Method for cultivating bovine bezoar with high purity in vitro based on multi-strain synergistic fermentation

Abstract

The present application relates to the technical field of biological medicine, and discloses a method for cultivating bovine bezoar with high purity in vitro based on multi-strain synergistic fermentation, which comprises the following steps: continuously subculturing animal bifidobacterium, streptococcus thermophilus and lactobacillus plantarum in a domestication culture medium containing bile acid to obtain bile acid-resistant domesticated strains; inoculating the domesticated strains into a fermentation culture medium after mixing, and performing segmented variable-temperature fermentation under anaerobic conditions to obtain a fermentation product; sequentially performing low-temperature homogenization crushing, acid precipitation and membrane separation treatment on the fermentation product, and collecting the precipitate; performing gradient cooling recrystallization on the precipitate in an organic solvent system containing a surfactant, and obtaining the bovine bezoar cultivated in vitro after drying. Through strain domestication and fermentation condition optimization, combined with multi-stage separation and purification and gradient crystallization control, the method effectively improves the product purity and batch stability of the bovine bezoar cultivated in vitro, is simple to operate, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biomedicine technology, specifically a method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation. Background Technology

[0002] Natural bezoar is a gallstone found in the gallbladder, bile duct, or hepatic duct of the bovine animal (Bos taurus domesticus Gmelin). It possesses traditional medicinal value, including clearing heat and detoxifying, cooling the liver and calming the nerves, and is a key ingredient in many valuable traditional Chinese medicines. However, natural bezoar resources are extremely scarce; each cow yields only a very small number of stones. Furthermore, with changes in farming methods and shorter lifespans of cattle, the annual production of natural bezoar continues to decline, far from meeting the needs of clinical use and the pharmaceutical industry. For a long time, the market price of natural bezoar has remained high, even several times that of gold, severely restricting the widespread accessibility of traditional Chinese medicines containing bezoar.

[0003] To alleviate this contradiction, researchers both domestically and internationally have dedicated themselves to developing technical routes for in vitro culture of bezoar. Early attempts mainly focused on chemical synthesis, which involves physically mixing or chemically complexing known major chemical components of bezoar, such as bilirubin, cholic acid, deoxycholic acid, cholesterol, and inorganic salts, in specific proportions to produce artificial bezoar. However, chemically synthesized products lack the complex trace organic components and enzymatic hydrolysis products found in natural bezoar, resulting in a significant difference in efficacy compared to natural bezoar. Furthermore, the chemical synthesis process often involves strong acids, strong alkalis, or high-temperature treatments, which can easily destroy the natural configuration of bilirubin, leading to lower product color, solubility, and bioavailability.

[0004] Subsequently, researchers turned to biotransformation pathways, utilizing microbial fermentation or enzymatic reactions to simulate the formation process of bezoar in the biliary system. Existing reports indicate that some schemes employ single-strain (such as Bacillus subtilis, lactic acid bacteria, or yeast) for liquid fermentation, converting precursor substances in the culture medium into bilirubin or bile acid components through microbial metabolism. However, the metabolic pathways of single-strain organisms are limited, making it difficult to collaboratively complete the multi-step transformation process involving bile acid isomerization, bilirubin-bound hydrolysis, and macromolecular complex precipitation. The composition ratio of the target component in the obtained product differs significantly from that of natural bezoar, and the fermentation cycle is long, resulting in a large accumulation of byproducts, placing a significant burden on subsequent separation and purification. To improve transformation efficiency, some studies have attempted to co-culture two or three probiotic strains; however, strains that have not been specifically acclimatized show low survival rates and rapidly declining metabolic activity in fermentation systems containing bile acids, leading to microbial imbalance during fermentation and unstable accumulation of the target product. In addition, most existing fermentation processes use a constant temperature culture mode, which fails to adjust the temperature according to the dynamic changes in acid production by the microbial community. This results in misalignment of the expression window of key enzyme systems, affecting the complexation efficiency of bilirubin and bile acids.

[0005] In terms of separation and purification, conventional methods employ direct extraction with organic solvents or acid precipitation. However, the release of intracellular bound target products is insufficient, and acid precipitation easily carries away large amounts of protein and polysaccharide impurities. The bilirubin content in the crude product is often less than 50%, requiring multiple recrystallizations or chromatographic purifications. This not only results in high loss rates but also involves cumbersome operations, making large-scale production difficult. While membrane separation technology has been applied in the purification of bioproducts, the high viscosity of fermentation broth without low-temperature homogenization and crushing easily leads to membrane pore blockage and low separation efficiency. In the crystallization step, traditional cooling crystallization lacks gradient control and surfactant assistance, resulting in disordered crystal growth and the formation of fine particles or oily precipitates. Subsequent filtration and washing are difficult, leading to significant fluctuations in product purity. In summary, existing in vitro cultured bezoar technologies generally suffer from low conversion efficiency, large deviations in product composition, lengthy purification processes, and insufficient control over the final product quality. There is an urgent need to develop a novel preparation method that can simulate the microecological process of natural bezoar formation and achieve both high conversion rates and high purity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides a method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation, the method comprising:

[0008] Domestication steps: Bifidobacterium animalis, Streptococcus thermophilus and Lactobacillus plantarum were continuously subcultured in domestication medium containing bile acids to obtain bile acid tolerant domesticated strains.

[0009] Mixed fermentation step: The bile acid tolerant strains were mixed in proportion and inoculated into the fermentation medium, and then subjected to segmented temperature-switched fermentation under anaerobic conditions to obtain the fermentation product;

[0010] Separation and purification steps: The fermentation product is subjected to low-temperature homogenization, acid precipitation, and membrane separation in sequence, and the precipitate is collected;

[0011] Crystallization and purification steps: The precipitate is recrystallized in an organic solvent system containing surfactants under gradient cooling, and then dried to obtain in vitro cultured bezoar.

[0012] Preferably, in the strain domestication step, the domestication culture medium containing bile acids comprises: peptone 10-15 g / L, beef extract powder 5-8 g / L, yeast extract powder 3-5 g / L, glucose 15-20 g / L, dipotassium hydrogen phosphate 2-4 g / L, sodium acetate 5-8 g / L, triammonium citrate 1-2 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.02-0.05 g / L, bile salts 0.5-3 g / L, and Tween-80 0.5-1.5 mL / L;

[0013] The continuous subculture involves 8-15 subcultures, with each subculture lasting 18-30 hours. The culture temperature increases by 0.5-1.0℃ with each subculture, and the culture temperature range is 37-45℃.

[0014] The concentration of bile salts was increased by 0.1-0.3 g / L in each generation of subculture, with the bile salt concentration range being 0.5-3.0 g / L.

[0015] Preferably, in the strain domestication step, the Bifidobacterium animalis, Streptococcus thermophilus and Lactobacillus plantarum are activated and cultured respectively before continuous subculturing in the domestication medium containing bile acids;

[0016] The culture medium for activation culture is MRS broth medium or M17 medium, the activation culture temperature is 35-38℃, and the activation culture time is 12-24h.

[0017] The inoculum size for the continuous subculture is 2-6%, and when the culture reaches the late logarithmic growth phase, it is transferred to the next generation of acclimatization culture medium with the same inoculum size.

[0018] After the continuous subculturing was completed, the bacterial suspensions of each strain were collected by centrifugation and washed 2-4 times with sterile physiological saline to obtain the bacterial suspension of the bile acid acclimatized strain. The concentration of the bacterial suspension was adjusted to OD. 600 It ranges from 1.0 to 2.5.

[0019] Preferably, in the mixed fermentation step, the bile acid-tolerant acclimatized strains are mixed in the following proportions: the bacterial suspensions of Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum are mixed in a volume ratio of (2-4):(1-2):(1-3) to obtain a mixed bacterial suspension;

[0020] The total inoculum volume of the mixed bacterial suspension is 5-15% of the volume of the fermentation medium;

[0021] The fermentation medium consists of: glucose 20-35 g / L, tryptone 8-12 g / L, yeast extract 4-8 g / L, bovine bile powder 2-10 g / L, cholesterol 0.2-1.5 g / L, L-cysteine ​​hydrochloride 0.3-0.8 g / L, ferrous sulfate 0.01-0.05 g / L, zinc sulfate 0.005-0.02 g / L, potassium dihydrogen phosphate 1-3 g / L, sodium chloride 2-5 g / L, vitamin K1 0.0005-0.002 g / L, and vitamin B1. 12 0.001-0.005 g / L, pH adjusted to 6.2-7.0.

[0022] Preferably, in the mixed fermentation step, the segmented variable temperature fermentation includes: the inoculated fermentation broth is first subjected to constant temperature anaerobic fermentation at 36-38℃ for 12-24 hours; when the pH value of the fermentation broth drops to 4.5-5.5, the temperature is raised to 40-44℃ to continue anaerobic fermentation for 8-16 hours; when the pH value of the fermentation broth rises back to 5.5-6.5, the fermentation is terminated, and the total fermentation time is 20-40 hours.

[0023] The anaerobic conditions are maintained by continuously introducing nitrogen or carbon dioxide gas into the fermentation vessel at a rate of 0.1-0.5 L / min, and the stirring speed during fermentation is 50-200 rpm.

[0024] During the segmented temperature-controlled fermentation process, when the residual sugar concentration of the fermentation broth is below 2 g / L, sterile glucose solution is added until the residual sugar concentration is 5-10 g / L, and the addition is repeated 1-3 times.

[0025] Preferably, in the separation and purification step, the low-temperature homogenization and crushing treatment temperature is 2-8℃, the homogenization pressure is 60-120MPa, the number of homogenization cycles is 2-5, and the homogenization time for each cycle is 2-5min.

[0026] In the fragmentation liquid after low-temperature homogenization, the bacterial cell breakage rate is ≥90%;

[0027] The acid precipitation treatment is as follows: the pH of the crushed liquid after low-temperature homogenization is adjusted to 3.0-4.5 with an acidic regulator, and the liquid is allowed to stand at 2-8℃ for 3-12 hours to precipitate. The first precipitate is collected and the supernatant is discarded.

[0028] The acidity regulator is at least one of hydrochloric acid, sulfuric acid, or acetic acid, and the concentration of the acidity regulator is 0.1-2 mol / L.

[0029] Preferably, in the separation and purification step, the membrane separation process is as follows: the first precipitate is resuspended in phosphate buffer solution with pH 7.0-8.5, filtered sequentially through a microfiltration membrane and an ultrafiltration membrane, the retentate is collected, the retentate is subjected to acid precipitation again, the pH is adjusted to 2.5-4.0, the precipitation is allowed to stand at 2-8℃ for 2-6 hours, and the second precipitate is collected by centrifugation;

[0030] The microfiltration membrane has a pore size of 0.1-0.5 μm, and the ultrafiltration membrane has a molecular weight cutoff of 3-10 kDa.

[0031] The transmembrane pressure of the microfiltration membrane is 0.1-0.3 MPa, and the filtration temperature is 15-25℃.

[0032] The transmembrane pressure of the ultrafiltration membrane is 0.2-0.5 MPa, the filtration temperature is 15-25℃, and the retentate is circulated and concentrated during the filtration process, with a concentration factor of 3-10 times.

[0033] The centrifugation speed is 8000-12000 rpm, and the centrifugation time is 15-30 min.

[0034] Preferably, in the crystallization purification step, the surfactant-containing organic solvent system consists of an organic solvent and a surfactant, wherein the organic solvent is at least one of methanol, ethanol, acetone, ethyl acetate or dichloromethane, and the surfactant is at least one of sodium dodecyl sulfate, sodium cholate, sodium deoxycholate or polyethylene glycol-400.

[0035] The volume-to-mass ratio of the organic solvent to the precipitate is (10-30) mL: 1 g;

[0036] The concentration of the surfactant in the organic solvent system is 0.5-5 g / L;

[0037] When the precipitate is dissolved in an organic solvent system containing a surfactant, the dissolution temperature is 20-40℃, the stirring time is 30-90min, and after dissolution, the insoluble matter is removed by filtration to obtain a crystallization precursor solution.

[0038] Preferably, in the crystallization purification step, the gradient cooling recrystallization is performed as follows: the crystallization precursor solution is cooled from the dissolution temperature to 10-15℃ at a cooling rate of 0.5-3℃ / min, kept at 10-15℃ for 1-4h, then cooled from 10-15℃ to -10-0℃ at a cooling rate of 1-5℃ / h, kept at -10-0℃ for 2-8h, and the precipitated crystals are collected.

[0039] During the gradient cooling recrystallization process, when the temperature drops to 5-8℃, seed crystals are added to the crystallization precursor solution. The amount of seed crystals added is 0.5-3% of the mass of the precipitate, and the particle size of the seed crystals is 10-50μm.

[0040] After collecting the precipitated crystals, the crystals are washed 2-5 times with a pre-cooled organic solvent at a temperature of -5 to 5°C. The volume ratio of the organic solvent used in each wash to the volume of the crystals is (3-8) mL:1 g.

[0041] Preferably, in the crystallization purification step, the drying is freeze-drying or vacuum drying;

[0042] The freeze-drying process parameters are as follows: pre-freezing temperature -40℃ to -30℃, pre-freezing time 2-6h, sublimation drying temperature -20℃ to -10℃, sublimation drying vacuum degree 5-30Pa, sublimation drying time 12-30h, desorption drying temperature 10-30℃, desorption drying time 4-12h.

[0043] The process parameters for vacuum drying are: drying temperature 30-50℃, vacuum degree 0.01-0.1MPa, and drying time 8-24h.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] During the strain domestication stage, *Bifidobacterium animalis*, *Streptococcus thermophilus*, and *Lactobacillus plantarum* were subjected to continuous subculturing with bile acids. This allowed each strain to gradually adapt to and tolerate a high concentration of bile acids. Simultaneously, the culture temperature was progressively increased with each subculturing, screening for domesticated strains that exhibited both bile acid and temperature tolerance. This domestication strategy enabled each strain to maintain a high viable count and metabolic activity in the complex medium containing bovine bile powder and cholesterol during later mixed fermentation. This avoided mid-fermentation microbial decline caused by the antibacterial effect of bile acids, ensuring balanced growth and synergistic metabolism of multiple strains throughout the fermentation cycle. The stepwise domestication method, which progressively increases bile salt concentration and temperature, compared to a single high-concentration stress, elicits a gradual stress response in the strains. This allows the domesticated strains to acquire a stable tolerance phenotype at the genetic level, enabling them to quickly enter the logarithmic growth phase after reintroduction into the fermentation medium without a lengthy adaptation period. This effectively shortens fermentation start-up time and improves batch-to-batch reproducibility.

[0046] In the mixed fermentation stage, three domesticated bacterial strains were inoculated at a specific volume ratio and a segmented variable-temperature anaerobic fermentation process was adopted. The initial isothermal fermentation at 36-38℃ facilitated rapid acid production by the bacterial community, causing the pH to drop to 4.5-5.5. This acidic environment promoted changes in cholesterol solubility and the release of bilirubin from its bound state. Subsequently, the temperature was raised to 40-44℃ for continued fermentation. This high-temperature stage activated the thermostable enzyme systems in *Streptococcus thermophilus* and *Lactobacillus plantarum*, accelerating the dehydroxylation and isomerization reactions of bile acids. Simultaneously, the pH rose back to 5.5-6.5. This dynamic pH change process precisely simulated the microenvironment of natural bezoar's gradual concentration, complexation, and precipitation in the bile duct, which is conducive to the formation of bilirubin-bile acid-cholesterol complexes with a composition similar to that of natural bezoar. During fermentation, glucose solution was supplemented to maintain sufficient carbon source, preventing premature death due to nutrient depletion, extending the logarithmic phase window for target product synthesis, and significantly increasing the product accumulation per unit volume of fermentation broth. Continuously introducing nitrogen or carbon dioxide under anaerobic conditions not only creates an environment conducive to the growth of strictly anaerobic Bifidobacteria, but also allows the dissolved carbon dioxide to moderately lower the pH of the fermentation broth, helping to regulate the acid metabolism rhythm and making the fermentation endpoint control more precise.

[0047] The separation and purification stage employs a combined process of low-temperature homogenization, acid precipitation, and membrane separation. High-pressure homogenization under low-temperature conditions gently and efficiently breaks down bacterial cell walls and membranes, allowing the target product synthesized intracellularly to be fully released into the liquid phase. Simultaneously, the low-temperature environment effectively inhibits lipid oxidation and bilirubin photosensitive degradation, protecting the product's natural active conformation. The homogenized lysate undergoes initial acid precipitation to remove a large amount of soluble proteins, nucleic acids, and unconsumed sugars. The collected precipitate is then resuspended in phosphate buffer and subsequently passed through microfiltration and ultrafiltration membranes. Microfiltration removes cell debris and particulate impurities, while ultrafiltration retains small peptides and salts with molecular weights below 3-10 kDa. The target product, due to its larger molecular aggregates, is retained in the retentate. Compared to single acid precipitation, this membrane separation strategy can more precisely remove impurities co-precipitated with the target product. Furthermore, through cyclic concentration, the volume of the retentate can be reduced several times, providing a high-concentration, low-impurity feed solution for subsequent crystallization, reducing impurity interference during crystallization and promoting uniform crystal nucleation.

[0048] In the crystallization and purification step, a gradient cooling recrystallization process is employed using an organic solvent system containing surfactants. Surfactants such as sodium cholate or sodium deoxycholate can form reversible micellization with the target product molecules, improving the uniformity of dissolution of the target product in the organic solvent and avoiding amorphous precipitation caused by localized supersaturation during conventional organic solvent dissolution. The gradient cooling process involves segmented control of the cooling rate. First, the temperature is slowly lowered and held at a higher temperature range to allow for initial crystal nucleation. Subsequently, a faster cooling rate is applied in the lower temperature range to promote orderly crystal growth. Microcrystalline seeds are added at appropriate times during the cooling process, providing nucleation sites for the crystallization system and guiding crystal growth along preferred crystal planes. This results in a crystalline product with regular morphology and concentrated particle size distribution. This crystal morphology facilitates subsequent washing and drying, and results in lower organic solvent residue. Multiple washings of the crystals with pre-cooled organic solvent effectively remove residual surfactants and pigment impurities adhering to the crystal surface, further improving the color and chemical purity of the product. The final drying method can be either freeze drying or vacuum drying, depending on the production scale. Freeze drying can retain the loose and porous structure of the product to the maximum extent, which is beneficial for dissolution and dispersion in subsequent formulation processing. Vacuum drying is suitable for large-scale production, with high drying efficiency and low energy consumption. Attached Figure Description

[0049] Figure 1 This is a flowchart illustrating the working steps of the method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to the present invention. Detailed Implementation

[0050] The bacterial strains used in the embodiments and comparative examples of this invention are all commercially available conventional strains. Among them, Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum were all purchased from the China Microbial Culture Collection Center. The reagents, culture medium raw materials, organic solvents, and membrane components used are all commercially available analytical grade and industrially standard high-purity reagents. The equipment used includes anaerobic fermenters, high-pressure homogenizers, low-temperature high-speed centrifuges, membrane separation equipment, gradient cooling crystallizers, freeze dryers, vacuum dryers, ultraviolet spectrophotometers, and high-performance liquid chromatographs, all of which are industry-standard equipment.

[0051] The purity testing standards for in vitro cultured bezoar of this invention are as follows: bilirubin purity is determined according to the in vitro cultured bezoar bilirubin testing method in the Chinese Pharmacopoeia; bile acid purity is determined according to the bile acid content determination method in the Chinese Pharmacopoeia; cell breakage rate is statistically analyzed by microscopic counting method; pH and residual sugar concentration during fermentation are monitored in real time by online detection equipment.

[0052] Example 1

[0053] See appendix Figure 1 This invention provides a method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation, comprising:

[0054] Activation and culture of bacterial strains:

[0055] Three original bacterial strains—Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum—were selected and activated using MRS broth medium. The activation temperature was set at 37℃, and the activation time was 18 hours. After the activation, activated bacterial suspensions with excellent growth and no contamination were obtained and used for later use.

[0056] Bacterial strain acclimation culture to bile acid tolerance:

[0057] Prepare an acclimatization culture medium containing bile acids. The specific components of the culture medium are: peptone 12 g / L, beef extract 6 g / L, yeast extract 4 g / L, glucose 18 g / L, dipotassium hydrogen phosphate 3 g / L, sodium acetate 6 g / L, triammonium citrate 1.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.03 g / L, bile salts 0.5-3.0 g / L, and Tween-80 1.0 mL / L.

[0058] The three activated bacterial strains were inoculated into the aforementioned acclimatization medium and passaged for 10 generations, with an inoculum size of 4% per generation. During the passage, the culture temperature was increased by 0.8℃ with each generation, ranging from 37 to 45℃; the bile salt concentration was increased by 0.2 g / L with each generation, ranging from 0.5 to 3.0 g / L; and the culture time for each generation was 24 hours. After each strain reached the late logarithmic growth phase, it was transferred to the next generation of acclimatization medium with the same inoculum size.

[0059] After subculturing and domestication, the bacterial suspensions of each strain were placed in a low-temperature centrifuge and centrifuged at 8000 rpm for 20 min to collect the bacterial precipitate. The precipitate was washed three times with sterile physiological saline to remove residual culture medium and metabolic impurities, and then diluted to prepare bacterial suspensions. The OD600 value of the bacterial suspensions was adjusted to 2.0 to obtain bacterial suspensions of bile acid-resistant domesticated strains of Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum.

[0060] Multi-strain mixed fermentation:

[0061] Three domesticated bacterial suspensions were uniformly mixed in a volume ratio of 3:1:2 (Bifidobacterium animalis: Streptococcus thermophilus: Lactobacillus plantarum) to obtain a mixed bacterial suspension. A fermentation medium was prepared with the following components: glucose 28 g / L, tryptone 10 g / L, yeast extract 6 g / L, ox bile powder 6 g / L, cholesterol 0.8 g / L, L-cysteine ​​hydrochloride 0.5 g / L, ferrous sulfate 0.03 g / L, zinc sulfate 0.01 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 3.5 g / L, vitamin K1 0.001 g / L, and vitamin B12 0.003 g / L. The pH of the medium was adjusted to 6.6 using sodium hydroxide and hydrochloric acid solutions.

[0062] The mixed bacterial suspension was inoculated into the sterilized fermentation medium at an inoculum volume of 10% of the fermentation medium, and then placed in an anaerobic fermenter for segmented variable-temperature anaerobic fermentation. Nitrogen gas was continuously introduced to maintain the anaerobic environment during the fermentation process at an aeration rate of 0.3 L / min and a stirring speed of 120 rpm.

[0063] The fermentation process was divided into two stages: Stage 1 involved anaerobic fermentation at a constant temperature of 37℃ for 18 hours, during which the pH of the fermentation broth decreased to 5.0. Stage 2 involved raising the temperature to 42℃ and continuing anaerobic fermentation for another 12 hours, during which the pH of the fermentation broth rose back to 6.0, at which point the fermentation was terminated. The total fermentation time was 30 hours. Residual sugar concentration was monitored in real time during fermentation. When the residual sugar concentration fell below 2 g / L, sterile glucose solution was added to raise the residual sugar concentration to 8 g / L. In this example, sugar was added twice.

[0064] Separation and purification:

[0065] After fermentation, the fermentation broth was transferred to a high-pressure homogenizer and homogenized under a low temperature of 5℃ and a homogenization pressure of 90MPa. The homogenization cycle was repeated 3 times, with each cycle lasting 3 minutes. The cell breakage rate after homogenization was 95.2%.

[0066] Using 1 mol / L hydrochloric acid as an acid regulator, the pH of the crushed liquid was adjusted to 3.8, and the mixture was allowed to settle at 4℃ for 8 hours. The first precipitate was collected, and the supernatant was discarded.

[0067] The first precipitate was fully resuspended in phosphate buffer (pH 7.8) and then subjected to microfiltration and ultrafiltration. Microfiltration used a 0.22 μm microfiltration membrane with a transmembrane pressure of 0.2 MPa and a filtration temperature of 20 °C. Ultrafiltration used a 5 kDa molecular weight cutoff ultrafiltration membrane with a transmembrane pressure of 0.35 MPa and a filtration temperature of 20 °C. The mixture was circulated and concentrated 6 times, and the ultrafiltration retentate was collected. The pH of the retentate was adjusted back to 3.2, and the mixture was allowed to settle at 4 °C for 4 h. It was then centrifuged at 10,000 rpm for 20 min, and the second precipitate was collected, completing the initial separation and purification.

[0068] Crystallization and purification:

[0069] An organic solvent system containing a surfactant was prepared, using anhydrous ethanol as the organic solvent and sodium cholate as the surfactant, with a surfactant concentration of 2.5 g / L. The volume-to-mass ratio of the organic solvent to the second precipitate was 20 mL: 1 g. The precipitate was added to the system and stirred at 30 °C for 60 min to dissolve. Insoluble impurities were removed by filtration, yielding a clear crystallization precursor solution.

[0070] The precursor solution was treated using a gradient cooling recrystallization process: first, the temperature was lowered from 30℃ to 12℃ at a cooling rate of 1.5℃ / min, and then held at 12℃ for 2 hours; when the system temperature dropped to 6℃, 2% of the precipitate mass of bezoar seed crystals with a particle size of 10-50μm were added; then, the temperature was lowered from 12℃ to -5℃ at a cooling rate of 3℃ / h, and then held at -5℃ for 5 hours to allow for crystallization.

[0071] After crystallization, the crystals were collected by filtration and washed four times with pre-cooled anhydrous ethanol at 0℃, with an organic solvent to crystal volume ratio of 5mL:1g each time, to remove residual impurities from the crystal surface. Finally, a vacuum drying process was used at 40℃, a vacuum degree of 0.05MPa, and a drying time of 16h to obtain high-purity in vitro cultured bezoar product.

[0072] Testing revealed that the in vitro cultured bezoar prepared in this embodiment had a bilirubin purity of 94.6% and a bile acid purity of 97.2%.

[0073] Example 2

[0074] Activation and culture of bacterial strains:

[0075] Three original bacterial strains—Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum—were selected and activated using M17 medium. The activation culture temperature was 35℃, and the activation time was 24 hours, yielding activated bacterial solutions free of contaminants and exhibiting excellent activity, which were then used for later use.

[0076] Bacterial strain acclimation culture to bile acid tolerance:

[0077] Prepare an acclimatization culture medium containing bile acids. The specific components of the culture medium are: peptone 10 g / L, beef extract 8 g / L, yeast extract 3 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 8 g / L, triammonium citrate 1 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.02 g / L, bile salts 0.5-3.0 g / L, and Tween-80 0.5 mL / L.

[0078] The three activated bacterial strains were inoculated into acclimatization medium and passaged for eight generations, with an inoculum size of 2% per generation. The passage temperature was increased by 0.5℃ with each generation, ranging from 37 to 41℃; the bile salt concentration was increased by 0.1 g / L with each generation, ranging from 0.5 to 3.0 g / L; each generation was cultured for 30 hours, and each strain was transferred to the next generation after reaching the late logarithmic growth phase.

[0079] After subculturing and domestication, the bacterial cells of each strain were collected by centrifugation at 9000 rpm for 18 min, washed twice with sterile physiological saline, diluted to prepare a bacterial suspension, and the OD600 value was adjusted to 1.0 to obtain a bacterial suspension of bile acid tolerant domesticated strains.

[0080] Multi-strain mixed fermentation:

[0081] Three types of domesticated bacterial suspensions were mixed in a volume ratio of 2:2:1:Bifidobacterium animalis:Streptococcus thermophilus:Lactobacillus plantarum to obtain a mixed bacterial suspension. Fermentation medium was prepared as follows: glucose 20 g / L, tryptone 12 g / L, yeast extract 4 g / L, ox bile powder 2 g / L, cholesterol 1.5 g / L, L-cysteine ​​hydrochloride 0.3 g / L, ferrous sulfate 0.05 g / L, zinc sulfate 0.005 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 2 g / L, vitamin K1 0.002 g / L, and vitamin B12 0.001 g / L. The pH was adjusted to 6.2.

[0082] The mixed bacterial suspension was inoculated at 5% of the culture medium volume. Carbon dioxide was introduced to maintain an anaerobic environment at a rate of 0.1 L / min, and the stirring speed was 50 rpm. Segmented variable-temperature fermentation was performed: fermentation at 36℃ for 24 h until the pH dropped to 4.8; then fermentation at 40℃ for 16 h until the pH rose back to 5.8, for a total fermentation time of 40 h. When the residual sugar level fell below 2 g / L during fermentation, glucose was added to bring the level up to 5 g / L, with a total of one glucose addition.

[0083] Separation and purification:

[0084] The fermentation broth was homogenized at 2℃ and 60MPa for 2 min at low temperature, and the cycle was repeated twice, with a cell disruption rate of 91.3%. The pH of the disrupted broth was adjusted to 3.0 with 0.1 mol / L acetic acid, and the mixture was allowed to stand at 2℃ for 12 h to settle. The first precipitate was then collected.

[0085] The precipitate was resuspended in pH 7.0 phosphate buffer and filtered through a 0.1 μm microfiltration membrane and a 3 kDa ultrafiltration membrane. The transmembrane pressure for microfiltration was 0.1 MPa and for ultrafiltration was 0.2 MPa. The filtration temperature was 15 °C, and the precipitate was concentrated three times. The pH of the retentate was adjusted to 2.5, and the mixture was allowed to stand at 2 °C for 6 h to precipitate. The precipitate was then centrifuged at 8000 rpm for 30 min, and the second precipitate was collected.

[0086] Crystallization and purification:

[0087] An organic solvent system was prepared using a 1:1 volume ratio of methanol and acetone, with polyethylene glycol-400 added as a surfactant at a concentration of 0.5 g / L. The volume ratio of organic solvent to precipitate was 10 mL:1 g. The mixture was stirred at 20 °C for 90 min, and then filtered to remove impurities, yielding the crystallization precursor solution.

[0088] Gradient cooling process: Cool from 20℃ to 10℃ at a rate of 0.5℃ / min and hold for 4h; when cooling to 5℃, add 0.5% of the precipitate and 10-50μm seed crystals; then cool to -10℃ at a rate of 1℃ / h and hold for 8h to crystallize.

[0089] The crystals were collected and washed twice with a mixed organic solvent pre-cooled at -5℃, 3 mL: 1 g each time. Freeze-drying was then performed: pre-freezing temperature -35℃, pre-freezing time 6 h; sublimation drying temperature -15℃, vacuum degree 5 Pa, drying time 30 h; and desorption drying temperature 10℃, drying time 12 h, to obtain the in vitro cultured bezoar product.

[0090] Testing revealed that the in vitro cultured bezoar prepared in this embodiment had a bilirubin purity of 88.2% and a bile acid purity of 92.5%.

[0091] Example 3

[0092] Activation and culture of bacterial strains:

[0093] All three bacterial strains were activated using MRS broth medium at 38℃ for 12 hours to obtain activated bacterial solutions that met the activity standards.

[0094] Bacterial strain acclimation culture to bile acid tolerance:

[0095] The acclimatization culture medium consisted of: peptone 15 g / L, beef extract 5 g / L, yeast extract 5 g / L, glucose 15 g / L, dipotassium hydrogen phosphate 4 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, bile salts 0.5-3.0 g / L, and Tween-80 1.5 mL / L.

[0096] The bacterial strain was passaged 15 times consecutively, with an inoculum size of 6% per generation. The culture temperature was increased by 1.0℃ with each generation, ranging from 37 to 45℃. The bile salt concentration was increased by 0.3 g / L with each generation, ranging from 0.5 to 3.0 g / L. Each generation was cultured for 18 hours, and the strain was transferred at the late logarithmic growth phase. After acclimatization, the bacterial cells were collected by centrifugation at 10,000 rpm for 15 minutes, washed four times with sterile physiological saline, and the OD600 value of the bacterial suspension was adjusted to 2.5 to obtain the acclimatized strain.

[0097] Multi-strain mixed fermentation:

[0098] The bacterial suspension was mixed in a volume ratio of 4:1:3, and the total inoculum volume was 15% of the culture medium volume. The fermentation medium components were: glucose 35 g / L, tryptone 8 g / L, yeast extract 8 g / L, ox bile powder 10 g / L, cholesterol 0.2 g / L, L-cysteine ​​hydrochloride 0.8 g / L, ferrous sulfate 0.01 g / L, zinc sulfate 0.02 g / L, potassium dihydrogen phosphate 1 g / L, sodium chloride 5 g / L, vitamin K1 0.0005 g / L, and vitamin B12 0.005 g / L. The pH was adjusted to 7.0.

[0099] Nitrogen gas was introduced into the fermenter at a rate of 0.5 L / min, and the stirring speed was 200 rpm. Fermentation was carried out in stages: fermentation at 38℃ for 12 hours until the pH dropped to 5.5; then fermentation was continued at 44℃ for 8 hours until the pH rose back to 6.5, for a total fermentation time of 20 hours. Sugar was replenished to 10 g / L when the residual sugar level fell below 2 g / L, and this was repeated three times.

[0100] Separation and purification:

[0101] The cells were homogenized and disrupted 5 times at 8℃ and 120MPa for 5 minutes each time, resulting in a cell disruption rate of 96.8%. The pH of the disruption solution was adjusted to 4.5 with 2mol / L sulfuric acid, and the mixture was allowed to stand at 8℃ for 3 hours to settle. The first precipitate was then collected.

[0102] The precipitate was resuspended in pH 8.5 phosphate buffer and filtered through a 0.5 μm microfiltration membrane and a 10 kDa ultrafiltration membrane. The transmembrane pressure for microfiltration was 0.3 MPa and for ultrafiltration was 0.5 MPa. The filtration temperature was 25 °C, and the concentration was increased 10-fold. The pH of the retentate was adjusted to 4.0, and the mixture was allowed to stand at 8 °C for 2 h to precipitate. The mixture was then centrifuged at 12,000 rpm for 15 min, and the second precipitate was collected.

[0103] Crystallization and purification:

[0104] Ethyl acetate was used as the organic solvent, and sodium deoxycholate (5 g / L) was used as the surfactant. The volume-to-mass ratio of solvent to precipitate was 30 mL: 1 g. The solution was stirred at 40 °C for 30 min, and then filtered to remove impurities to obtain the crystallization precursor solution.

[0105] Gradient cooling: Cool from 40℃ to 15℃ at a rate of 3℃ / min and hold for 1h; add 3% seed crystals by mass of precipitate when cooling to 8℃; cool to 0℃ at a rate of 5℃ / h and hold for 2h to crystallize.

[0106] Wash five times with ethyl acetate pre-cooled at 5℃, each time using 8 mL:1 g. Then, vacuum dry at 50℃ and 0.1 MPa for 8 hours to obtain the final product.

[0107] Testing revealed that the in vitro cultured bezoar prepared in this embodiment had a bilirubin purity of 97.5% and a bile acid purity of 98.8%.

[0108] Comparative Example 1 (without bile acid tolerance acclimation)

[0109] The process of this comparative example is basically the same as that of Example 1. The only difference is that the three strains were only activated and cultured in a conventional manner, and no bile acid gradient subculturing treatment was carried out. The activated original strains were directly mixed and inoculated for fermentation according to the proportion of Example 1. The other activation, fermentation, separation and purification, crystallization and drying process parameters are exactly the same as those of Example 1.

[0110] Specific steps: The bacterial strain was activated at 37℃ for 18 hours using MRS broth medium, and then passaged and domesticated without gradient temperature increase or gradient bile salt extraction; the bacterial solution was directly mixed at a volume ratio of 3:1:2, and 10% of the inoculum was inoculated into the same fermentation medium. The same segmented variable temperature anaerobic fermentation, low-temperature homogenization and crushing, acid precipitation, membrane separation, gradient cooling recrystallization and vacuum drying process was used to finally prepare in vitro cultured bezoar samples.

[0111] The comparative sample was tested and found to have a bilirubin purity of 72.3% and a bile acid purity of 81.5%.

[0112] Comparative Example 2 (using constant temperature fermentation, without staged temperature variation)

[0113] The process of this comparative example is basically the same as that of Example 1, except that the mixed fermentation stage adopts constant temperature fermentation throughout the process and the segmented temperature change process is cancelled. The parameters for the domestication, activation, separation and purification, crystallization and drying of other strains are completely the same as those of Example 1.

[0114] Specific steps: The strain domestication, activation, and mixed inoculation processes are the same as in Example 1; the fermentation process is carried out at a constant 37°C for 30 hours of anaerobic fermentation, with the nitrogen aeration rate, stirring speed, and sugar supplementation rules remaining unchanged, and no secondary fermentation with increased temperature is performed; the subsequent crushing, precipitation, membrane separation, crystallization, and drying processes are completely replicated in Example 1.

[0115] The comparative sample was tested and found to have a bilirubin purity of 79.6% and a bile acid purity of 85.2%.

[0116] Comparative Example 3 (Conventional natural cooling crystallization, without gradient cooling + no surfactant added)

[0117] The process of this comparative example is basically the same as that of Example 1, except that the gradient cooling process is cancelled in the crystallization and purification stage, and conventional natural cooling crystallization is adopted. In addition, no surfactant is added to the organic solvent system. All other process parameters are completely consistent with those of Example 1.

[0118] Specific steps: The initial strain domestication, activation, mixed fermentation, separation and purification steps are exactly the same as in Example 1; during the crystallization stage, anhydrous ethanol is used as the pure solvent to dissolve the precipitate. After dissolving at 30°C for 60 minutes and filtering to remove impurities, the precipitate is directly cooled to room temperature and then allowed to stand at low temperature to crystallize. No segmented gradient cooling is set, no seed crystals are added, and no surfactants are added. The subsequent washing and drying processes remain unchanged.

[0119] The comparative sample was tested and found to have a bilirubin purity of 83.1% and a bile acid purity of 89.4%.

[0120] The experimental data comparison and analysis are as follows:

[0121] Table 1. Comparison of core process variables between each embodiment and the comparative example.

[0122] Example 1 Gradient temperature increase + gradient bile salt acclimation (10 generations) Pre-fermentation at 37℃ + staged variable-temperature fermentation at 42℃ Two-stage gradient cooling + seed-induced crystallization Add sodium cholate (2.5g / L) Example 2 Gradient temperature increase + gradient bile salt acclimation (8 generations) Pre-fermentation at 36℃ + staged variable-temperature fermentation at 40℃ Two-stage gradient cooling + seed-induced crystallization Add polyethylene glycol-400 (0.5 g / L) Example 3 Gradient temperature increase + gradient bile salt acclimation (15 generations) Pre-fermentation at 38℃ + staged variable-temperature fermentation at 44℃ Two-stage gradient cooling + seed-induced crystallization Add sodium deoxycholate (5g / L) Comparative Example 1 No domestication, conventional activation Same as Example 1, segmented temperature-controlled fermentation Gradient cooling crystallization as in Example 1 Add sodium cholate (2.5g / L) Comparative Example 2 Same as the domestication process in Example 1 Fermentation at a constant temperature of 37℃ throughout the entire process Gradient cooling crystallization as in Example 1 Add sodium cholate (2.5g / L) Comparative Example 3 Same as the domestication process in Example 1 Same as Example 1, segmented temperature-controlled fermentation Natural cooling and gradient-free crystallization No additives

[0123] As shown in Table 1, all three examples fully employ the complete core process of gradient strain domestication, segmented variable-temperature fermentation, and gradient cooling crystallization with surfactant-assisted crystallization. The process parameters are adjusted only within a specific range to verify the universality and optimality of the parameter range of this invention. Comparative Example 1 removes the strain domestication step as a single variable; Comparative Example 2 removes the segmented variable-temperature fermentation step as a single variable; and Comparative Example 3 removes the gradient crystallization and surfactant-assisted crystallization steps as a single variable. Through the single-variable control method, the contribution of each core process step to product quality can be accurately quantified, eliminating interference from multiple variables. The experimental data possess rigorous comparability and persuasiveness.

[0124] Table 2: Comparison of cell breakage rate and fermentation cycle parameters of each experimental group

[0125] Example 1 95.2 30 6.0 2 Example 2 91.3 40 5.8 1 Example 3 96.8 20 6.5 3 Comparative Example 1 82.6 30 5.2 2 Comparative Example 2 88.1 30 5.4 2 Comparative Example 3 95.0 30 6.0 2

[0126] The data shows that the gradient acclimatization process of the present invention can significantly improve the cell activity and pressure resistance. After gradient acclimatization, the cell breakage rate of Examples 1, 2, and 3 is higher than 91%. Among them, Example 3 has the most passages and the most complete gradient acclimatization, with a cell breakage rate of 96.8%. In contrast, the cell breakage rate of the unacclimatized Comparative Example 1 is only 82.6%. This indicates that bile acid tolerance acclimatization can effectively enhance the adaptability of the strain to the high bile salt and high osmotic pressure environment of the fermentation system, improve the cell proliferation efficiency and the accumulation of intracellular effective products, and thus improve the release rate of effective components after breakage.

[0127] Regarding the fermentation cycle, Example 3 exhibited the optimal parameter ratio and the highest synergistic metabolic efficiency of the microorganisms, completing fermentation in just 20 hours, significantly improving fermentation efficiency. Example 2, with parameters biased towards the lower limit of the range, had a slower metabolic rate and the longest fermentation cycle. Comparative Example 2, which underwent isothermal fermentation, showed a lower pH at the fermentation endpoint, indicating metabolic imbalance under isothermal conditions, accumulation of acidic metabolites, poor metabolic integrity, and insufficient synthesis of effective products. Comparative Example 3 maintained unchanged fermentation and crushing processes; therefore, the crushing rate and fermentation parameters were essentially the same as in Example 1, with no significant differences in data, further verifying the single-variable nature.

[0128] Table 3: Purity Test Results of In Vitro Cultured Bezoar Products in Each Experimental Group

[0129] Example 1 94.6 97.2 High purity 2.1 Example 2 88.2 92.5 Medium to high purity 4.3 Example 3 97.5 98.8 Ultra-high purity 1.2 Comparative Example 1 72.3 81.5 ordinary purity 8.7 Comparative Example 2 79.6 85.2 ordinary purity 6.5 Comparative Example 3 83.1 89.4 medium purity 4.9

[0130] The purity data of the finished products show that the entire process of this invention can stably prepare high-purity in vitro cultured bezoar. The purity of bilirubin in the three examples is ≥88%, and the purity of bile acid is ≥92%, which is far superior to the comparative samples. Among them, Example 3 is the optimal combination of process parameters, with sufficient domestication and subculturing, reasonable fermentation ratio, and precise crystallization process, achieving a bilirubin purity of 97.5% and a bile acid purity of 98.8%, with only 1.2% impurity residue, resulting in the best product quality. Example 2 uses the lower limit of the parameter range, and the purity indicators are slightly lower than the other two examples, but are still significantly better than the products produced by conventional processes.

[0131] In Comparative Example 1, after removing the strain domestication process, the strain could not adapt to the high bile salt fermentation environment, resulting in a significant decrease in the strain's synergistic metabolic capacity, insufficient synthesis of effective products, and an increase in miscellaneous metabolites. The impurity residue reached 8.7%, and the purity of bilirubin and bile acids was significantly reduced, directly demonstrating that tolerance to bile acid gradient domestication is a fundamental prerequisite for ensuring high yield and high purity of products. Comparative Example 2 used isothermal fermentation, lacking a segmented temperature-controlled metabolic regulation mechanism. The synergistic metabolic process of low-temperature proliferation in the early stage and high-temperature conversion in the later stage could not be completed, resulting in low product conversion efficiency and a significant decrease in purity. Comparative Example 3, without surfactant assistance and gradient cooling crystallization, resulted in disordered crystal precipitation and severe impurity encapsulation, with a purity significantly lower than the process of this invention, verifying the core role of the crystallization purification process.

[0132] Table 4: Analysis of the Contribution Rate of Core Processes to Product Purity Improvement

[0133] Bacterial strains adapted to bile acid acclimatization Example 1 vs Comparative Example 1 22.3 15.7 48.2 Segmented variable-temperature fermentation Example 1 vs Comparative Example 2 15.0 12.0 32.6 Gradient cooling + surfactant crystallization Example 1 vs Comparative Example 3 11.5 7.8 19.2

[0134] Calculations based on the purity improvement of a single variable reveal a clear hierarchical contribution relationship among the three core processes of this invention. The strain bile acid tolerance acclimation process contributes the most, reaching 48.2%, making it the key to improving product purity. This process, by gradually increasing bile salt concentration and culture temperature, directionally acclimates the strain's stress resistance and bile acid conversion ability, thereby improving the efficiency of effective product synthesis from the source and significantly reducing the generation of impurities. The segmented variable-temperature fermentation process contributes 32.6%, precisely matching the metabolic needs of multiple strains at different growth stages through segmented temperature control. The initial low temperature enables rapid strain proliferation, ensuring microbial biomass, while the subsequent high temperature promotes metabolic transformation, efficiently synthesizing effective active ingredients such as bilirubin and bile acids, thus optimizing the metabolic pathway.

[0135] Gradient cooling coupled with surfactant-assisted crystallization contributes 19.2% to the final purification process, serving as a key technology for terminal purification. Surfactants optimize the polarity of the solvent system, reducing impurity adsorption and encapsulation. Gradient cooling combined with seed induction enables ordered and uniform crystal precipitation, significantly improving crystal purity and product uniformity. These three core processes work synergistically to improve quality and efficiency throughout the entire process, from strain adaptation and product synthesis to purification. Each is indispensable, collectively ensuring the ultra-high purity of in vitro cultured bezoar.

[0136] Based on the experimental data from the above embodiments and comparative examples, it is evident that the present invention employs a complete process involving multi-strain bile acid-resistant gradient acclimatization, segmented temperature-controlled synergistic fermentation, multi-stage separation and purification, and surfactant-assisted gradient cooling recrystallization. Compared to traditional methods involving single-strain fermentation, isothermal fermentation, and natural crystallization, this invention possesses significant technical advantages. Through a multi-strain synergistic adaptation fermentation system, the present invention fully leverages the metabolic complementary advantages of Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum. Gradient acclimatization enhances the strains' stress resistance and transformation capacity. Segmented temperature control precisely regulates the growth and metabolic processes of the microbial community. Further impurities are removed through multi-stage membrane separation and gradient crystallization purification, ultimately resulting in a stable high-purity in vitro cultured bezoar.

[0137] The process parameters of this invention are reasonably controllable and highly repeatable. The three embodiments cover the entire parameter gradient range of the claims, and all can produce high-quality products with bilirubin purity of over 85% and bile acid purity of over 90%. Under optimal process conditions, the product purity can reach the industry's highest standards. The experimental data of each comparative example fully verify the innovation and necessity of the core process of this invention. Removing any core process will lead to a significant decrease in product purity, an increase in impurity content, and a reduction in fermentation metabolic efficiency, fully demonstrating that the technical solution of this invention has outstanding substantive features and significant technological progress, and is suitable for industrial-scale production.

[0138] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation, characterized in that, The method includes: Domestication steps: Bifidobacterium animalis, Streptococcus thermophilus and Lactobacillus plantarum were continuously subcultured in domestication medium containing bile acids to obtain bile acid tolerant domesticated strains. Mixed fermentation step: The bile acid tolerant strains were mixed in proportion and inoculated into the fermentation medium, and then subjected to segmented temperature-switched fermentation under anaerobic conditions to obtain the fermentation product; Separation and purification steps: The fermentation product is subjected to low-temperature homogenization, acid precipitation, and membrane separation in sequence, and the precipitate is collected; Crystallization and purification steps: The precipitate is recrystallized in an organic solvent system containing surfactants under gradient cooling, and then dried to obtain in vitro cultured bezoar.

2. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 1, characterized in that, In the strain domestication step, the domestication culture medium containing bile acids is composed of: peptone 10-15 g / L, beef extract powder 5-8 g / L, yeast extract powder 3-5 g / L, glucose 15-20 g / L, dipotassium hydrogen phosphate 2-4 g / L, sodium acetate 5-8 g / L, triammonium citrate 1-2 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.02-0.05 g / L, bile salts 0.5-3 g / L, and Tween-80 0.5-1.5 mL / L; The continuous subculture involves 8-15 subcultures, with each subculture lasting 18-30 hours. The culture temperature increases by 0.5-1.0℃ with each subculture, and the culture temperature range is 37-45℃. The concentration of bile salts was increased by 0.1-0.3 g / L in each generation of subculture, with the bile salt concentration range being 0.5-3.0 g / L.

3. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 1, characterized in that, In the strain domestication step, the animal bifidobacterium, thermophilic streptococcus, and plant lactobacillus are activated and cultured respectively before being continuously passaged in the domestication medium containing bile acids; The culture medium for activation culture is MRS broth medium or M17 medium, the activation culture temperature is 35-38℃, and the activation culture time is 12-24h. The inoculum size for the continuous subculture is 2-6%, and when the culture reaches the late logarithmic growth phase, it is transferred to the next generation of acclimatization culture medium with the same inoculum size. After the continuous subculturing was completed, the bacterial suspensions of each strain were collected by centrifugation and washed 2-4 times with sterile physiological saline to obtain the bacterial suspension of the bile acid acclimatized strain. The concentration of the bacterial suspension was adjusted to OD. 600 It ranges from 1.0 to 2.

5.

4. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 1, characterized in that, In the mixed fermentation step, the bile acid tolerant strains are mixed in the following proportions: the bacterial suspensions of Bifidobacterium animalis, Streptococcus thermophilus, and Lactobacillus plantarum are mixed in a volume ratio of (2-4):(1-2):(1-3) to obtain a mixed bacterial suspension; The total inoculum volume of the mixed bacterial suspension is 5-15% of the volume of the fermentation medium; The fermentation medium consists of: glucose 20-35 g / L, tryptone 8-12 g / L, yeast extract 4-8 g / L, bovine bile powder 2-10 g / L, cholesterol 0.2-1.5 g / L, L-cysteine ​​hydrochloride 0.3-0.8 g / L, ferrous sulfate 0.01-0.05 g / L, zinc sulfate 0.005-0.02 g / L, potassium dihydrogen phosphate 1-3 g / L, sodium chloride 2-5 g / L, vitamin K1 0.0005-0.002 g / L, and vitamin B1. 12 0.001-0.005 g / L, pH adjusted to 6.2-7.

0.

5. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 4, characterized in that, In the mixed fermentation step, the segmented variable temperature fermentation includes: the inoculated fermentation broth is first subjected to constant temperature anaerobic fermentation at 36-38℃ for 12-24 hours; when the pH value of the fermentation broth drops to 4.5-5.5, the temperature is raised to 40-44℃ to continue anaerobic fermentation for 8-16 hours; when the pH value of the fermentation broth rises back to 5.5-6.5, the fermentation is terminated, and the total fermentation time is 20-40 hours. The anaerobic conditions are maintained by continuously introducing nitrogen or carbon dioxide gas into the fermentation vessel at a rate of 0.1-0.5 L / min, and the stirring speed during fermentation is 50-200 rpm. During the segmented temperature-controlled fermentation process, when the residual sugar concentration of the fermentation broth is below 2 g / L, sterile glucose solution is added until the residual sugar concentration is 5-10 g / L, and the addition is repeated 1-3 times.

6. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 1, characterized in that, In the separation and purification step, the low-temperature homogenization and crushing process is carried out at a temperature of 2-8°C, a homogenization pressure of 60-120 MPa, a homogenization cycle of 2-5 times, and a homogenization time of 2-5 minutes for each cycle. In the fragmentation liquid after low-temperature homogenization, the bacterial cell breakage rate is ≥90%; The acid precipitation treatment is as follows: the pH of the crushed liquid after low-temperature homogenization is adjusted to 3.0-4.5 with an acidic regulator, and the liquid is allowed to stand at 2-8℃ for 3-12 hours to precipitate. The first precipitate is collected and the supernatant is discarded. The acidity regulator is at least one of hydrochloric acid, sulfuric acid, or acetic acid, and the concentration of the acidity regulator is 0.1-2 mol / L.

7. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 6, characterized in that, In the separation and purification step, the membrane separation process is as follows: the first precipitate is resuspended in phosphate buffer solution with pH 7.0-8.5, filtered sequentially through a microfiltration membrane and an ultrafiltration membrane, the retentate is collected, the retentate is acid-precipitated again, the pH is adjusted to 2.5-4.0, the precipitation is allowed to stand at 2-8℃ for 2-6 hours, and the second precipitate is collected by centrifugation. The microfiltration membrane has a pore size of 0.1-0.5 μm, and the ultrafiltration membrane has a molecular weight cutoff of 3-10 kDa. The transmembrane pressure of the microfiltration membrane is 0.1-0.3 MPa, and the filtration temperature is 15-25℃. The transmembrane pressure of the ultrafiltration membrane is 0.2-0.5 MPa, the filtration temperature is 15-25℃, and the retentate is circulated and concentrated during the filtration process, with a concentration factor of 3-10 times. The centrifugation speed is 8000-12000 rpm, and the centrifugation time is 15-30 min.

8. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 1, characterized in that, In the crystallization purification step, the surfactant-containing organic solvent system consists of an organic solvent and a surfactant. The organic solvent is at least one of methanol, ethanol, acetone, ethyl acetate, or dichloromethane, and the surfactant is at least one of sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, or polyethylene glycol-400. The volume-to-mass ratio of the organic solvent to the precipitate is (10-30) mL: 1 g; The concentration of the surfactant in the organic solvent system is 0.5-5 g / L; When the precipitate is dissolved in an organic solvent system containing a surfactant, the dissolution temperature is 20-40℃, the stirring time is 30-90min, and after dissolution, the insoluble matter is removed by filtration to obtain a crystallization precursor solution.

9. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 8, characterized in that, In the crystallization purification step, the gradient cooling recrystallization is as follows: the crystallization precursor solution is cooled from the dissolution temperature to 10-15℃ at a cooling rate of 0.5-3℃ / min, kept at 10-15℃ for 1-4h, and then cooled from 10-15℃ to -10-0℃ at a cooling rate of 1-5℃ / h, kept at -10-0℃ for 2-8h, and the precipitated crystals are collected. During the gradient cooling recrystallization process, when the temperature drops to 5-8℃, seed crystals are added to the crystallization precursor solution. The amount of seed crystals added is 0.5-3% of the mass of the precipitate, and the particle size of the seed crystals is 10-50μm. After collecting the precipitated crystals, the crystals are washed 2-5 times with a pre-cooled organic solvent at a temperature of -5 to 5°C. The volume ratio of the organic solvent used in each wash to the volume of the crystals is (3-8) mL:1 g.

10. The method for high-purity in vitro culture of bezoar based on multi-strain synergistic fermentation according to claim 1, characterized in that, In the crystallization purification step, the drying is either freeze-drying or vacuum drying; The freeze-drying process parameters are as follows: pre-freezing temperature -40℃ to -30℃, pre-freezing time 2-6h, sublimation drying temperature -20℃ to -10℃, sublimation drying vacuum degree 5-30Pa, sublimation drying time 12-30h, desorption drying temperature 10-30℃, desorption drying time 4-12h. The process parameters for vacuum drying are: drying temperature 30-50℃, vacuum degree 0.01-0.1MPa, and drying time 8-24h.

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