Fermentation method and fermentation equipment of combined bile acid

Abstract

The invention relates to the technical field of preparation of bile acid, in particular to a fermentation method and fermentation equipment of combined bile acid. The fermentation method comprises the following steps: mixing a calcium chloride solution and a fermentation seed solution containing recombinant escherichia coli engineering bacteria to obtain a mixed bacteria solution; mixing sorbitol cholate, sodium taurate and water to obtain a mixed raw material; mixing the mixed bacterial liquid with the mixed raw material to obtain a fermentation raw material; fermenting the fermentation raw material under the condition of circulating electric shock to activate the recombinant escherichia coli engineering bacteria of the fermentation raw material to obtain fermentation liquor; wherein the circulating electric shock comprises a plurality of electric shock sections and a plurality of intermittent sections, the voltage of the electric shock sections is sinusoidal voltage, and the intermittent sections are non-electric shock sections. According to the fermentation method, the catalytic performance of generating tauroursodeoxycholic acid under the catalysis of recombinant escherichia coli engineering bacteria is effectively improved through the steps of increasing cell permeability through a calcium chloride solution, promoting raw material entering and product releasing through circulating electric shock, relieving feedback inhibition and the like.

Description

Technical Field

[0001] The present application relates to the technical field of bile acid preparation, and in particular to a fermentation method and fermentation equipment for conjugated bile acid. Background Art

[0002] Bile acids are the main component of bile. They are a general term for bile acids synthesized in the liver from cholesterol through a series of enzymatic reactions. Based on their state, bile acids can be divided into free bile acids and conjugated bile acids. Free bile acids include cholic acid, deoxycholic acid, and chenodeoxycholic acid, while conjugated bile acids are the product of free bile acids conjugated with glycine or taurine. Conjugated bile acids are generally more water-soluble and exist in the body as sodium salts. Therefore, conjugated bile acids are more stable than free bile acids.

[0003] At present, a mixed solution of sorbitan bile acid ester, sodium taurate and water can be used as raw materials, and then recombinant Escherichia coli engineered bacteria can be used as the strain to prepare conjugated bile acids such as tauroursodeoxycholic acid through fermentation. However, since the cells of the recombinant Escherichia coli engineered bacteria have selective permeability and contain a large amount of tauroursodeoxycholic acid in the bacterial cells, it is difficult for the recombinant Escherichia coli engineered bacteria to catalyze fermentation efficiently, resulting in a low level of catalytic performance in the final fermentation. Summary of the Invention

[0004] The present application provides a fermentation method and fermentation equipment for conjugated bile acid to solve the following technical problem: how to improve the catalytic performance of preparing tauroursodeoxycholic acid by fermentation.

[0005] In a first aspect, the present invention provides a method for fermenting conjugated bile acids, the method comprising:

[0006] Mixing the calcium chloride solution and the fermentation seed liquid containing the recombinant Escherichia coli engineered bacteria to obtain a mixed bacterial liquid;

[0007] mixing sorbitan bile acid ester, sodium taurate and water to obtain a mixed raw material;

[0008] mixing the mixed bacterial liquid and the mixed raw material to obtain a fermentation raw material;

[0009] The fermentation raw material is fermented under cyclic electric shock conditions to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain fermentation liquid; wherein the cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a non-electric shock segment.

[0010] Optionally, the duration of a single electric shock segment is 5 minutes to 10 minutes, and the voltage of a single electric shock segment is -2kV to 2kV.

[0011] Optionally, a single cycle of the sinusoidal voltage is 5ms to 20ms.

[0012] Optionally, the intermittent period is 15 minutes to 60 minutes.

[0013] Optionally, the enzyme activity of the fermentation seed liquid is ≥2.3 U / mL.

[0014] Optionally, the mass m1 of the sorbitan bile ester, the mass m2 of the sodium taurate and the mass m3 of the water satisfy the relationship: m1:m2:m3=(5-20):(5-20):(78-100).

[0015] Optionally, the fermentation raw material is fermented under cyclic electric shock conditions to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain a fermentation liquid; wherein the cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a non-electric shock segment, and then includes:

[0016] The fermentation liquid is centrifuged to obtain tauroursodeoxycholic acid; wherein the centrifugation temperature is 0°C to 15°C, the centrifugation speed is 6000rpm to 15000rpm, and the centrifugation time is 5min to 20min.

[0017] In a second aspect, the present application provides a fermentation device for conjugated bile acid, comprising:

[0018] Fermentation tanks;

[0019] The circulating electric shock part includes a circulating pipe and an electric shock sleeve. The electric shock sleeve is arranged on the surface of the circulating pipe. The feed port of the circulating pipe is connected to the bottom discharge port of the fermentation tank, and the discharge port of the circulating pipe is connected to the top feed port of the fermentation tank. The electric shock sleeve is used to perform cyclic electric shock on the circulating pipe.

[0020] Optionally, a timing valve is provided at the bottom of the fermentation tank, and the timing valve is used to periodically discharge the fermentation liquid; the opening and closing time difference of the timing valve is the same as the working time of the electric shock sleeve.

[0021] Optionally, the circulating electric shock unit further includes a feeding tank, which is connected to the circulating pipeline and is arranged downstream of the electric shock sleeve.

[0022] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0023] The present application provides a fermentation method for conjugated bile acids. The fermentation method first mixes a fermentation seed liquid containing recombinant Escherichia coli engineered bacteria with a calcium chloride solution. The calcium chloride solution is used to increase the permeability of the cells of the recombinant Escherichia coli engineered bacteria to increase the cells' ability to uptake sorbitan bile acid ester and sodium taurate. In addition, cyclic electric shock is used during the fermentation process. The sinusoidal voltage electric shock segment of the cyclic electric shock can generate an instantaneous high-intensity induced electric field in the fermentation raw material. In the high-intensity induced electric field, the potential difference between the inside and outside of the cell membrane of the recombinant Escherichia coli engineered bacteria increases. The cell membrane at the high potential difference will experience electroporation, causing a large number of channels in the cell membrane to be opened. This can promote the entry of raw material molecules such as sorbitol bile acid ester and sodium taurate into the bacterial cells in large quantities. At the same time, a large amount of tauroursodeoxycholic acid produced by the bacterial cells is also released into the fermentation liquid, which can relieve the feedback inhibition of the enzyme catalytic reaction in the bacterial cells, thereby effectively promoting the enzyme catalytic effect of the recombinant Escherichia coli engineered bacteria, so as to fully improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid. In addition, an intermittent section is set in the cyclic electric shock to avoid damage to the recombinant Escherichia coli engineered bacteria caused by continuous electric shock, and at the same time, sufficient time is provided for the enzyme catalysis of the recombinant Escherichia coli engineered bacteria, so as to further improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0026] Figure 1 A schematic diagram of the main process of a fermentation method for conjugated bile acid provided in an embodiment of the present application;

[0027] Figure 2 The following is a schematic diagram showing a fermentation process of a conjugated bile acid provided in an embodiment of the present application;

[0028] Figure 3 A schematic diagram of the structure of a conjugated bile acid fermentation device provided in an embodiment of the present application;

[0029] Figure 4 A schematic diagram of a sinusoidal voltage used in a conjugated bile acid fermentation method provided in Example 1 of the present application;

[0030] Figure 5 A schematic diagram of voltage changes of an induced electric field generated by a fermentation method for conjugated bile acid according to Example 1 of the present application is provided;

[0031] Among them, 1-fermentation tank, 2-circulation pipeline, 3-electric shock sleeve, 4-timing valve, 5-feeding tank, 6-pressure-resistant silicone rubber layer, 7-peristaltic pump. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0033] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values within the range; for example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited number (fractional or integer) within the indicated range.

[0034] As used herein, the terms "including," "comprising," and the like mean "including but not limited to." Relational terms such as "first" and "second" are used solely to distinguish one entity or operation from another, and do not necessarily require or imply any actual relationship or order between these entities or operations. "And / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, "A and / or B" can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, "plurality" means two or more; "at least one," "at least one of the following," or similar expressions, refers to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c," or "at least one of a, b, and c," can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can each be single or plural. "Parts" notation, such as parts by weight and parts by mass, indicates the proportional relationship between components. In this article, the parameters described by ratio should be understood as the first term of the proportional formula, in the order in which they are described, and the proportional figures should be understood as the second term. For example, if the mass ratio of substances A, B, and C is 1:2:3, then substances A, B, and C should correspond to the proportional figures in the proportional formula in the order in which they are described, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0035] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0036] It should be noted that although CN202210851363.0 discloses the preparation process and fermentation process of recombinant Escherichia coli engineered bacteria, this technology is subject to the limitations of the bacterial cells of the recombinant Escherichia coli engineered bacteria themselves, making it difficult to have high catalytic efficiency and enzyme activity, resulting in a low level of catalytic performance in the final fermentation.

[0037] Therefore, how to improve the catalytic performance of preparing tauroursodeoxycholic acid by fermentation is a technical problem that urgently needs to be solved.

[0038] Figure 1 The main flow chart of a fermentation method for conjugated bile acid provided in an embodiment of the present application is exemplarily shown;

[0039] like Figure 1 As shown, the present invention provides a fermentation method for conjugated bile acid, which comprises:

[0040] S1. The calcium chloride solution and the fermentation seed liquid containing the recombinant Escherichia coli engineered bacteria were mixed to obtain a mixed bacterial liquid;

[0041] S2. The sorbitan bile acid ester, sodium taurate and water are mixed to obtain a mixed raw material;

[0042] S3. The mixed bacterial solution and the mixed raw material are mixed to obtain a fermentation raw material;

[0043] S4. Fermenting the fermentation raw material under cyclic electric shock conditions to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain fermentation liquid; wherein the cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a segment without electric shock.

[0044] It should be noted that the recombinant Escherichia coli engineered bacteria are derived from the applicant's self-made strains. For the specific preparation process, please refer to CN202210851363.0.

[0045] It should be noted that the mass concentration of the recombinant Escherichia coli engineered bacteria in the fermentation seed liquid containing the recombinant Escherichia coli engineered bacteria is ≥10 mg / mL.

[0046] It should be noted that the specific sinusoidal voltage changes in this electric shock section are as follows: Figure 4 shown.

[0047] The present invention provides a fermentation method for conjugated bile acid. The fermentation method effectively improves the catalytic performance of recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid through specific steps and conditions. The specific analysis is as follows:

[0048] First, the method mixes a fermentation seed solution containing recombinant E. coli with a calcium chloride solution. The calcium chloride solution plays a key role here, increasing the permeability of the recombinant E. coli cells. This step aims to enhance the cells' ability to absorb raw material molecules such as sorbitan bile esters and sodium taurate, providing ample substrate for the subsequent fermentation process.

[0049] Next, during the fermentation process, cyclic electroporation technology was used. The characteristic of cyclic electroporation is that it contains multiple electroporation segments and intermittent segments. During the electroporation segment, the high-intensity induced electric field formed by the sinusoidal voltage increases the potential difference between the inside and outside of the cell membrane of the recombinant E. coli engineered bacteria. When the potential difference reaches a certain level, electroporation occurs in the cell membrane, that is, a large number of channels are formed on the cell membrane. These channels provide a path for raw material molecules such as sorbitan bile ester and sodium taurate to enter the bacterial cells, and also allow tauroursodeoxycholic acid produced in the bacterial cells to be smoothly released into the fermentation broth.

[0050] Electroporation not only facilitates the entry of raw material molecules and the release of products, but also relieves the feedback inhibition of enzyme-catalyzed reactions within bacterial cells. Normally, the accumulation of products within cells inhibits enzyme activity, thereby reducing catalytic efficiency. However, through electroporation, the products are promptly released outside the cell, avoiding this feedback inhibition and enabling the enzyme to continue to catalyze the reaction efficiently.

[0051] Furthermore, the intermittent periods during the cyclic electroporation also play a crucial role. They provide time for the recombinant E. coli to recover and perform enzyme catalysis, preventing damage to the cells from continuous electroporation. During these periods, the cells can utilize the ingested raw material molecules for enzyme-catalyzed reactions, producing more tauroursodeoxycholic acid. This cyclic electroporation method not only maintains cell activity but also increases product yield and catalytic efficiency.

[0052] In summary, the embodiments of the present application provide a fermentation method for conjugated bile acids, which effectively improves the catalytic performance of recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid by increasing cell permeability with a calcium chloride solution, promoting the entry of raw materials and product release through cyclic electric shock, and relieving feedback inhibition.

[0053] In some optional embodiments, the duration of a single electric shock segment is 5 minutes to 10 minutes, and the voltage of a single electric shock segment is -2 kV to 2 kV;

[0054] In these embodiments, the duration of a single electric shock segment can be 5 minutes to 10 minutes, and the voltage of a single electric shock segment can be -2kV to 2kV, so that the electric shock segment has sufficient time and voltage to promote sufficient electroporation of the bacterial cells of the recombinant Escherichia coli engineered bacteria in the fermentation raw material, thereby effectively promoting the enzyme catalytic effect of the recombinant Escherichia coli engineered bacteria to fully improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid.

[0055] The duration of the single shock segment can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0056] The voltage of the single shock segment may be -2 kV, -1.5 kV, -1.0 kV, -0.5 kV, 0 kV, 0.5 kV, 1.0 kV, 1.5 kV, or 2.0 kV.

[0057] In some optional embodiments, a single cycle of the sinusoidal voltage is 5ms to 20ms;

[0058] In these optional embodiments, a single cycle of the sinusoidal voltage can be 5ms to 20ms, which indicates that the sinusoidal voltage has a sufficiently fast change cycle, and the sufficiently fast change cycle can prompt the fermentation raw material to generate an induced electric field of sufficient intensity, thereby prompting the bacterial cells of the recombinant Escherichia coli engineered bacteria in the fermentation raw material to undergo sufficient electroporation, thereby effectively promoting the enzyme catalytic effect of the recombinant Escherichia coli engineered bacteria, and thereby fully improving the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid.

[0059] A single period of the sinusoidal voltage may be 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 15 ms or 20 ms.

[0060] In some optional embodiments, the intermittent period is 15 min to 60 min;

[0061] In these embodiments, the intermittent period can be 15 minutes to 60 minutes, so that the intermittent period has a sufficient amount of time. An intermittent period of sufficient time can avoid damage to the recombinant E. coli engineered bacteria caused by continuous electric shock, and at the same time provide sufficient time for the enzyme catalysis of the recombinant E. coli engineered bacteria, so as to further improve the catalytic performance of the recombinant E. coli engineered bacteria in producing tauroursodeoxycholic acid.

[0062] The duration of the rest period can be 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0063] In some optional embodiments, the enzyme activity of the fermentation seed liquid is ≥2.3 U / mL.

[0064] In these embodiments, the enzyme activity of the fermentation seed liquid is ≥2.3U / mL, which ensures that the fermentation seed liquid has a sufficient amount of recombinant Escherichia coli engineered bacteria to facilitate the subsequent cyclic electric shock and promote the sufficient electroporation of the bacterial cells of the recombinant Escherichia coli engineered bacteria in the fermentation raw material, thereby effectively promoting the enzyme catalytic effect of the recombinant Escherichia coli engineered bacteria to fully improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid.

[0065] In some optional embodiments, the mass m1 of the sorbitan bile ester, the mass m2 of the sodium taurate, and the mass m3 of the water satisfy the relationship: m1:m2:m3=(5-20):(5-20):(78-100);

[0066] In these embodiments, the mass ratio of sorbitan bile acid ester, sodium taurate and water can be (5-20):(5-20):(78-100), so that the mixed raw materials have sufficient amounts of sorbitan bile acid ester and sodium taurate to facilitate subsequent fermentation, thereby obtaining a fermentation broth containing sufficient tauroursodeoxycholic acid.

[0067] The mass m1 of the sorbitan bile ester can be 5, 6, 7, 8, 9, 10, 15 or 20.

[0068] The mass m2 of the sodium taurate can be 5, 6, 7, 8, 9, 10, 15 or 20.

[0069] The value of the water mass m3 can be 78, 79, 80, 85, 90, 95 or 100.

[0070] Figure 2 The following is a schematic diagram showing a fermentation process of a conjugated bile acid provided in an embodiment of the present application;

[0071] In some optional embodiments, such as Figure 2 As shown, the fermentation raw material is fermented under the condition of cyclic electric shock to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain a fermentation liquid; wherein the cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a non-electric shock segment, which then includes:

[0072] S5. The fermentation broth is filtered to obtain a filtrate;

[0073] S6. dialyzing the filtrate to obtain tauroursodeoxycholic acid;

[0074] In these embodiments, the fermentation broth is filtered and dialyzed to separate the tauroursodeoxycholic acid in the fermentation broth to obtain pure and sufficient tauroursodeoxycholic acid.

[0075] Figure 3 The following is a schematic diagram of the structure of a fermentation device for a conjugated bile acid provided in an embodiment of the present application;

[0076] Based on a general inventive concept, such as Figure 3 As shown, the embodiment of the present application provides a fermentation device for conjugated bile acid, the fermentation device comprising:

[0077] Fermentation tank 1;

[0078] The circulating electric shock part includes a circulating pipe 2 and an electric shock sleeve 3. The electric shock sleeve 3 is arranged on the surface of the circulating pipe 2. The feed port of the circulating pipe 2 is connected to the bottom discharge port of the fermentation tank 1, and the discharge port of the circulating pipe 2 is connected to the top feed port of the fermentation tank 1. The electric shock sleeve 3 is used to perform cyclic electric shock on the circulating pipe 2.

[0079] It should be noted that the fermentation tank 1 can be a stirred fermentation tank 1, and the stirred tank can also be provided with a compressed air pipeline, and the air outlet of the compressed air pipeline is arranged at the bottom of the fermentation tank 1 to promote sufficient fermentation.

[0080] It should be noted that a pressure-resistant silicone rubber layer 6 is provided at the contact portion between the circulation pipe 2 and the electric shock sleeve 3 to prevent the electric shock sleeve 3 from electrifying the circulation pipe 2 or even the fermentation tank 1 and affecting the safety of other equipment.

[0081] It should be noted that the circulation pipe 2 can be a flat pipe to enable the fermentation liquid electrocuted by the electrocuting sleeve 3 to generate an electric field of sufficient intensity.

[0082] It should be noted that the time for the fermentation liquid to flow through the circulation pipe 2 is the same as the working time of the electric shock sleeve 3.

[0083] It should be noted that, in order to promote the flow of the fermentation liquid in the circulation pipe 2, a non-contact peristaltic pump 7 can be provided upstream of the electric shock sleeve 3 to provide power for the flow of the fermentation liquid.

[0084] The system is implemented based on the above method. The specific steps of the method can refer to the above embodiments. Since the system adopts part or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0085] In some optional embodiments, a timing valve 4 is provided at the bottom of the fermentation tank 1, and the timing valve 4 is used to periodically discharge the fermentation liquid; the opening and closing time difference of the timing valve 4 is the same as the working time of the electric shock sleeve 3;

[0086] In these embodiments, a timing valve 4 is provided at the bottom of the fermentation tank 1, and the opening and closing time difference of the timing valve 4 is the same as the working time of the electric shock sleeve 3. The circulation time of the fermentation liquid in the circulation pipe 2 can be controlled by switching the timing valve 4, so that the electric shock sleeve 3 can fully shock the fermentation liquid in the circulation pipe 2, so as to prompt the fermentation raw materials to generate an induced electric field of sufficient intensity, thereby prompting the bacterial cells of the recombinant Escherichia coli engineered bacteria in the fermentation raw materials to fully undergo electroporation, so as to effectively promote the enzyme catalytic effect of the recombinant Escherichia coli engineered bacteria, and thereby fully improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid.

[0087] It should be noted that the timing valve 4 can be a D971X type DN80 timing electric switching valve.

[0088] It should be noted that the closing time of the timing valve 4 and the next opening and closing time are the working time of the intermittent section.

[0089] In some optional embodiments, the circulating electric shock unit further includes a feeding tank 5, the feeding tank 5 is connected to the circulating pipe 2, and the feeding tank 5 is arranged downstream of the electric shock sleeve 3;

[0090] In these embodiments, a feeding tank 5 is introduced into the circulating electric shock section and is arranged downstream of the electric shock sleeve 3, which can prevent the newly added material from affecting the progress of the circulating electric shock, so as to fully improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid.

[0091] The present application is further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national / industry standards. If there are no corresponding national / industry standards, the methods are carried out in accordance with commonly used international standards, conventional conditions, or conditions recommended by the manufacturer.

[0092] Example 1

[0093] 1. Preparation of fermentation seed liquid:

[0094] The recombinant E. coli engineered bacteria were cultured in 60 mL of sterile LB medium to obtain a seed bacterial suspension; the culture conditions were a constant temperature of 37.0°C, a culture time of 8 hours, and a shaker at 240 rpm;

[0095] The seed suspension was inoculated into 3 L of sterile LB liquid medium for fermentation to obtain a fermentation seed liquid. The process parameters for the first 0 to 3 hours of fermentation were as follows: fermentation temperature of 37.0°C, sterile air flow rate of 1.5 L / min, stirring at 200 rpm, and a tank pressure of 0.02 MPa. The process parameters for the first 4 to 20 hours of fermentation were as follows: fermentation temperature of 18.0°C, sterile air flow rate of 3.0 L / min, stirring at 300 rpm, and a tank pressure of 0.02 MPa. At the 4th hour of fermentation, 100 mL of a sterile solution of 0.1% IPTG was added as an inducer. Throughout the fermentation process, sterile 10% ammonia and 5% dilute sulfuric acid were used to maintain a stable pH of 7.15±0.05. After the fermentation was cultured for 12 hours, samples were taken every hour and the cell density OD of the fermentation process was measured at 600 nm. 600 Values and hBAT8 enzyme activity.

[0096] After 14 hours of fermentation, the test results of the fermentation seed liquid were OD = 10.5 and hBAT8 enzyme activity = 2.3 U / mL, which met the usage standards. Then the following process was carried out:

[0097] like Figure 2 As shown, a fermentation method for conjugated bile acid comprises:

[0098] S1. The pH of the fermentation seed solution containing the recombinant E. coli engineered bacteria was lowered to 6.2-6.5, and then 300 mL of a sterile 10% calcium chloride solution was added to the fermenter at one time and mixed to obtain a mixed bacterial solution;

[0099] S2. The sorbitan bile acid ester, sodium taurate and water are mixed to obtain a mixed raw material;

[0100] S3. The mixed raw material was added to the mixed bacterial solution at a rate of 20 mL / h to obtain a fermentation raw material;

[0101] S4. Half an hour after the addition of step S3 is completed, the cyclic electric shock is turned on, and the fermentation raw material is fermented under the conditions of the cyclic electric shock to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain a fermentation broth; wherein the cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a non-electric shock segment;

[0102] S5. The fermentation broth is filtered to obtain a filtrate;

[0103] S6. The filtrate is dialyzed to obtain tauroursodeoxycholic acid.

[0104] The duration of a single electric shock segment is 10 minutes, and the voltage of a single electric shock segment is -2kV to 2kV.

[0105] A single cycle of the sinusoidal voltage is 10ms.

[0106] The interval time is 60 minutes.

[0107] The mass ratio of sorbitan bile acid ester, sodium taurate and water is 16:6:100.

[0108] like Figure 3 As shown, a fermentation device for conjugated bile acid comprises:

[0109] Fermentation tank 1;

[0110] The circulating electric shock part includes a circulating pipe 2 and an electric shock sleeve 3. The electric shock sleeve 3 is arranged on the surface of the circulating pipe 2. The feed port of the circulating pipe 2 is connected to the bottom discharge port of the fermentation tank 1, and the discharge port of the circulating pipe 2 is connected to the top feed port of the fermentation tank 1. The electric shock sleeve 3 is used to perform circulating electric shock on the circulating pipe 2.

[0111] A timing valve 4 is provided at the bottom of the fermentation tank 1 , and the timing valve 4 is used to periodically discharge the fermentation liquid; the opening and closing time difference of the timing valve 4 is the same as the working time of the electric shock sleeve 3 .

[0112] The circulating electric shock unit further includes a feeding tank 5 , which is connected to the circulating pipeline 2 and is arranged downstream of the electric shock sleeve 3 .

[0113] At this time, the opening and closing time of the timing valve 4 and the working time of the electric shock sleeve 3 are both 10 minutes apart.

[0114] Example 2

[0115] Based on the content disclosed in Example 1, the following modifications are further made:

[0116] The duration of a single shock session was 5 minutes.

[0117] At this time, the opening and closing time of the timing valve 4 and the working time of the electric shock sleeve 3 are both 5 minutes apart.

[0118] Example 3

[0119] Based on the content disclosed in Example 1, the following modifications are further made:

[0120] The duration of a single shock session was 7 minutes.

[0121] At this time, the opening and closing time of the timing valve 4 and the working time of the electric shock sleeve 3 are both 5 minutes apart.

[0122] Comparative Example 1

[0123] Based on the content disclosed in Example 1, the following modifications are further made:

[0124] Do not use cyclic shock.

[0125] Comparative Example 2

[0126] Based on the content disclosed in Example 2, the following modifications are further made:

[0127] The voltage of a single electric shock segment is -3kV to 3kV.

[0128] Comparative Example 3

[0129] Based on the content disclosed in Example 2, the following modifications are further made:

[0130] The voltage of a single electric shock segment is -1kV to 1kV.

[0131] Related experiments and effect data:

[0132] 1. The potential of the fermentation liquid at the outlet of the circulation pipeline of Example 1 was calculated. The results are as follows: Figure 5 This indicates that the cyclic electric shock did generate an induced electric field in the fermentation broth.

[0133] 2. After 24 h and 36 h of fermentation culture in each example and comparative example, the cell density OD value and hBAT8 enzyme activity of the fermentation broth were detected, and the final conversion rate (yield) of tauroursodeoxycholic acid after 36 h was also detected. The results are shown in Table 1.

[0134] Table 1 Results of fermentation culture of various examples and comparative examples

[0135]

[0136] As can be seen from Table 1, the electric shock segments of the various embodiments after cyclic electric shocks of different times have the advantages of promoting the entry of raw materials and the release of products and relieving feedback inhibition, and effectively improving the conversion rate of tauroursodeoxycholic acid produced by the recombinant Escherichia coli engineered bacteria to 90% or more. This shows that the fermentation method of a conjugated bile acid provided in the embodiments of the present application can effectively improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid.

[0137] In addition, although Comparative Example 1 does not use the cyclic electric shock operation, it can promote the normal catalytic production of tauroursodeoxycholic acid products by the recombinant Escherichia coli engineered bacteria, but its conversion rate is low; in addition, Comparative Example 2 uses a higher voltage in the electric shock section, which may cause partial death of the recombinant Escherichia coli engineered bacteria, affecting the process of catalytic production of tauroursodeoxycholic acid by the recombinant Escherichia coli engineered bacteria; in addition, Comparative Example 3 uses a lower voltage in the electric shock section, which can partially improve the catalytic performance of the recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid, but the improvement effect is not obvious, and the conversion rate of tauroursodeoxycholic acid cannot reach 90% or above.

[0138] In summary, the embodiments of the present application provide a fermentation method for conjugated bile acids, which effectively improves the catalytic performance of recombinant Escherichia coli engineered bacteria in producing tauroursodeoxycholic acid by increasing cell permeability with a calcium chloride solution, promoting the entry of raw materials and product release through cyclic electric shock, and relieving feedback inhibition.

[0139] In addition, the present application provides a method for fermenting conjugated bile acids, which can maximize the activity of the hBAT8 enzyme catalyzed by recombinant Escherichia coli engineered bacteria to produce tauroursodeoxycholic acid to 33.7 U / mL, and can convert 21.8 g / L of raw materials after 36 hours of fermentation culture, ultimately making the conversion rate of tauroursodeoxycholic acid reach more than 90%.

[0140] In addition, the embodiment of the present application provides a conjugated bile acid fermentation system, which can be formed by simply modifying a traditional fermentation tank, with low overall modification cost and simple operation.

[0141] The foregoing is merely a detailed description of the present invention, intended to enable those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein, but rather is intended to conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A fermentation method for conjugated bile acid, comprising: Mixing the calcium chloride solution and the fermentation seed liquid containing the recombinant Escherichia coli engineered bacteria to obtain a mixed bacterial liquid; mixing sorbitan bile acid ester, sodium taurate and water to obtain a mixed raw material; mixing the mixed bacterial liquid and the mixed raw material to obtain a fermentation raw material; The fermentation raw material is fermented under cyclic electric shock conditions to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain fermentation liquid; wherein the cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a non-electric shock segment.

2. The fermentation method according to claim 1, wherein the duration of a single electric shock segment is 5 min to 10 min, and the voltage of a single electric shock segment is -2 kV to 2 kV. The fermentation method according to claim 1 , wherein a single cycle of the sinusoidal voltage is 5 ms to 20 ms. The fermentation method according to claim 1 , wherein the intermittent period is 15 to 60 minutes. The fermentation method according to claim 1 , wherein the enzyme activity of the fermentation seed liquid is ≥2.3 U / mL.

6. The fermentation method according to claim 1, wherein the mass m1 of the sorbitan bile ester, the mass m2 of the sodium taurate, and the mass m3 of the water satisfy the relationship: m1:m2:m3=(5-20):(5-20):(78-100).

7. The fermentation method according to claim 1, wherein the fermentation raw material is fermented under cyclic electric shock conditions to activate the recombinant Escherichia coli engineered bacteria in the fermentation raw material to obtain a fermentation liquid; wherein, The cyclic electric shock includes multiple electric shock segments and multiple intermittent segments, the voltage of the electric shock segment is a sinusoidal voltage, and the intermittent segment is a non-electric shock segment, and then includes: The fermentation liquid is centrifuged to obtain tauroursodeoxycholic acid; wherein the centrifugation temperature is 0°C to 15°C, the centrifugation speed is 6000rpm to 15000rpm, and the centrifugation time is 5min to 20min.

8. A fermentation device for conjugated bile acid, comprising: Fermentation tank (1); A circulating electric shock unit comprises a circulating pipe (2) and an electric shock sleeve (3), wherein the electric shock sleeve (3) is arranged on the surface of the circulating pipe (2), the feed port of the circulating pipe (2) is connected to the bottom discharge port of the fermentation tank (1), and the discharge port of the circulating pipe (2) is connected to the top feed port of the fermentation tank (1), and the electric shock sleeve (3) is used to perform cyclic electric shock on the circulating pipe (2).

9. The fermentation equipment according to claim 8, wherein a timing valve (4) is provided at the bottom of the fermentation tank (1), and the timing valve (4) is used to periodically discharge the fermentation liquid; the opening and closing time difference of the timing valve (4) is the same as the working time of the electric shock sleeve (3).

10. The fermentation equipment according to claim 8, wherein the circulating electric shock part further comprises a feeding tank (5), the feeding tank (5) is connected to the circulating pipeline (2), and the feeding tank (5) is arranged downstream of the electric shock sleeve (3).

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