve described in the first aspect above;

[0060] Step 4) coating the bezoar core: 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);

[0061] Step 5) 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), to obtain a crude bezoar; preferably, the step 5) further comprises a drying step.

[0062] Preferably, the preparation process of the crude bezoar includes a detection process until the coated particles are obtained and the diameter is continuously increased to any value or interval in the range of 1-5 cm, preferably any value or interval in the range 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, which is a spherical or quasi-spherical shape, that is, the crude bezoar.

[0063] Preferably, the calcium ion donor includes one or more of calcium chloride, calcium sulfate, calcium nitrate, calcium lactate or calcium hydroxide.

[0064] Preferably, the B vitamins include one or more of B1, B2, B3, B5, B6, B7, B9 or B12.

[0065] Preferably, the components 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 comprising B1:B2:B3:B9=2:1:1:1) and 20.5% of bezoar core, accounting for the final total weight.

[0066] Preferably, the preparation of the coated fine powder in step 2) includes fully mixing and grinding into fine powder under a sterile, light-proof environment, preferably also including a sieving step, and more preferably sieving with 200 mesh.

[0067] Preferably, the method further comprises further processing the crude bezoar product, and the further processing comprises the following steps:

[0068] Step 6) rotating and vibrating the crude bezoar product in a container to obtain a bezoar semi-finished product;

[0069] Step 7) wetting the bezoar semi-finished product with the coating concentrate obtained in step 3) and then drying;

[0070] Step 8) Repeat the above step 7) at least 3 times, preferably any value or range between 3 and 10 times (e.g., 3, 4, 5, 6, 7, 8, 9 or 10), to obtain in vitro cultured bezoar.

[0071] 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 10-20 days (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days).

[0072] Preferably, the container comprises a solid fermentation culture device for in vitro cultivation of bezoar.

[0073] Preferably, the measured water content of the in vitro cultured bezoar is less than 10%, preferably less than 6%;

[0074] Preferably, the drying method includes dehumidification drying, vacuum drying, freeze drying or hot air drying, etc.

[0075] 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).

[0076] In a specific embodiment of the present invention, the morphology or detection indexes of bezoar cultured in vitro at various stages are as follows:

[0077] Bezoar core: brown-red precipitate (i.e., calcium bilirubin precipitate);

[0078] Crude bezoar: spherical or quasi-spherical particles of 1-5 cm (preferably 2-3 cm), with a non-wet outer surface and no particle dust falling off;

[0079] Bezoar semi-finished product: smooth surface, with fine cracks on the exterior;

[0080] In vitro cultured bezoar: The surface is smooth and there are no obvious visible cracks.

[0081] The ninth aspect of the present invention provides an in vitro cultured bezoar, which is prepared by the method described in the eighth aspect.

[0082] 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.

[0083] 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".

[0084] The benefits of the present invention are:

[0085] 1. The Lactobacillus breve described in the present application is isolated from traditional fermented foods and is a Generally Reconized as Safe (GRAS) strain. The present application uses edible-grade probiotic Lactobacillus breve to replace pathogenic bacteria or conditional pathogens such as Escherichia coli for bovine bile fermentation. The fermented product does not contain substances harmful to human health, which is conducive to ensuring the safety of the medical beauty products or pharmaceutical products produced by the product.

[0086] 2. The short growth-promoting Lactobacillus described in the present application can produce glucuronidase and a certain amount of bile salt hydrolase, which can decompose the bound bilirubin in the bovine bile fermentation broth into free bilirubin. Under acidic conditions, the free bilirubin and cholesterol co-precipitate, making the bilirubin and calcium ions in the bile fermentation broth easier to be freed, increasing the probability of combining to form bilirubin calcium.

[0087] 3. The glucuronidase produced by the short growth-promoting Lactobacillus described in the present application during the fermentation process decomposes the bound bilirubin in the bile into free bilirubin. At the same time, the short growth-promoting Lactobacillus also utilizes the protein and carbohydrate macromolecules in the bile and reduces the pH of the bile fermentation liquid, thereby promoting the further secretion of glucuronidase and maintaining the continuity of activity.

[0088] Strain deposit information:

[0089] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0090] Address of the preservation unit: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province;

[0091] Deposit number: GDMCC No.63910;

[0092] Deposit date: October 23, 2023;

[0093] Classification name: Levilactobacillus brevis. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0095] Figure 1 The colony morphology and Gram staining microscopic examination images of strain ZY-001 on MRS solid medium;

[0096] Figure 2 To detect the β-glucuronidase activity of strain ZY-001 by MUG method;

[0097] Figure 3 is the growth curve of strain ZY-001;

[0098] Figure 4 An in vitro cultured bezoar product prepared by the method described in this application;

[0099] Figure 5 The invention relates to an in vitro cultured bezoar product prepared by the method in the prior art. DETAILED DESCRIPTION

[0100] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 making creative work are within the scope of protection of the present invention.

[0101] Example 1 Isolation, screening and taming of strains for tolerance to bovine bile

[0102] Step 1: Isolation of strains

[0103] Use a disposable sterile syringe to collect the juice from traditional kimchi, and dilute it with physiological saline in a gradient. Take the kimchi juice sample diluted by an appropriate multiple and evenly spread it on MRS agar medium, culture it at 37±1℃ for 24-48h, observe the morphology of single colonies on the plate, and select colonies that conform to the morphology of lactic acid bacteria (colonies are round, medium-sized, convex, slightly white, and moist). Perform Gram staining and contact enzyme tests on the isolated and purified colonies. The rod-shaped strains that are positive for Gram staining and negative for contact enzyme tests are preliminarily judged to be lactobacilli, and a total of 289 strains that are preliminarily judged to be lactobacilli are obtained. The strains preliminarily judged to be lactobacilli can be stored at 4℃ on MRS slant for a short period of time, and 30% glycerol cryopreservation tubes can be made for long-term storage and stored at -80℃.

[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] Step 2: Screening of Bezoar Transformation Strains

[0107] Step 1 obtained 289 strains that were initially judged to be lactobacillus. After activating these strains 3 times, 4 ml of bacterial solution was inoculated in 16 ml of MRS broth medium containing 25% sterilized ox bile, and a control group of 4 mL of MRS medium was added to 16 mL of MRS medium containing 25% sterilized ox bile, and cultured at 37 ° C for 72 h. Free bilirubin and conjugated bilirubin in the fermentation broth were separated, and the total bilirubin, conjugated bilirubin and free bilirubin content in each strain culture were determined according to the quality inspection method specified in the in vitro cultivation of bezoar in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181), and the ratio of conjugated bilirubin to free bilirubin was calculated, and 29 lactobacillus strains with a ratio of conjugated bilirubin to free bilirubin greater than 50% were obtained. The bilirubin conversion rate of each strain is shown in Table 1 below.

[0108] Table 1

[0109] serial number Bilirubin conversion rate (%) serial number Bilirubin conversion rate (%) L2 58％ L177 58％ L14 63％ L183 51％ L27 52％ L195 74％ L39 76％ L207 69％ L51 59％ L219 53％ L68 61％ L231 77％ L75 71％ L238 79％ L87 50％ L243 62％ L99 67％ L252 56％ L112 79％ L267 68％ L123 55％ L273 73％ L135 60％ L277 80％ L147 82％ L279 70％ L159 57％ L282 66％ L175 69％

[0110] The preparation method of the MRS medium containing 25% sterilized ox bile is as follows: weigh each component according to the MRS formula, fully dissolve it with an appropriate amount of distilled water, slowly add 25 mL of sterilized and filtered ox bile to the solution, and distilled water to make the volume to 100 mL to prepare 25% ox bile-MRS medium.

[0111] 3. Step 3: Adaptation of strains to cow bile tolerance

[0112] The 29 strains screened above were further domesticated for tolerance to ox bile.

[0113] The liquid culture medium for domesticating lactobacilli tolerant to ox bile is based on MRS medium and is prepared by adding an appropriate amount of ox bile. The formula is as follows: relative to a volume of 1000 mL, it contains 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate (MgSO4·7H2O), 0.05 g / L manganese sulfate (MnSO4·H2O), 2.0 g / L potassium dihydrogen phosphate, 20.0 g / L glucose, and Tween-80. 1mL / L, stir and dissolve an appropriate amount of distilled water, on this basis, add 100mL, 200mL, 300mL, 400mL, 500mL, 600mL, 700mL, 800mL of sterilized and filtered ox bile respectively, make up to 1000ml with distilled water or purified water, prepare ox bile acclimation medium with ox bile addition of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% (volume fraction), adjust the pH to 6.2±0.2, and sterilize at 121℃ for 20 minutes before use.

[0114] The solid culture medium for domesticating lactic acid bacteria tolerant to ox bile is prepared by adding 15 g / L agar to the above liquid culture medium formula for domesticating lactic acid bacteria tolerant to ox bile, adjusting the pH to 6.2±0.2, and sterilizing by high pressure at 121°C for 20 minutes before use.

[0115] After activating the above 29 strains three times, the bacterial liquid was inoculated in an acclimation medium containing 10% ox bile-MRS at an inoculum volume ratio of 5%, and cultured at 37°C until turbidity was observed. Then, 100 μL of the culture was spread on an MRS solid acclimation medium containing 10% ox bile. After culture at 37°C for 48 hours, a single colony with good growth was selected and preserved for later use, and inoculated into an acclimation medium containing 20% ​​ox bile. After cultured at 37°C until turbidity was observed, 100 μL of the culture was spread on an MRS solid acclimation medium containing 20% ​​ox bile. After cultured at 37°C for 48 hours, a single colony with good growth was selected and preserved for later use, and inoculated into an acclimation medium containing 30% ox bile. The above steps were repeated in sequence, and the content of 10% ox bile was gradually increased, so that the strain was acclimated to survive and reproduce in 80% ox bile-MRS medium. After domestication, three strains of lactobacillus were obtained that could survive and reproduce in 80% ox bile-MRS medium and were numbered L68, L175, and L238.

[0116] The three lactic acid bacteria strains isolated above were inoculated into 5 ml of MRS medium containing 60% ox bile, and cultured at 37°C for 48 h. Then, 5 ml of the bacterial solution was inoculated into 95 ml of sterilized ox bile and cultured at 30 rpm. The bacterial density (OD600 ), with OD 600 The value is the vertical axis, and the culture time is the horizontal axis to make a growth curve (taking L238 as an example, Figure 3 The survival time of Lactobacillus No. L238 in cattle bile was greater than 120 hours, with a stagnant period of 0-25 hours, followed by a late logarithmic growth period, and a maximum OD of 48 hours was reached. 600 It reached a maximum of 1.1, and then slowly decreased to about 1. Among the three lactic acid bacteria investigated, L238 had the shortest delay period, the largest growth rate and the maximum bacterial concentration in bovine bile, and had good growth performance and bile tolerance (see Table 2 below).

[0117] Table 2

[0118] strain Delay period (h) <![CDATA[Growth rate (OD 600 h -1 )]]> <![CDATA[Maximum cell concentration (OD 600 )]]> L68 35 0.038 0.92 L175 30 0.033 0.82 L238 25 0.046 1.10

[0119] Example 2 Identification of ZY-001 strain

[0120] The strain L238 obtained in Example 1, which has the ability to transform bezoar and tolerate 80% ox bile, and has a short delay period, a fast growth rate and the largest bacterial concentration, was selected and named ZY-001 and identified.

[0121] 1. Colony morphology identification: strain ZY-001 was inoculated on MRS solid medium and cultured at 37°C for 24 hours. Single colonies were slightly white, medium-sized, raised, smooth and moist circles with mostly irregular edges (such as Figure 1 Under microscopic examination, strain ZY-001 is Gram-positive and appears as straight or slightly curved short rods, sometimes single, sometimes in pairs or in chains (e.g. Figure 1 B).

[0122] 2. Molecular identification of strains: 16S rDNA sequencing was performed on the short growth-promoting Lactobacillus ZY-001 provided by the present invention, and the sequence was compared on NCBI (https: / / blast.ncbi.nlm.nih.gov) and found that the sequence had a homology of 99.93% with Levilactobacillus brevis; the sequence was compared on EZBioCloud (https: / / www.ezbiocloud.net) and found that the sequence had a homology of 99.93% with Levilactobacillus brevis. The comparison results showed that the strain with the highest sequence homology of strain ZY-001 was short growth-promoting Lactobacillus. Combined with the morphological and 16S rDNA sequence comparison results, the strain was determined to be short growth-promoting Lactobacillus (Levilactobacillus brevis). Deposited in Guangdong Provincial Microbiological Culture Collection Center, deposit number: GDMCC No: 63910, deposit date: October 23, 2023, address: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0123] The 16s rDNA sequencing sequence is shown in SEQ ID NO: 1.

[0124] SEQ ID NO: 1:

[0125]

[0126] Example 3 Detection of β-glucuronidase activity of strain ZY-001

[0127] β-glucuronidase can promote the conversion of conjugated bilirubin in bile into free bilirubin, but the expression of β-glucuronidase in different microorganisms is not the same. For example, Lactobacillus brevis KU15153 (see non-patent literature: Probiotic characterization of Lactobacillus brevis KU15153 showing antimicrobial and antioxidant effect isolated from kimchi), Lactobacillus brevis KT16-2 (see non-patent literature: Probiotic Potential and Gluten Hydrolysis Activity of Lactobacillus brevis KT16-2), and Lactobacillus brevis S82 (see non-patent literature: Characterization of Probiotic Properties of Antifungal Lactobacillus Strains Isolated from Traditional Fermenting Green Olives) cannot express β-glucuronidase. Some model strains of Lactobacillus brevis, such as ATCC 393 or ATCC 33323, although the strains carry the gene encoding β-glucuronidase, these two strains cannot express β-glucuronidase with enzyme activity. (See non-patent literature: Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH).

[0128] After ZY-001 was activated three times, it was inoculated into β-glucuronidase activity screening medium (MRS-MUG) at a volume ratio of 5%. After culturing at 37°C for 24 hours, it was observed under 366nm wavelength ultraviolet light, and blue-white fluorescence was seen, indicating that ZY-001 had β-glucuronidase activity.

[0129] The β-glucuronidase activity screening medium (MRS-MUG) is a medium in which 4-methylumbellifery-β-D-Glucuronide (MUG) is added to a final concentration of 350 μg / mL. The β-glucuronidase produced by microorganisms can hydrolyze MUG to release the fluorescent substance 4-methylumbellifery, which produces blue-white fluorescence under a 366nm ultraviolet lamp (see the results). Figure 2 ).

[0130] Example 4 Effect of fermentation time on the conversion of conjugated bilirubin to free bilirubin

[0131] Pretreatment of cow bile: Filter and sterilize fresh cow bile to remove impurities and bacteria, and set aside.

[0132] Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve with appropriate amount of distilled water, slowly add 50mL of the above-mentioned pretreated ox bile to the solution, dilute to 100mL with distilled water, prepare 50% ox bile-MRS medium, inoculate ZY-001 into 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage;

[0133] The seed liquid of the above-mentioned strain was inoculated into sterile bovine bile culture medium at a ratio of 20 mL of culture per 80 mL of sterile bovine bile, and then cultured at 37°C and 80 rpm, and samples were taken at 36h, 48h, 60h, 72h, 84h, 96h, 108h, and 120h, respectively. According to the quality inspection method specified in the in vitro cultivation of bezoar in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181), the total bilirubin content, conjugated bilirubin, and free bilirubin content in the fermented bovine bile were detected, and the ratio of conjugated bilirubin converted to free bilirubin was calculated, where the conjugated bilirubin conversion rate = (C1-C2) / C1*100%, where C1 is the conjugated bilirubin content in bovine bile before fermentation, and C2 is the conjugated bilirubin content in bovine bile after fermentation.

[0134] The results are shown in Table 3 below. Compared with unfermented bovine bile (serial number 0), the amount of free bile acid in the bovine bile fermented with ZY-001 as the strain increased in the samples at each sampling time investigated, and the conversion rate continued to change with the extension of the fermentation time. When the fermentation reached 72 hours, the conversion rate of conjugated bilirubin to free bilirubin could reach 75%, and the conversion rate was subsequently measured to fluctuate between 72% and 76%, indicating that the conversion of conjugated bilirubin in bovine bile had reached the end point when the fermentation reached 72 hours. Considering factors such as evaporation during the fermentation process, the preferred fermentation time is 72 hours.

[0135] Table 3

[0136]

[0137]

[0138] Example 5 Effect of fermentation temperature on the conversion of conjugated bilirubin to free bilirubin

[0139] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0140] Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve with appropriate amount of distilled water, slowly add 50mL of the above-mentioned pretreated ox bile to the solution, dilute to 100mL with distilled water, prepare 50% ox bile-MRS medium, inoculate ZY-001 into 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage;

[0141] The seed liquid of the above-mentioned strain was inoculated into sterile bovine bile culture medium at a ratio of 20 mL of culture per 80 mL of sterile bovine bile, and then cultured at 25°C, 30°C, 37°C and 40°C at 80 rpm for 96 hours, respectively, to separate the free bilirubin and conjugated bilirubin in the fermentation broth. According to the quality inspection method specified in the in vitro cultivation of bezoar in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181), the total bilirubin content, conjugated bilirubin and free bilirubin content in the fermented bovine bile were tested, and the ratio of conjugated bilirubin converted to free bilirubin was calculated.

[0142] The results are shown in Table 4 below. Compared with unfermented bovine bile (serial number 0), the free bilirubin content of bovine bile fermented with ZY-001 strain increased at each temperature examined. Among them, the conversion rate of bound bilirubin to free bilirubin was the highest at 37°C for 72h, reaching 74%.

[0143] Table 4

[0144]

[0145] Example 6 Effect of initial pH of fermentation system on bilirubin conversion capacity of ZY-001

[0146] Pretreatment of cattle bile: obtain fresh cattle bile, filter and sterilize it to remove impurities and bacteria, and adjust the pH to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 respectively for use.

[0147] Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve with appropriate amount of distilled water, slowly add 50mL of the above-mentioned pretreated ox bile to the solution, dilute to 100mL with distilled water, prepare 50% ox bile-MRS medium, inoculate ZY-001 into 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage;

[0148] The seed liquid of the above-mentioned strains was inoculated into sterile bovine bile culture medium with pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, at a ratio of 20 mL of culture per 80 mL of sterile bovine bile, and cultured at 37°C and 80 rpm for 96 h to separate the free bilirubin and conjugated bilirubin in the fermentation broth. The bilirubin content of the bovine bile before and after fermentation was tested according to the quality inspection method specified in the in vitro cultivation of bezoar in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, page 181).

[0149] The results are shown in Table 5 below. Compared with unfermented bovine bile (serial number 0), the bovine bile fermented with ZY-001 as the strain had an increased free bilirubin content at each initial pH examined. When the initial pH was 6.5, the conversion rate of bound bilirubin to free bilirubin was the highest, reaching 73%.

[0150] Table 5

[0151]

[0152]

[0153] Example 7 ZY-001 Fermentation of cattle bile to prepare calcium bilirubin precipitate

[0154] Filter and sterilize fresh ox bile to remove impurities and bacteria, and set aside.

[0155] E. coli fermented cow bile:

[0156] Scheme 1: Inoculate Escherichia coli ATCC25922 into 50% ox bile-MRS sterile culture medium and culture at 37°C to the late logarithmic growth stage; inoculate the Escherichia coli seed liquid into sterile ox bile at a ratio of 200 mL of culture per 800 mL of sterile ox bile, and ferment at 37°C for 72 hours to obtain Escherichia coli fermented ox bile; add 2000 mL of saturated calcium hydroxide solution to the Escherichia coli fermented ox bile, stir evenly and boil at room temperature and pressure for 30 minutes, filter and wash to obtain 8.2 g (dry weight) of brown-red precipitate.

[0157] Scheme 2: Inoculate Escherichia coli ATCC25922 into 50% ox bile-MRS sterile culture medium and culture at 37°C to the late logarithmic growth stage; inoculate the Escherichia coli seed liquid into sterile ox bile at a ratio of 200 mL of culture per 800 mL of sterile ox bile, and ferment at 37°C for 72 hours to obtain Escherichia coli fermented ox bile; add 2000 mL of saturated calcium hydroxide solution to the Escherichia coli fermented ox bile, stir thoroughly at room temperature and pressure, and filter and wash to obtain 3.1 g (dry weight) of brown-red precipitate.

[0158] ZY-001 Fermented Ox Bile:

[0159] Scheme A: Inoculate ZY-001 into 50% ox bile-MRS sterile culture medium and culture at 37°C to the late logarithmic growth stage; inoculate the above ZY-001 culture solution into sterile ox bile at a ratio of 200 mL of culture per 800 mL of sterile ox bile, and ferment at 80 rpm at 37°C for 72 h to obtain ZY-001 fermented ox bile; add 2000 mL of saturated calcium hydroxide solution to the ZY-001 fermented ox bile, stir evenly and boil at room temperature and pressure for 30 min; filter and wash to obtain 8.6 g (dry weight) of brown-red precipitate.

[0160] Scheme B: Inoculate ZY-001 into 50% ox bile-MRS sterile culture medium and culture at 37°C to the late logarithmic growth stage; inoculate the above ZY-001 culture solution into sterile ox bile at a ratio of 200 mL of culture per 800 mL of sterile ox bile, and ferment at 80 rpm at 37°C for 96 hours to obtain ZY-001 fermented ox bile; add 2000 mL of saturated calcium hydroxide solution to the ZY-001 fermented ox bile, and stir thoroughly at room temperature and pressure; filter and wash to obtain 8.5 g (dry weight) of brown-red precipitate.

[0161] The results show that compared with the existing method of fermenting bovine bile using Escherichia coli as the strain, when fermenting with Escherichia coli, the difference between Scheme 1 and Scheme 2 is only whether the step of heating and boiling is included after stirring, but the yield difference is more than twice. However, when using the ZY-001 of the present application to ferment bovine bile, a higher yield can be obtained regardless of the fermentation time and whether heating and boiling are performed after stirring. This shows that the strain ZY-001 of the present invention is suitable for a wider range of process production, and the strain of the present invention is a probiotic and will not produce harmful bacterial toxins produced by pathogenic bacteria such as Escherichia coli, and there is no safety risk for medical beauty or pharmaceutical products.

[0162] Example 8 Preparation of Bezoar Cultivated in Vitro

[0163] The preparation method of in vitro cultured bezoar comprises the following steps:

[0164] 1. Preparation of bezoar core: Obtain calcium bilirubin precipitate according to the method described in Example 7, and adjust the pH to neutral by hydrolysis to obtain bezoar core.

[0165] 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 7 to make up to 100% of the final total weight, grind into fine powder in a clean and sterile dark room or in a red light dark room and mix well.

[0166] 3. Prepare coating concentrate: inoculate ZY-001 into 50% ox bile-MRS sterile culture medium and culture at 37°C to the late logarithmic growth stage; inoculate the above ZY-001 culture solution into sterile ox bile at a ratio of 200 mL of culture per 800 mL of sterile ox bile, and ferment at 80 rpm for 72 h at 37°C to obtain ZY-001 fermented ox bile; adjust the pH of ZY-001 fermented ox bile to 6.8±0.2, and vacuum concentrate under clean and sterile conditions, control the temperature to 28°C±2°C, and the vacuum degree to be above -0.085Mpa; concentrate until the appearance of the mixed fermentation liquid is obviously attached to the wall of a glass vacuum rotator and obvious stringing is 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.

[0167] 4. Package bezoar core: under clean, sterile, luminous conditions, the bezoar core obtained in step 1 is placed in an in vitro culture bezoar solid fermentation solid culture device, first fully moistened with the coating concentrated solution obtained in the above-mentioned step 3, then fully spherically packaged with the fully mixed ground powder obtained in the above-mentioned step 2, then fully moistened with the above-mentioned coating concentrated solution, then fully spherically packaged with the above-mentioned fully mixed ground powder, so moistened and packaged 5 times (and the last time is packaged), moved into a clean silica gel dryer and dried for more than 6 hours, took out and observed that the outer surface was not moistened and no particle dust fell off; the dried packaged particles were fully moistened with the coating concentrated solution again, and the above process was repeated until the packaged particles were continuously proliferated and grown into a spherical or spherical in vitro cultured bezoar crude product with a diameter of between 2cm and 3cm, and the outer surface was not moistened and no particle dust fell off.

[0168] 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.

[0169] 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.

[0170] 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):

[0171] Appearance: spherical or quasi-spherical, 2cm-3cm in diameter. Smooth surface, yellow-red to brownish yellow. Light, crisp, with concentric layers on the cross section; fragrant, bitter and sweet, with a cool feeling; measured moisture content is 4%; the bilirubin content of the in vitro cultured bezoar obtained by the above method is 41%, the bile acid content is 8%, and the free bilirubin content is 0.04% at OD 453 The absorbance at the bottom is 0.21. The above determination indicators are in compliance with the provisions of the in vitro cultured bezoar in the Chinese Pharmacopoeia (2020, Part I, Medicinal Materials and Decoction Pieces, Page 181).

[0172] 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. In the process of continuous wetting and wrapping, 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 4 , and the in vitro cultured bezoar obtained by conventional means of the prior art is shown in Figure 5 .

[0173] 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.

[0174] 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 short growth-promoting lactobacillus (Levilactobacillus brevis), characterized in that The described short growth-promoting lactobacillus was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the collection number GDMCCNo:63910.

2. A method for culturing the Lactobacillus breve according to claim 1, characterized in that: The culture method comprises using a culture medium, wherein the culture medium contains ox bile with a volume fraction of at least 10%.

3. The culture method according to claim 2, characterized in that The culture medium contains 10%-50% of ox bile in volume fraction.

4. Use of the Lactobacillus breve described in claim 1 in fermenting bovine bile, converting conjugated bilirubin into free bilirubin, preparing calcium bilirubin, and / or preparing in vitro cultured bezoar.

5. A method for converting conjugated bilirubin into free bilirubin, characterized in that: The method comprises introducing a microorganism into a system containing bound bilirubin, wherein the microorganism comprises the Lactobacillus breve of claim 1; The method comprises fermenting a system containing conjugated bilirubin using a microorganism.

6. The method according to claim 5, characterized in that The method comprises filtering and sterilizing the system containing conjugated bilirubin to obtain a sterilized system.

7. The method according to claim 6, characterized in that The method comprises inoculating microorganisms into a seed culture medium to obtain a microorganism seed solution.

8. The method according to claim 7, characterized in that The method comprises connecting the microbial seed liquid to the sterilization system to obtain a fermentation system.

9. The method according to claim 8, characterized in that The initial pH of the fermentation system is 4-8.

10. The method according to claim 8, characterized in that The temperature of the fermentation system is 25-40°C.

11. The method according to any one of claims 5 to 10, characterized in that: The fermentation time is at least 30 hours.

12. The method according to claim 11, characterized in that The fermentation time is 30-150h.

13. The method according to any one of claims 5 to 10, characterized in that: The system comprising conjugated bilirubin comprises ox bile.

14. A fermentation product obtained by the method according to any one of claims 5 to 13.

15. The fermentation product according to claim 14, characterized in that The fermentation product includes a system with high free bilirubin.

16. A method for preparing calcium bilirubin, characterized in that: The preparation method comprises introducing a microorganism into a system containing bound bilirubin, wherein the microorganism comprises the Lactobacillus breve described in claim 1; The preparation method further comprises adding calcium ions; The preparation method comprises fermenting a system containing conjugated bilirubin using a microorganism; The system comprising conjugated bilirubin comprises ox bile.

17. The preparation method according to claim 16, characterized in that: The preparation method comprises filtering and sterilizing the system containing bound bilirubin to obtain a sterilized system.

18. The preparation method according to claim 17, characterized in that: The preparation method comprises inoculating microorganisms into a seed culture medium to obtain a microorganism seed solution.

19. The preparation method according to claim 18, characterized in that: The preparation method comprises connecting the microbial seed liquid to the sterilization system to obtain a fermentation system.

20. The preparation method according to claim 19, characterized in that: The initial pH of the fermentation system is 4-8.

21. The preparation method according to claim 19, characterized in that: The temperature of the fermentation system is 25-40°C.

22. The preparation method according to any one of claims 16 to 21, characterized in that: The fermentation time is at least 30 hours.

23. The preparation method according to claim 22, characterized in that: The fermentation time is 30-150h.

24. The preparation method according to any one of claims 16 to 21, characterized in that: The adding of calcium ions includes adding in the middle or late stage of fermentation or after the end of fermentation.

25. The preparation method according to claim 24, characterized in that: Calcium ions are added before or after adjusting the pH of the fermentation system to alkaline, or calcium ions are added while adjusting the pH of the fermentation system to alkaline.

26. The preparation method according to claim 25, characterized in that: After adding calcium ions, the process also includes the steps of stirring, separating, filtering and washing.

27. The preparation method according to claim 26, characterized in that: A drying step is also included.

28. The preparation method according to claim 26, characterized in that: The stirring includes stirring at normal temperature and pressure, or stirring under heating.

29. The preparation method according to claim 27, characterized in that: The drying includes dehumidification drying, vacuum drying, freeze drying or hot air drying.

30. The preparation method according to any one of claims 16 to 21, characterized in that: The calcium ions are added by adding a calcium ion donor, wherein the calcium ion donor includes one or more of calcium oxide, calcium sulfate, calcium chloride or calcium hydroxide.

31. A mixture comprising calcium bilirubin, characterized in that The mixture containing calcium bilirubin is obtained by the preparation method described in any one of claims 16-30.

32. A method for preparing in vitro cultured bezoar, characterized in that: The method comprises preparing calcium bilirubin using the preparation method of any one of claims 16 to 30, or the mixture containing calcium bilirubin according to claim 31, and performing the following steps: Step 1) preparing bezoar core: washing calcium bilirubin or a mixture containing calcium bilirubin with water and adjusting the pH to neutral to obtain bezoar core; Step 2) preparing coated fine powder: taking part of the bezoar core obtained in step 1), adding bile acid, deoxycholic acid, taurine, cholesterol, bilirubin, ferrous sulfate, a calcium ion donor, zinc sulfate, magnesium sulfate, ascorbic acid and B vitamins, mixing evenly and grinding into fine powder to obtain coated fine powder; the calcium ion donor includes one or more of calcium chloride, calcium sulfate, calcium nitrate, calcium lactate or calcium hydroxide; the B vitamins include one or more of B1, B2, B3, B5, B6, B7, B9 or B12; Step 3) preparing a coating concentrate: fermenting bovine bile using a microorganism, and concentrating the fermentation liquid to obtain a coating concentrate; the microorganism comprises the Lactobacillus breve described in claim 1; Step 4) coating the bezoar core: 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.

33. The method according to claim 32, characterized in that The concentration in step 3) includes low-temperature vacuum concentration.

34. The method according to claim 32, characterized in that The step 5) includes repeating the above step 4) 3-10 times.

35. The method according to claim 32, characterized in that The step 5) further comprises a drying step.

36. The method according to any one of claims 32-35, characterized in that: The method further comprises further processing the crude bezoar product, wherein the further processing comprises the following steps: Step 6) rotating and vibrating the crude bezoar product in a container to obtain a bezoar semi-finished product; Step 7) wetting the bezoar semi-finished product with the coating concentrate obtained in step 3) and then drying; Step 8) Repeat the above step 7) at least 3 times to obtain in vitro cultured bezoar.

37. The method according to claim 36, characterized in that The step 8) comprises repeating the above step 7) 3-10 times.

38. The method according to claim 36, characterized in that The conditions of the rotational vibration in step 6) are: the temperature is set to 35-40°C, the relative humidity is set to 40-50%, the rotational vibration is 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.

39. The method according to claim 38, characterized in that Rotate and vibrate once every 10 minutes, for 10-20 seconds each time, and rotate in the opposite direction next time.

40. The method according to claim 38, characterized in that Cultivate for 10-20 days.

41. The method according to any one of claims 32-35, characterized in that: The measured water content of the in vitro cultured bezoar is less than 10%.

42. The method according to claim 41, characterized in that The measured water content of the in vitro cultured bezoar is less than 6%.

43. The method according to claim 35, characterized in that The drying includes drying in a dryer for at least 1 hour.

44. The method according to claim 43, characterized in that The drying comprises drying in a dryer for 1 to 10 hours.

45. 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 32-44.

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