Biological fermentation of chlorophyllin hydrochloride extraction process and method for synthesizing bilirubin

Abstract

This invention provides a process for extracting biliverdin hydrochloride through bio-fermentation and a method for synthesizing bilirubin. By studying the complex system of biliverdin obtained through microbial bacterial fermentation, a process for separating and purifying biliverdin was established, and the obtained biliverdin was further used to synthesize bilirubin. This process is simple to operate, uses common resins, employs solvents with low toxicity that are easily recovered and reused, utilizes low-energy membrane separation equipment for concentration, and uses conventional reaction reagents as reducing agents. The process has low operating costs, is environmentally friendly, and yields high-purity and stable-yield biliverdin and bilirubin products, demonstrating excellent prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically, it relates to a process for extracting biliverdin hydrochloride by bio-fermentation and a method for synthesizing bilirubin. Background Technology

[0002] Biliverdin is a bile pigment of trimethylbilirubin, a precursor of bilirubin. It is a dark green chromophore found in abundance in the bile of herbivores and is a product of normal hemoglobin metabolism. It appears as dark green flaky or columnar crystals, blackening and decomposing above 300°C. Commercially available biliverdin is usually biliverdin hydrochloride, soluble in methanol, ether, chloroform, and carbon disulfide.

[0003] Biliverdin has four subunits: α, β, γ, and δ. Due to the high selectivity of the carbon atom at the α-position of heme, 95%–97% of the biliverdin isomers in mammals are biliverdin-IXα, while the remaining 3%–5% are biliverdin-IXβ / δ. The former is catalyzed by biliverdin reductase A, and the latter by biliverdin reductase B. During the conversion of biliverdin to bilirubin, two biliverdin molecules bind to biliverdin reductase. One biliverdin molecule accepts a hydrogen atom donated by reduced nicotinamide adenine dinucleotide phosphate, while the other biliverdin molecule donates a proton to the first biliverdin molecule through a neighboring arginine residue. Then, biliverdin reductase hydrolyzes the γ-methyl bridge in the biliverdin molecule, ultimately producing bilirubin.

[0004] Biliverdin is the final product of the breakdown of heme in fish, amphibians, reptiles and birds and their excrement. Therefore, biliverdin can be separated and purified using the bile and blood of these animals.

[0005] Seok et al. improved the yield of precursor heme by overexpressing the rate-limiting enzyme in the heme synthesis pathway in *C. glutamicum* and optimizing the module, and then overexpressed HO to drive carbon metabolism to produce biliverdin. Yan Sihan et al. constructed a recombinant strain *E. coli* BL21 / pETDuet-gdhAEc-blc / hoCt, which shortened the transformation time and further increased biliverdin yield, representing the highest reported level of biliverdin synthesis by biological methods to date, but its industrial application has not yet been observed. Zhao Xinrui et al. utilized a heme oxygenase mutant to efficiently catalyze the biosynthesis of biliverdin, achieving source screening and rational modification of key enzymes for biliverdin synthesis, solving the previous problem of low biliverdin yield, with a yield of 13.41 mg / L, but its industrial application has also not been observed.

[0006] Bilirubin possesses a wide range of physiological activities, including antioxidant properties, and can inhibit the oxidation of linoleic acid and phospholipids. Bilirubin is one of the main raw materials for in vitro cultured bezoar. The Chinese Pharmacopoeia (2015 edition) stipulates that the bilirubin IXα content in in vitro cultured bezoar shall not be less than 35.0%. The method for testing bilirubin IXα content in the Chinese Pharmacopoeia is the ultraviolet method, specifying that the content of bilirubin IXα detected by ultraviolet light under the bilirubin IXα item shall not be less than 90%. Bilirubin IXα is a compound that is unstable at room temperature (FEBS Lett. 18, 315, 1971), capable of undergoing a series of complex reactions at room temperature, including oxidation, isomerization, disproportionation, degradation, polymerization, and intramolecular "gate closing."

[0007] Currently, all bilirubin IXα on the market is extracted from animal bile (mainly cattle and pigs) using chloroform. It contains a considerable amount of unknown impurities, including small amounts of high-molecular-weight impurities (detected at the origin by TLC, but not showing a peak by HPLC). The low-content bilirubin IXα extracted (although reaching the 90% concentration stipulated in the Chinese Pharmacopoeia) must be further refined before it can be used for in vitro culture of bezoar. China has had over 30 years of industrial-scale bilirubin IXα extraction. Although the industry is quite mature, the molecular structure of bilirubin IXα in the bile of slaughtered animals changes constantly, making it difficult to achieve a bilirubin IXα content of over 97%. Furthermore, bile resources are limited, making it difficult to meet market demand. Summary of the Invention

[0008] The purpose of this invention is to provide a process for extracting biliverdin hydrochloride through bio-fermentation and a method for synthesizing bilirubin.

[0009] To achieve the objectives of this invention, in a first aspect, this invention provides a process for extracting biliverdin hydrochloride through bio-fermentation, wherein the fermentation broth of biliverdin-producing engineered Escherichia coli (biliverdin fermentation broth) is sequentially subjected to solvation treatment, first filtration, resin adsorption and desorption of the filtrate, membrane concentration, acid precipitation crystallization, second filtration, dissolution and recrystallization of crude biliverdin hydrochloride, and dehydration, impurity removal and drying of refined biliverdin hydrochloride powder.

[0010] Furthermore, the solvent used in the solvation treatment is selected from at least one of methanol, ethanol, isopropanol, or acetone (industrial grade).

[0011] Furthermore, the fermentation broth of the biliverdin-producing engineered Escherichia coli was mixed with the solvent at a volume ratio of (0.5-1):1; The bacterial content of the fermentation broth was 10 × 10⁻⁶. 8 -30×10 8 CFU / mL.

[0012] Furthermore, the first filtration process employs methods such as plate and frame filters, disc centrifuges, and ceramic membranes.

[0013] Furthermore, the second filtration is carried out using methods such as disc centrifuges, tubular centrifuges, and plate and frame filters.

[0014] Furthermore, macroporous nonpolar resins are used for resin adsorption, preferably D312, HZ816, XR919, and LX110; before resin adsorption, the concentration of the filtrate is adjusted to 40-60% with methanol or ethanol.

[0015] Furthermore, the solvent used for analysis was a 70-90% methanol or ethanol aqueous solution.

[0016] Furthermore, membrane concentration uses nanofiltration membranes with a molecular weight range of 200-300 Da.

[0017] Furthermore, the feed solution obtained from membrane concentration is adjusted to the isoelectric point pH 4-7 (preferably pH 5-6) with hydrochloric acid for acid precipitation crystallization.

[0018] Further, the crude biliverdin hydrochloride was dissolved in methanol and adjusted to pH 9-11 with sodium hydroxide. The solution was then filtered through a Buchner funnel, and the resulting filtrate was adjusted to pH 5-6 with hydrochloric acid and recrystallized.

[0019] Furthermore, the solvent used for dehydration and impurity removal is methanol or ethanol.

[0020] Furthermore, the drying conditions are: vacuum drying in the dark, at 45°C for 12 hours.

[0021] Secondly, the present invention provides a method for synthesizing bilirubin. The method involves preparing biliverdin hydrochloride according to the above process, dissolving it in methanol at a ratio of 1 g:(23-28) mL (i.e., methanol usage is approximately 23-28 BV), and mixing it with sodium borohydride at a molar ratio of 1:(0.5-0.8) (preferably a molar ratio of biliverdin to sodium borohydride of 1:0.6) at room temperature for 2-4 hours (preferably 3 hours). After the reaction is complete, water of approximately 5-12 times (preferably 10 times) of the total volume of the reaction solution is added, along with dichloromethane of approximately 10-100 times (preferably 100 times) of the total volume of the reaction solution. The mixture is extracted and separated, the dichloromethane phase is collected, dehydrated with anhydrous sodium sulfate, filtered, concentrated, and crystallized to obtain bilirubin.

[0022] The objectives of this invention can also be further achieved using the following technical measures.

[0023] This invention provides a method for extracting biliverdin and synthesizing bilirubin through bio-fermentation, comprising the following steps: (1) Biliverdin fermentation broth was treated with solvation to change the fluid properties of the fermentation broth and the state of the products.

[0024] (2) The solvated filtrate is enriched with biliverdin by adsorption with low-concentration solvent resin and desorption with high-concentration solvent.

[0025] (3) The eluent is concentrated through a membrane to further enrich biliverdin and obtain a high-concentration biliverdin solution.

[0026] (4) The biliverdin concentrate was crystallized by acid precipitation isoelectric point crystallization to obtain crude biliverdin hydrochloride.

[0027] (5) The crude product was filtered by pre-laying perlite and then washed with solvent.

[0028] (6) Recrystallization: The coarse powder is dissolved by solvent and alkali adjustment, and then acid precipitation isoelectric point crystallization is used to obtain the refined biliverdin hydrochloride powder.

[0029] (7) The wet powder is dehydrated and impurities removed by solvent and further purified.

[0030] (8) Dry the refined powder by low-temperature vacuum drying in the dark to obtain the finished biliverdin product (biliverdin hydrochloride).

[0031] (9) Biliverdin is dissolved in methanol and reduced with sodium borohydride to obtain bilirubin precipitate.

[0032] (10) Filter to obtain precipitate, dissolve in water and dichloromethane, separate the liquid, collect the dichloromethane phase, dehydrate with anhydrous sodium sulfate, concentrate and crystallize.

[0033] (11) Filter to obtain bilirubin wet powder, dehydrate and remove impurities with solvent (such as methanol or ethanol), and dry in vacuum at 40-45℃ in the dark to obtain a high-purity bilirubin sample.

[0034] Furthermore, the solvent used for solvation treatment of the biliverdin fermentation broth is methanol or ethanol. The fermentation broth after biliverdin solvation is filtered using centrifugal ceramic membrane technology.

[0035] Furthermore, before resin adsorption, the solvent concentration (such as methanol or ethanol) needs to be adjusted to between 40-60% (the solvent is used to adjust the fermentation broth after solvation). The adsorption resin used is D312, and the desorption method is to use methanol or ethanol with a concentration of 70-90% for desorption.

[0036] Furthermore, the solvent is concentrated using a nanofiltration membrane with a molecular weight range of 200-300 Da, allowing most of the solvent to pass through and further enriching biliverdin.

[0037] Furthermore, hydrochloric acid is used for biliverdin acid precipitation crystallization, and the pH is adjusted to 4-7, preferably 5-6.

[0038] Furthermore, the solvent used for dehydrating and removing impurities from wet concentrate is methanol or ethanol.

[0039] Furthermore, the sodium borohydride required for the biliverdin reduction reaction is 1:0.5-1:0.8 (i.e., the molar ratio of biliverdin to sodium borohydride is 1:0.5-1:0.8), preferably 1:0.6.

[0040] Further, after the reaction is complete, add water at a volume of about 5-12 times (preferably 10 times) of the total volume of the reaction solution, and simultaneously add dichloromethane at a volume of about 10-100 times (preferably 100 times) of the total volume of the reaction solution to fully dissolve the bilirubin generated in the reaction and purify it to obtain the target product.

[0041] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention provides a method for extracting biliverdin and synthesizing bilirubin through bio-fermentation. By solvating the fermentation broth, the fluid properties and product state are altered, facilitating subsequent resin purification, crystallization, and recrystallization steps to obtain a high-purity biliverdin sample, which is then further synthesized into bilirubin. This method is suitable for industrial application, as the solvent is recyclable, waste liquid is minimal, it is relatively environmentally friendly, and production costs are controllable. Attached Figure Description

[0042] Figure 1 This is a biliverdin hydrochloride spectrum in a preferred embodiment of the present invention.

[0043] Figure 2 This is a bilirubin mass spectrum in a preferred embodiment of the present invention.

[0044] Figure 3 The 1H NMR spectrum of bilirubin is shown in a preferred embodiment of the present invention.

[0045] Figure 4 This is a chromatogram of bilirubin concentrate in a preferred embodiment of the present invention. Detailed Implementation

[0046] The fermentation broth for biliverdin produced by Escherichia coli is notoriously difficult to process, and currently there are no readily available and easily extracted extraction methods, either domestically or internationally. Furthermore, there are no reports of synthesizing bilirubin from biliverdin through bio-fermentation. This invention fully utilizes synthetic biology metabolic pathways and combines various purification methods to efficiently prepare high-purity industrial-grade biliverdin products, which can then be further synthesized into bilirubin.

[0047] The present invention adopts the following technical solution: This invention provides a method for extracting and synthesizing bilirubin from biliverdin through bio-fermentation, particularly the extraction and preparation of biliverdin under complex microbial metabolic conditions and its derivative synthesis technology.

[0048] The method for separating and purifying biliverdin provided by this invention includes the following steps: a) The fermentation broth for microbial synthesis and metabolism of biliverdin is first solvated and then filtered to obtain the filtrate.

[0049] b) Adjust the solvent concentration of the above filtrate, use resin adsorption, and desorb with high-concentration solvent to obtain the eluent.

[0050] c) The eluent is concentrated through a membrane to further enrich biliverdin and obtain a high-concentration biliverdin solution.

[0051] d) The biliverdin concentrate was crystallized by acid precipitation to obtain crude biliverdin hydrochloride.

[0052] e) The crude product from acid precipitation is filtered by pre-laying perlite and then top-washed with solvent.

[0053] f) The crude product was dissolved by solvent and alkali adjustment, and then subjected to secondary acid precipitation crystallization. The product was then filtered to obtain biliverdin hydrochloride concentrate.

[0054] g) The wet powder is dehydrated and impurities removed by solvent, and then dried at low temperature in a vacuum in the dark to obtain the finished product.

[0055] In step a) of the biliverdin fermentation broth solvation treatment, the solvent added is a hydrophilic solvent such as methanol, ethanol, isopropanol, or acetone. The purpose is to reduce the solution viscosity and improve the filtration permeability. This scheme preferably uses solvents that are environmentally friendly, have low market price, low evaporation loss, and low overall cost.

[0056] The above step a) involves filtering the fermentation broth after biliverdin solubilization using technologies such as plate and frame centrifuges, disc centrifuges, ceramic membranes, and centrifugal ceramic membranes. The preferred method is one that is simple to operate, easy to automate, and easy to clean.

[0057] In step b) above, the adsorption solvent needs to be adjusted to a concentration that the resin can just adsorb, and the desorption solvent needs to be adjusted to a concentration of the resin swelling and releasing products. Based on experience, the preferred concentrations are generally between 40-60% for the adsorption solvent and between 70-100% for the desorption solvent.

[0058] In step b) above, the adsorption resin used is a macroporous nonpolar resin such as D312, HZ816, XR919, and LX110. This scheme prefers resins produced by domestic enterprises with mature technology, low market price, and stable quality system.

[0059] Step c) above, concentration of the eluent is usually achieved through vacuum thermal concentration, membrane concentration, etc. Considering factors such as product heat resistance, energy saving, and automation, nanofiltration membrane concentration is preferred. Combining factors such as solvent and molecular weight, the molecular weight range of nanofiltration membrane is further optimized to 200-300 Da.

[0060] The acid precipitation crystallization of the concentrate in step d) above is based on the principle that biliverdin has low isoelectric point solubility, with the preferred isoelectric point being pH 4-7.

[0061] In step e) above, coarse product filtration can be carried out using disc centrifuges, tubular centrifuges, plate and frame filters, etc. The preferred option is simple to operate, low in equipment investment, and easy to clean. Plate and frame filters are further preferred. Considering the high viscosity of the product, a certain amount of filter aid is pre-spread.

[0062] In step g) above, the wet powder is dehydrated and impurities are removed. The wet powder has high viscosity and high water content, making it difficult to press dry by plate and frame filtration or vacuum filtration. Directly entering the drying oven will make drying difficult and cause it to clump together. Therefore, solvent dehydration is considered, and solvents that are environmentally friendly, have low market price, low evaporation loss, and low overall cost are preferred.

[0063] The present invention synthesizes bilirubin from biliverdin by dissolving it in methanol and directly adding sodium borohydride as a solid reducing agent to obtain crude bilirubin precipitate.

[0064] The bilirubin recrystallization steps of this invention are as follows: dissolve the crude bilirubin precipitate together with water and dichloromethane, separate the liquid and collect the dichloromethane phase, dehydrate it with anhydrous sodium sulfate, and concentrate and crystallize it.

[0065] This invention establishes a process for the separation and purification of biliverdin by studying the complex system of biliverdin obtained through microbial bacterial fermentation, and further synthesizes bilirubin from the obtained biliverdin. The process is simple to operate, uses common resins, employs solvents with low toxicity that are easily recyclable, utilizes low-energy membrane separation equipment for concentration, and uses conventional reaction reagents as reducing agents. It has low production and operating costs, is environmentally friendly, and yields high-purity and stable-yield biliverdin and bilirubin products, demonstrating excellent prospects for industrial application.

[0066] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0067] The biliverdin fermentation broth used in the following examples was obtained according to commonly used biological culture methods in the art (see the method disclosed in CN202410228043.9, Zhao Xinrui, Wang Ziwei, Zhou Jingwen, et al. Heme oxygenase mutant and its application in biliverdin synthesis). The fermentation strains used (including but not limited to BL21(DE3), C41(DE3), or C43(DE3)) were purchased from Beijing Panqiu Biotechnology Co., Ltd. The macroporous resin D312 used in the examples was purchased from Shandong Lukang Co., Ltd.; the centrifugal ceramic membrane was purchased from Jiangsu Lingdong Biotechnology Co., Ltd.; other instruments and raw materials were all commercially available products.

[0068] Example 1 20L of biliverdin fermentation broth was obtained from the bacterial culture (the bacterial content of the fermentation broth was approximately 30 × 10⁻⁶).8 The physicochemical properties of the biliverdin fermentation broth (CFU / mL) are as follows: It is a dark green viscous liquid with a centrifugation bacterial concentration of 21% and a pH of 6.4. The biliverdin potency was measured to be 109 μg / mL. 20 L of methanol was added to the biliverdin fermentation broth under stirring, and the mixture was vacuum filtered after 1 hour. After approximately 16 hours, 31 L of filtrate was obtained, which was a clear dark green liquid with an alcohol content of 43% and a potency of 36 μg / mL.

[0069] Example 2 10 L of the filtrate from Example 1 was taken, and the pH was adjusted to 4.5 with hydrochloric acid, causing the solution to become turbid. The precipitate was allowed to stand for 12 hours, and then filtered using a Buchner funnel, which proved very difficult. Subsequently, centrifugation was used to obtain a wet powder, which was then dried in a vacuum oven at 45°C for 12 hours to obtain 2 g of crude biliverdin hydrochloride powder. Analysis showed that the content of biliverdin hydrochloride in the crude powder was 12.8%.

[0070] Example 3 10 L of the filtrate from Example 1 was concentrated using a 1812 nanofiltration membrane with a molecular weight of 200 Da until the solution became slightly turbid, at which point it was stopped and discharged, with a measured volume of approximately 4 L. The pH of the solution was adjusted to 4.5 with hydrochloric acid, and the precipitate was allowed to stand for 12 hours. Filtering with a Buchner funnel was very difficult. Centrifugation was then used to obtain a wet powder, which was dried in a vacuum oven at 45°C for 12 hours to obtain 4 g of crude biliverdin hydrochloride powder. Analysis showed that the content of biliverdin hydrochloride in the crude powder was 8.3%.

[0071] Example 4 10 L of the filtrate from Example 1 was subjected to dynamic adsorption using approximately 1 L of D312 resin. The titer of the tailings was measured to be 2 μg / ml. After adsorption, the resin was eluented with 90% methanol to obtain 4.2 L of eluent, with a titer of 76 μg / ml. The eluent was concentrated using a 1812 nanofiltration membrane with a molecular weight of 200 Da until the solution became slightly turbid, at which point it was stopped and discharged, with a volume of approximately 1.3 L. The pH of the solution was adjusted to 4.5 with hydrochloric acid, and the mixture was allowed to precipitate for 12 hours. Perlite was pre-spread in a Buchner funnel, and then the crystallization precipitation solution was added and filtered. This process was relatively easy, yielding coarse powder containing perlite (coarse biliverdin hydrochloride powder). This wet coarse powder was dissolved in 0.5 L of methanol, and the pH was adjusted to 8.5 with sodium hydroxide. After thorough stirring and dissolution, the mixture was filtered through a Buchner funnel to obtain approximately 0.5 L of filtrate. The filtrate was adjusted to pH 4.5 with hydrochloric acid, allowed to precipitate for 12 hours, and then filtered through a Buchner funnel to obtain a wet powder of biliverdin hydrochloride. This powder was then dried in a vacuum oven at 45°C for 12 hours to obtain 0.3 g of refined biliverdin hydrochloride powder, which was found to have a content of 79.3%.

[0072] Example 5 23 L of biliverdin fermentation broth was obtained from bacterial culture. Its physicochemical properties were as follows: appearance: blackish-green viscous liquid; centrifugation concentration: 20%; pH: 6.6; biliverdin titer: 249 μg / ml. 23 L of methanol was added to the biliverdin fermentation broth under stirring. After 1 hour, the mixture was centrifuged and filtered through a ceramic membrane. After approximately 2 hours, 29 L of filtrate was obtained, which was a clear blackish-green liquid with an alcohol content of 49% and a titer of 148 μg / ml. Dynamic adsorption was performed using approximately 2.5 L of D312 resin, and the titer of the tail liquid was measured to be 6 μg / ml. After adsorption, the resin was desorbed using 90% methanol, yielding 6.5 L of eluent with a titer of 399 μg / ml. The eluent was concentrated using a 1812 nanofiltration membrane with a molecular weight of 200 Da until the solution became slightly turbid, at which point it was discharged, with a volume of approximately 3.3 L. The pH of the feed solution was adjusted to 4.5 with hydrochloric acid, and the mixture was allowed to precipitate for 12 hours. Perlite was pre-spread in a Buchner funnel, and then the crystallization precipitation solution was added and filtered. This process yielded a relatively easy crude powder of biliverdin hydrochloride containing perlite. This wet crude powder was dissolved in 2L of methanol, and the pH was adjusted to 8.5 with sodium hydroxide. After thorough stirring and dissolution, the mixture was filtered through a Buchner funnel to obtain approximately 2L of filtrate. The pH of this filtrate was adjusted to 4.5 with hydrochloric acid, and the mixture was allowed to precipitate for 12 hours. The filtrate was then filtered through a Buchner funnel to obtain a wet powder of biliverdin hydrochloride. This powder was dried in a vacuum oven at 45°C for 12 hours to obtain 2.8 grams of refined biliverdin hydrochloride powder, with a purity of 96.8%.

[0073] Example 6 21 L of biliverdin fermentation broth was obtained from bacterial culture. Its physicochemical properties were as follows: appearance: blackish-green viscous liquid; centrifugal bacterial concentration: 21%; pH: 6.5; biliverdin titer: 186 μg / ml. 30 L of methanol was added to the biliverdin fermentation broth under stirring. After 1 hour, the mixture was centrifuged and filtered through a ceramic membrane. After approximately 2 hours, 36 L of filtrate was obtained, which was a clear blackish-green liquid with an alcohol content of 55% and a titer of 115 μg / ml. Dynamic adsorption was performed using approximately 2.5 L of D312 resin, and the titer of the tail liquid was measured to be 3 μg / ml. After adsorption, the resin was desorbed using 80% methanol, yielding 7 L of eluent with a titer of 307 μg / ml. The eluent was concentrated using a 1812 nanofiltration membrane with a molecular weight of 200 Da until the solution became slightly turbid, at which point it was discharged, with a volume of approximately 4.5 L. The feed solution was adjusted to pH 4.7 with hydrochloric acid and allowed to precipitate for 12 hours. Perlite was pre-spread in a Buchner funnel, and then the crystallization precipitation solution was added and filtered to obtain coarse powder containing perlite. The powder was washed with an appropriate amount of methanol and filtered to dryness. The resulting wet coarse powder was dissolved in 3L of methanol, and the pH was adjusted to 10.5 with sodium hydroxide. After thorough stirring and dissolution, the solution was filtered through a Buchner funnel to obtain approximately 3L of filtrate. The filtrate was adjusted to pH 5.5 with hydrochloric acid, allowed to precipitate for 12 hours, filtered through a Buchner funnel, washed with an appropriate amount of methanol, and filtered to dryness to obtain wet powder. This wet powder was dried in a vacuum oven at 45°C for 12 hours to obtain 1.5g of refined biliverdin hydrochloride powder, with a purity of 98.3%.

[0074] Example 7 23 L of biliverdin fermentation broth was obtained from bacterial culture. Its physicochemical properties were as follows: appearance: blackish-green viscous liquid; centrifugal bacterial concentration: 22%; pH: 6.5; biliverdin titer: 266 μg / ml. 23 L of methanol was added to the biliverdin fermentation broth under stirring. After 1 hour, the mixture was centrifuged and filtered through a ceramic membrane. After approximately 2 hours, 30 L of filtrate was obtained, which was a clear blackish-green liquid with an alcohol content of 49% and a titer of 178 μg / ml. Dynamic adsorption was performed using approximately 2.5 L of D312 resin, and the titer of the tail liquid was measured to be 5 μg / ml. After adsorption, the resin was desorbed using 80% methanol, yielding 8.4 L of eluent with a titer of 458 μg / ml. The eluent was concentrated using a 1812 nanofiltration membrane with a molecular weight of 200 Da until the solution became slightly turbid, at which point it was discharged, with a volume of approximately 4.8 L. The pH of the feed solution was adjusted to 4.5 with hydrochloric acid, and the precipitate was allowed to set for 12 hours. Perlite was pre-spread in a Buchner funnel, and then the crystallization precipitation solution was added and filtered to obtain coarse powder containing perlite. The powder was washed with an appropriate amount of methanol and filtered to dryness. This wet coarse powder was dissolved in 3.5 L of methanol, and the pH was adjusted to 8.5 with sodium hydroxide. After thorough stirring and dissolution, the solution was filtered through a Buchner funnel to obtain approximately 3.5 L of filtrate. The pH of this filtrate was adjusted to 4.5 with hydrochloric acid, and the precipitate was allowed to set for 12 hours. The solution was filtered through a Buchner funnel, washed with an appropriate amount of methanol, and filtered to dryness to obtain wet powder. This powder was dried in a vacuum oven at 45°C for 12 hours to obtain 3.2 g of refined biliverdin hydrochloride powder, with a purity of 98.32%. The test results are shown below. Figure 1 .

[0075] Example 8 Weigh 1.5 g of biliverdin hydrochloride (the biliverdin product from Example 5), dissolve it in 40 ml of methanol, and add 1.2 g of sodium borohydride while stirring. React at room temperature for 4 hours. After the reaction is complete, filter and collect the solid phase. Then add 200 ml of water and 500 ml of dichloromethane, extract and separate the liquid, collect the dichloromethane phase, dehydrate with anhydrous sodium sulfate, filter, concentrate and crystallize to obtain 0.5 g of bilirubin sample, which was found to have a content of 88.8%.

[0076] Example 9 Weigh 1.5 g of biliverdin hydrochloride (the biliverdin product from Example 6), dissolve it in 40 ml of methanol, and add 0.9 g of sodium borohydride while stirring. The mixture is stirred at room temperature for 3 hours. After the reaction is complete, filter and collect the solid phase. Then add 200 ml of water and 500 ml of dichloromethane, extract and separate the liquid, collect the dichloromethane phase, dehydrate with anhydrous sodium sulfate, filter, concentrate and crystallize to obtain 0.6 g of bilirubin sample, with a purity of 97.5%.

[0077] Example 10 Weigh 1.5 g of biliverdin hydrochloride, dissolve it in 40 ml of methanol, and add 1.5 g of sodium borohydride while stirring. React at room temperature for 3 hours. After the reaction is complete, filter to obtain bilirubin precipitate. Add 50 ml of water and 500 ml of dichloromethane, extract and separate the liquid, collect the dichloromethane phase, dehydrate with anhydrous sodium sulfate, filter, concentrate and crystallize to obtain 0.2 g of bilirubin sample. The purity was determined to be 95.8%. MS test results are shown below. Figure 2 The results of the 1H NMR spectrum are shown below. Figure 3 .

[0078] Example 11 Weigh 1.5 g of crude biliverdin powder (the crude biliverdin powder from Example 6), dissolve it in 40 ml of methanol, and add 1.5 g of sodium borohydride while stirring. React at room temperature for 3 hours. After the reaction is complete, filter and collect the solid phase. Then add 500 ml of water and 5000 ml of dichloromethane, extract and separate the liquid, collect the dichloromethane phase, dehydrate with anhydrous sodium sulfate, filter, concentrate and crystallize to obtain 1.1 g of bilirubin sample, with a content of 99.6%. HPLC results are shown below. Figure 4 .

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A bio-fermentation process for extracting biliverdin hydrochloride, characterized in that, The fermentation broth of engineered Escherichia coli producing biliverdin is subjected to the following steps in sequence: solvation treatment, first filtration, resin adsorption and desorption of the filtrate, membrane concentration, acid precipitation crystallization, second filtration, dissolution and recrystallization of crude biliverdin hydrochloride, and dehydration, impurity removal and drying of refined biliverdin hydrochloride powder.

2. The process according to claim 1, characterized in that, The solvent used in the solvation treatment is selected from at least one of methanol, ethanol, isopropanol or acetone.

3. The process according to claim 2, characterized in that, The fermentation broth of engineered Escherichia coli producing biliverdin was mixed with the solvent at a volume ratio of (0.5-1):

1. The bacterial content of the fermentation broth was 10 × 10⁻⁶. 8 -30×10 8 CFU / mL.

4. The process according to claim 1, characterized in that, The first filtration stage uses plate and frame filters, disc centrifuges, or ceramic membranes; and / or, The second filtration is performed using a disc centrifuge, a tubular centrifuge, or a plate and frame filter press.

5. The process according to claim 1, characterized in that, Macroporous nonpolar resins are used for resin adsorption, preferably D312, HZ816, XR919, and LX110; before resin adsorption, the concentration of the filtrate is adjusted to 40-60% with methanol or ethanol; and / or, The solvent used for analysis is a 70-90% methanol or ethanol aqueous solution.

6. The process according to claim 1, characterized in that, Nanofiltration membranes are used for membrane concentration, with a molecular weight range of 200-300 Da.

7. The process according to claim 1, characterized in that, The feed solution obtained from membrane concentration is adjusted to the isoelectric point pH 4-7 with hydrochloric acid and then subjected to acid precipitation crystallization.

8. The process according to claim 1, characterized in that, The crude biliverdin hydrochloride was dissolved in methanol and the pH was adjusted to 9-11 with sodium hydroxide. The solution was filtered through a Buchner funnel, and the resulting filtrate was adjusted to pH 5-6 with hydrochloric acid and recrystallized.

9. The process according to any one of claims 1-8, characterized in that, The solvent used for dehydration and impurity removal is methanol or ethanol; and / or, The drying conditions are: vacuum drying in the dark, at 45°C for 12 hours.

10. A method for synthesizing bilirubin, characterized in that, Bilirubin hydrochloride was prepared according to the process described in any one of claims 1-9. It was dissolved in methanol at a ratio of 1 g:(23-28) mL and mixed with sodium borohydride at a molar ratio of 1:(0.5-0.8) at room temperature for 2-4 hours. After the reaction was completed, 5-12 times the total volume of water and 10-100 times the total volume of dichloromethane were added to the reaction solution. The mixture was extracted and separated, and the dichloromethane phase was collected. It was dehydrated with anhydrous sodium sulfate, filtered, concentrated and crystallized to obtain bilirubin.

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