Preparation method of combined bile acid for feed

Abstract

The invention discloses a preparation method of combined bile acid for feed, and the method comprises the following steps: S1, providing a catalyst which is whole cells of clostridium perfringens or bile salt hydrolase extracted from the whole cells of clostridium perfringens; s2, constructing a reaction system, and adding a bile acid substrate, an amino acid substrate and the catalyst into a buffer system with the pH value of 5.0-5.6; s3, under an anaerobic or micro-aerobic condition, carrying out a catalytic reaction at 35-40 DEG C to obtain a reaction solution containing the bound bile acid; and S4, carrying out post-treatment on the reaction liquid to obtain the combined bile acid product for the feed. Whole cells of clostridium perfringens or bile salt hydrolase (BSH / T) extracted from the clostridium perfringens are adopted as a catalyst, the catalyst has high catalytic activity and specificity in an acidic buffer system with the pH value of 5.0-5.6, the binding reaction of bile acid and various non-glycine / taurine amino acids can be efficiently catalyzed, the reaction conditions are mild, strong acid, strong base or high temperature and high pressure are not needed, and the method is suitable for industrial production. The method is suitable for large-scale production.

Description

Technical Field

[0001] This application belongs to the field of conjugated bile acid preparation technology, specifically relating to a method for preparing feed-grade conjugated bile acids. Background Technology

[0002] Conjugated bile acids are a class of bioactive molecules with important physiological functions in animals. They are mainly composed of bile acids linked to certain amino acids (such as glycine and taurine) via amide bonds. Their application in animal feed has received widespread attention, especially in improving fat digestion and absorption, enhancing intestinal health, and inhibiting the growth of pathogenic bacteria, demonstrating significant effects.

[0003] Currently, the production of conjugated bile acids for feed mainly relies on chemical synthesis or extraction from animal bile, which suffers from problems such as complex processes, high costs, limited product variety, and limited functionality. Chemical synthesis methods typically require strong acids and alkalis or high-temperature and high-pressure conditions, which are not only energy-intensive but may also introduce harmful residues. Natural extraction methods are limited by the source and purity of raw materials, making large-scale production difficult. In addition, traditional conjugated bile acids are mainly limited to glycine or taurine conjugated forms, and their antibacterial activity and digestive aid functions are still insufficient under certain conditions, making it difficult to meet the demands of modern animal husbandry for efficient, safe, and multifunctional feed additives.

[0004] Therefore, developing a simple, mild, and functionally diverse method for preparing feed-grade conjugated bile acids has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] This application provides a method for preparing feed-grade conjugated bile acids, aiming to solve the problems existing in the prior art.

[0006] A method for preparing feed-grade conjugated bile acids, the method comprising: S1: Provide a catalyst, wherein the catalyst is a whole cell of Clostridium perfringens or a bile salt hydrolase extracted therefrom; S2: Construct a reaction system by adding bile acid substrate, amino acid substrate, and the catalyst to a buffer system with a pH of 5.0-5.6; The amino acid substrate is an amino acid other than glycine and taurine, and the molar percentage of hydrophobic amino acids in the amino acid substrate is not less than 50%. S3: Under anaerobic or microaerobic conditions, a catalytic reaction is carried out at 35-40℃ to obtain a reaction solution containing conjugated bile acids; S4: Post-process the reaction solution to obtain a feed-grade conjugated bile acid product.

[0007] Optionally, the pH of the catalytic reaction is 5.3.

[0008] Optionally, in S2, the hydrophobic amino acid is selected from one or more of valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and proline; In the amino acid substrate, the molar percentage of hydrophobic amino acids is 70%-95%.

[0009] Optionally, in S2, the bile acid substrate is selected from one or more of cholic acid, chenodeoxycholic acid, and deoxycholic acid, in their free or pharmaceutically acceptable salt form. The concentration of the bile acid substrate in the reaction system is 10-100 mM.

[0010] Optionally, in step S2, the molar ratio of the bile acid substrate to the amino acid substrate is 1:1 to 1:5.

[0011] Optionally, in step S2, the catalyst is wet cells of Clostridium perfringens, and the amount added is 5-50 g / L on a wet weight basis.

[0012] Optionally, the catalyst is a BSH / T enzyme preparation, and its addition amount makes the enzyme activity in the reaction system 1-10 U / mL, where 1 U is defined as the amount of enzyme produced per minute under the conditions of pH 5.3 and 37°C. The amount of enzyme required for conjugated bile acids.

[0013] Optionally, in step S3, the catalytic reaction is carried out in a batch feeding manner: when the concentration of bile acid substrate in the reaction system is monitored to drop to 20%-40% of its initial concentration, bile acid substrate and amino acid substrate are added to the system.

[0014] Optionally, in step S4, the post-processing includes: heat-treating the reaction solution to terminate the reaction, and after solid-liquid separation, purifying, concentrating, and drying the supernatant; The purification was carried out using macroporous adsorption resin, with gradient elution using an ethanol aqueous solution with a concentration of 20%-80%.

[0015] Compared with the prior art, this application has at least the following beneficial effects: This application uses whole cells of Clostridium perfringens or bile salt hydrolase (BSH / T) extracted from it as a catalyst. It has high catalytic activity and specificity in an acidic buffer system with pH 5.0–5.6. It can efficiently catalyze the combination reaction of bile acids with a variety of non-glycine / taurine amino acids. The reaction conditions are mild and do not require strong acids or bases or high temperature and pressure, making it suitable for large-scale production.

[0016] This application also limits the molar percentage of hydrophobic amino acids (such as valine, leucine, phenylalanine, etc.) in the amino acid substrate to not less than 50%, preferably 70%–95%, so that the synthesized conjugated bile acids have stronger hydrophobicity and biomembrane penetration ability, thereby significantly improving their antibacterial activity and digestive function, which is superior to traditional glycine or taurine conjugated products.

[0017] The reaction can be carried out under anaerobic or microaerobic conditions to avoid oxidation side reactions; the batch feeding method can maintain the substrate concentration in the reaction system and improve the conversion rate and product yield; the post-treatment uses gradient elution with macroporous adsorption resin, which has good purification effect and is suitable for preparing feed grade products. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart illustrating a method for preparing feed-grade conjugated bile acids, provided as an embodiment of this application. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0020] This application provides a method for preparing conjugated bile acids for animal feed, comprising the following steps: First, a catalyst is provided, wherein the catalyst is the whole cell of Clostridium perfringens or bile salt hydrolase / transferase (BSH / T) extracted from it. Specifically, Clostridium perfringens can be a standard preserved strain, such as ATCC13124.

[0021] The strain was cultured in an enzyme-enhanced medium. This medium contained: 10-30 g / L tryptone, 10-20 g / L yeast extract, 3-8 g / L glucose, 3-8 g / L sodium chloride, 0.5-1.5 g / L L-cysteine ​​hydrochloride, and 0.3-1.0 g / L bile acid salts (such as sodium taurocholate) as an inducer. The initial pH of the medium was adjusted to 6.8-7.4. Culture was carried out under strictly anaerobic conditions, for example, using a medium filled to 85% capacity. 10% 5% The anaerobic workstation with mixed gas was incubated at 35-38℃ for 12-18 hours until the optical density of the culture medium at a wavelength of 600nm reached 1.0-1.6, which is the late logarithmic growth stage. The catalyst can be used in whole-cell or enzyme preparation form, wherein: The whole-cell catalyst is prepared by collecting the bacterial cells after culturing (e.g., 5000-8000×g, 10-15 minutes, 4-10℃). The collected wet bacterial cells are washed 1-3 times with a buffer solution (e.g., 0.05-0.15M sodium citrate buffer) with a pH of 5.0-5.6, preferably 5.3. The resulting wet bacterial sludge can be used directly as a catalyst. For ease of storage and transportation, the washed wet bacterial sludge can also be mixed with a protectant (e.g., 5-15% trehalose or glycerol), freeze-dried to prepare dry bacterial powder, and reconstituted with the appropriate buffer solution before use. The enzyme preparation catalyst is prepared by disrupting the wet bacterial cells obtained above using ultrasonic disruption, high-pressure homogenization, or enzymatic hydrolysis. After centrifugation to remove cell debris, a crude enzyme solution is obtained. For further purification, the crude enzyme solution can be subjected to ammonium sulfate fractionation precipitation. For example, the precipitate in the 30%-70% saturation range can be collected, and after reconstitution, a partially purified enzyme solution can be obtained. Alternatively, further purification can be carried out using methods such as anion exchange chromatography (e.g., DEAE-Sepharose medium) to obtain an enzyme preparation with significantly improved specific activity, which can be used in applications where high product purity is required. Subsequently, a reaction system was constructed, with bile acids and amino acids as reaction substrates. The bile acid substrates were selected from one or more of cholic acid, chenodeoxycholic acid, and deoxycholic acid, and could be used in the form of their free acid or pharmaceutically acceptable salts (such as sodium salts or potassium salts). The final concentration range in the reaction system was 10-100 mM. The amino acid substrate is an amino acid other than glycine and taurine, selected from one or more of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and proline. To impart enhanced antibacterial activity to the product, the molar percentage of hydrophobic amino acids (valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and proline) in the amino acid substrate is not less than 50%, preferably 70%-95%, and the molar ratio of bile acid substrate to amino acid substrate can be in the range of 1:1 to 1:5. The following provides specific examples. Combination 1 uses sodium deoxycholate (40-60mM) as a bile acid substrate and an equimolar mixture of L-leucine and L-phenylalanine (total molarity 2-4 times that of bile acids) as an amino acid substrate. Combination 1 has an antibacterial focus. Combination 2 uses a mixture of sodium cholate and sodium chenodeoxycholate (molar ratio 1:1, total concentration 30-50mM) as bile acid substrates and a mixture of L-methionine, L-valine and L-alanine in a molar ratio of 5:3:2 (total molar number 2-4 times that of bile acids) as amino acid substrates. Combination 2 has a more balanced function. Simultaneously, reaction conditions need to be controlled, including pH and buffer system, anaerobic environment and catalyst dosage; Specifically, the reaction is carried out under acidic conditions, using a buffer system with a pH of 5.0-5.6 to maintain the acidity of the reaction solution, with pH 5.3 being the optimal choice. Suitable buffers include citrate-phosphate buffer, sodium citrate buffer, etc., with concentrations typically ranging from 20-100 mM. Furthermore, to maintain optimal catalyst activity, the reaction should be carried out in an anaerobic or slightly aerobic environment. This can be achieved by introducing an inert gas (such as nitrogen or argon) into the reaction vessel to replace the oxygen, or by adding an appropriate reducing agent (such as 0.01%-0.05% sodium mercaptoacetate). When using whole-cell catalysts, the addition amount, based on wet cell weight, can be 5-50 g / L (reaction volume); when using enzyme preparations, the addition amount should ensure that the BSH / T enzyme activity in the reaction system reaches 1-10 U / mL. Here, enzyme activity unit (U) is defined as: the amount of 1 unit of enzyme activity produced per minute at pH 5.3 and 37℃. The amount of enzyme required for conjugated bile acids; Simultaneously, the progress of the catalytic reaction needs to be monitored. The constructed reaction system is placed at 35-40℃ and the catalytic reaction is carried out with stirring. The reaction time is usually 2-12 hours. Under these conditions, the reaction usually reaches a high conversion rate within 4-6 hours. To improve substrate utilization and final yield, a batch feeding strategy can be adopted. That is, during the reaction, when the concentration of bile acid substrate is monitored to drop to 20%-40% of the initial concentration, a certain amount of bile acid and amino acid substrate is added to the system to continue the reaction. The reaction process can be monitored using methods conventional in the art; For example, thin-layer chromatography can be used for rapid monitoring: using a silica gel plate with an appropriate developing solvent system (such as chloroform:methanol:acetic acid:water = 60-70:20-30:3-5:3-5 volume ratio), the extent of the reaction can be estimated by observing the changes in the substrate spots and product spots; For precise quantification, high performance liquid chromatography (HPLC) can be used: a reversed-phase C18 column is used, with acetonitrile and an aqueous solution containing a small amount of acid (such as 0.1% formic acid) as the mobile phase for gradient elution, and a UV detector or evaporative light scattering detector is used to quantitatively analyze substrate consumption and product formation. Finally, a reaction solution containing conjugated bile acids was obtained; After the reaction is completed, the reaction solution is heat-treated (e.g., 80-90℃ for 10-20 minutes) to terminate the reaction and inactivate any bacteria that may be present. Then, the solid matter is removed by centrifugation or filtration to obtain a clear reaction supernatant. To obtain feed-grade product, the supernatant can be purified. A preferred method is to use macroporous adsorption resin for adsorption and elution. For example, the supernatant is passed through an HPD-100 macroporous adsorption resin column at a certain flow rate. First, it is washed with water to remove water-soluble impurities. Then, it is eluted with ethanol-water solutions of increasing concentration (e.g., 20-40%, 40-60%, and 60-80% ethanol-water solutions in sequence). The elution fraction rich in target bound bile acids (usually mainly the 40-80% ethanol elution fraction) is collected. The collected target fractions are combined and concentrated under reduced pressure to a suitable consistency at an appropriate temperature (e.g., ≤65°C). Then, it is dried by spray drying or other means to finally obtain a light yellow to light brown powder product. The obtained product was analyzed by liquid chromatography-mass spectrometry and its main components were confirmed to be one or more novel conjugated bile acids formed by the combination of the bile acids and the amino acids through amide bonds. The novel conjugated bile acid has the following characteristic functional indicators: For antibacterial activity, the micro-broth dilution method should be used for determination. The minimum inhibitory concentration (MIC) of the novel conjugated bile acid against common intestinal pathogens (such as Escherichia coli K88, Salmonella ATCC14028, and Clostridium perfringens) is lower than the MIC value of sodium glycocholate or sodium taurocholate at the same molar concentration under the same test conditions. Regarding its digestive activity, in an in vitro simulated digestion model (such as a system containing pancreatic lipase and a pH of approximately 6.5), the novel conjugated bile acid can enhance the hydrolysis rate of fat emulsions (such as lard emulsions), and its effect is superior to that of conventional conjugated bile acids of the same concentration.

[0022] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A method for preparing a feed conjugated bile acid, characterized by, The method comprises: S1: providing a catalyst, which is a whole cell of Clostridium perfringens or a bile salt hydrolase extracted therefrom; S2: constructing a reaction system, adding a bile acid substrate, an amino acid substrate and the catalyst in a buffer system with a pH of 5.0-5.6; wherein the amino acid substrate is an amino acid other than glycine and taurine, and the molar proportion of hydrophobic amino acids in the amino acid substrate is not less than 50%; S3: performing a catalytic reaction under anaerobic or micro-aerobic conditions at 35-40°C to obtain a reaction solution containing conjugated bile acids; S4: post-treating the reaction solution to obtain a feed conjugated bile acid product.

2. The method for preparing a feedable conjugated bile acid according to claim 1, characterized by, The pH of the catalytic reaction is 5.

3.

3. The method for preparing a feedable conjugated bile acid according to claim 1, characterized by, In S2, the hydrophobic amino acid is selected from one or more of valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and proline; In the amino acid substrate, the molar proportion of hydrophobic amino acids is 70%-95%.

4. The method for preparing a feedable conjugated bile acid according to claim 1, characterized by, In S2, the bile acid substrate is selected from one or more of cholic acid, chenodeoxycholic acid, and deoxycholic acid, in free or pharmaceutically acceptable salt form; The concentration of the bile acid substrate in the reaction system is 10-100 mM.

5. The method for preparing a feedable conjugated bile acid according to claim 1, characterized by, In S2, the molar ratio of the bile acid substrate to the amino acid substrate is 1:1 to 1:

5.

6. The method for preparing a feedworthy conjugated bile acid according to claim 1, characterized by, In S2, the catalyst is a wet cell of Clostridium perfringens, and the addition amount is 5-50 g / L by wet weight.

7. The method for preparing a feedworthy conjugated bile acid according to claim 1, characterized by, The catalyst is BSH / T enzyme preparation, and the added amount is such that the enzyme activity in the reaction system is 1-10 U / mL, wherein 1 U is defined as the amount of enzyme that catalyzes the production of 1 Enzyme amount required for conjugated bile acids.

8. The method for preparing a feedworthy conjugated bile acid according to claim 1, characterized by, In S3, the catalytic reaction is performed in a fed-batch manner: when it is monitored that the concentration of the bile acid substrate in the reaction system decreases to 20%-40% of the initial concentration, the bile acid substrate and the amino acid substrate are added to the system.

9. The method for preparing feed-grade conjugated bile acids according to claim 1, characterized in that, In S4, the post-treatment comprises: heat-treating the reaction solution to terminate the reaction, after solid-liquid separation, purifying, concentrating and drying the supernatant; The purification is performed using a macroporous adsorption resin, and gradient elution is performed using an ethanol aqueous solution with a concentration of 20%-80%.

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