Preparation method of high-quality chicken gallbladder paste

Abstract

This invention relates to the field of natural product raw material preparation technology, specifically to a method for preparing high-quality chicken bile extract. In this technical solution, chicken bile is concentrated to obtain a first concentrated material. This first concentrated material is then extracted with diethyl ether, and the aqueous phase is separated. The aqueous phase is concentrated to obtain a second concentrated material, which is mixed with anhydrous ethanol, allowed to stand, and then filtered to obtain the supernatant. The supernatant is then concentrated to obtain chicken bile extract. The chicken bile extract prepared by this method can have a taurine chenodeoxycholic acid content of up to 50% or more. This solution can solve the technical problems of high impurity content and unsatisfactory taurine chenodeoxycholic acid content in existing chicken bile extracts. This technical solution, through ether extraction and ethanol precipitation, increases the content of the active ingredient in chicken bile extract, thereby improving the quality of the chicken bile extract, reducing subsequent processing steps, and meeting market demand for high-quality chicken bile extract, thus having ideal application and promotion prospects.

Description

Technical Field

[0001] This invention relates to the field of natural product raw material preparation technology, specifically to a method for preparing high-quality chicken bile extract. Background Technology

[0002] Chicken bile extract is a major raw material for extracting taurine chenodeoxycholic acid (TCDCA) or producing chenodeoxycholic acid (CDCA), etc., and has enormous market value. It is an important industrial and pharmaceutical raw material. Currently available chicken bile extracts are mainly made by melting and breaking frozen chicken bile, filtering out the bile, and concentrating it. Chicken bile extract obtained by directly concentrating bile contains a large amount of impurities such as protein, fat, nucleic acids, and inorganic salts, which greatly hinders the subsequent purification of the active ingredients, making it difficult to meet the purity standards of the final product and significantly increasing production costs. Therefore, high-quality chicken bile extract with fewer impurities is in high demand and short supply in the market.

[0003] Current technologies mostly employ ethyl acetate extraction to process bile, which is then concentrated to produce chicken bile extract. However, the yield, content, and purity of taurine chenodeoxycholic acid in chicken bile extract obtained through ethyl acetate extraction have certain shortcomings. There is an urgent need to develop a new method for purifying poultry bile to improve the quality of chicken bile extract, reduce subsequent processing steps, and meet market demand for high-quality chicken bile extract. Summary of the Invention

[0004] The present invention aims to provide a method for preparing high-quality chicken bile paste, so as to solve the technical problems of high impurity content and unsatisfactory content of the active ingredient taurine chenodeoxycholic acid in the existing chicken bile paste.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing high-quality chicken bile extract, which is used to enrich bile acids in chicken bile extract; concentrating chicken bile to obtain a first concentrated material, extracting the first concentrated material with ether, separating the phases and taking the aqueous phase; concentrating the aqueous phase to obtain a second concentrated material, mixing it with anhydrous ethanol, allowing it to stand, filtering and taking the supernatant; concentrating the supernatant to obtain chicken bile extract.

[0007] This technical solution also provides a method for preparing high-quality chicken bile paste.

[0008] Furthermore, the volume ratio of ether to chicken bile is 1:1.

[0009] Furthermore, the volume ratio of anhydrous ethanol to chicken bile is 1:1.

[0010] Furthermore, the volume of the first concentrated material is one-quarter that of chicken bile.

[0011] Furthermore, the volume of the second concentrated material is one-fifth that of chicken bile.

[0012] Furthermore, after mixing the second concentrated material with anhydrous ethanol, it is allowed to stand at 10°C for 10-12 hours.

[0013] Furthermore, the chicken bile is obtained by the following method: take a frozen chicken gallbladder, cut it into 3-5 mm thin slices; dissolve it at a temperature of 4-10℃, and then filter to obtain the clear liquid to obtain chicken bile.

[0014] Furthermore, the bile acid is mainly composed of TCDCA.

[0015] Furthermore, the TCDCA content in chicken bile extract is >50%.

[0016] The technical principle and beneficial effects of this technical solution are as follows:

[0017] The main component of chicken bile is taurine chenodeoxycholic acid (approximately 50%), along with other bile acids (approximately 20%), lipids (approximately 20%), proteins, nucleic acids, amino acids, and inorganic salts. Bile acids (including taurine chenodeoxycholic acid and other bile acids) are the target active ingredient; other substances (including lipids) need to be removed to improve the quality of the chicken bile extract. This invention employs an extraction method. Ether and water are immiscible, and most lipids are soluble in ether but not in water. The bile acids we need are readily soluble in water but insoluble in ether. A certain amount of ether is added to the bile, mixed thoroughly, and after the ether and water separate into layers, the lower aqueous phase is removed, effectively removing most of the lipids and other impurities. Then, proteins and other substances in the chicken bile are removed by ethanol precipitation, ultimately obtaining chicken bile extract. The chicken bile extract obtained by this method has a high content of active ingredients, effectively improving the quality of the intermediate product and enhancing its market competitiveness.

[0018] In this technical solution, the selection of diethyl ether is crucial for effectively removing impurities. Most industrial extraction processes use ethyl acetate, but in practical applications for purifying chicken bile, the inventors found that ethyl acetate extraction was not ideal. They actively explored other extraction solvents and ultimately found diethyl ether, which significantly improved extraction efficiency compared to other organic solvents. In the purification of chicken bile extract, diethyl ether offers the advantages of higher purity and minimal loss of effective substances after extraction.

[0019] In this technical solution, concentrating the bile to 1 / 4 of its original volume before adding ether extraction allows for more complete extraction, extracting substances more suited to the organic phase. It also effectively removes substances soluble in both the aqueous and organic phases, resulting in higher purity of TCDCA, which is insoluble in the organic phase. After extraction and before alcohol precipitation, the ether in the aqueous phase needs to be evaporated and removed. Ether remaining in the aqueous phase can affect the alcohol precipitation process and product quality. Furthermore, the order of ether and ethanol treatment significantly impacts the final product quality. Through extensive research, the inventors have established the most effective process for processing chicken bile, effectively improving product quality.

[0020] In summary, ether extraction significantly improves the purity and yield of taurine chenodeoxycholic acid. This technical solution, through ether extraction and alcohol precipitation, increases the content of active ingredients in chicken bile extract, thereby improving its quality, reducing subsequent processing steps, and meeting market demand for high-quality chicken bile extract. This technical solution provides a simple treatment of chicken bile to obtain high-quality raw material chicken bile extract, enhancing our company's competitive advantage among similar raw material pharmaceutical manufacturers. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0022] Example 1:

[0023] (1) Chicken bile extraction

[0024] Chicken bile jelly is made by quick-freezing the gallbladder after slaughtering and removing the internal organs at the slaughterhouse. The chicken bile jelly is sliced ​​into 3-5mm thin slices using a slicer. The slices are then transferred to a thawing tank, which is jacketed with steam (or hot water, or circulating water), and the temperature inside the tank is controlled at 4-10℃. Once the chicken bile jelly slices have partially melted, stirring is started until completely melted. The melted liquid is then filtered through a drum sieve, and the clear liquid after filtration is taken as chicken bile.

[0025] (2) Chicken bile extraction

[0026] Chicken bile was concentrated to 1 / 4 volume using conventional vacuum concentration (80℃, -0.09MPa). The concentrated bile was then transferred to an extraction tank, and an equal volume of ether was added to the original bile (e.g., if the supernatant contained 4000L of bile, it was concentrated to 1000L, and then 4000L of ether was added). After stirring for 1 hour, the mixture was allowed to stand for 1 hour. The lower aqueous phase was then discharged through the bottom outlet. The mixture was then concentrated under reduced pressure (40℃, -0.09MPa) to 1 / 5 volume of the original bile, removing the ether from the aqueous phase. An equal volume of ethanol was added to the original bile, the temperature was lowered to 10℃, and the mixture was stirred until homogeneous. The mixture was then allowed to stand overnight (10-12 hours), filtered to remove the alcohol precipitate, and the supernatant was concentrated under reduced pressure (40℃, -0.09MPa) to obtain chicken bile extract at 1 / 5 volume of the original bile. This extract was then packaged into 10kg bags and stored at -18℃.

[0027] The main components of chicken bile include taurine chenodeoxycholic acid (approximately 50%), along with other bile acids (approximately 20%), lipids (approximately 20%), proteins, nucleic acids, amino acids, and inorganic salts. Bile acids (including taurine chenodeoxycholic acid and other bile acids) are the desired active ingredients, while lipids are the largest impurities that need to be removed. This invention employs an extraction method. Organic solvents are immiscible with water, and most lipids are soluble in organic solvents but not in water. The bile acids we need are readily soluble in water but insoluble in organic solvents. A certain amount of organic solvent is added to concentrated chicken bile, mixed thoroughly, and after the organic solvent and water separate into layers, the lower aqueous phase is collected, effectively removing most of the lipids. Through extensive research, the inventors discovered that using diethyl ether as an organic solvent significantly improves the purification and extraction effect compared to other solvents (e.g., conventional organic solvents such as ethyl acetate and petroleum ether). Then, ethanol is used to treat the chicken bile, precipitating proteins and other impurities, thus improving the quality of the chicken bile extract.

[0028] Experimental Example 1: Comparison of Extraction Effects of Different Extractants

[0029] Prepare multiple 100ml portions of chicken bile (preparation process as described in Example 1), and concentrate each portion to 25ml using vacuum concentration. The control group was treated without any organic solvent. After stirring for 1 hour and standing for 1 hour, the bile was dried into powder (drying at 80°C until moisture content ≤5%), weighed, and the TCDCA content was determined. The experimental group was treated with an equal volume of organic solvent to extract the chicken bile. After stirring for 1 hour and standing for 1 hour, the lower aqueous phase was collected, dried into powder, weighed, and the TCDCA content was determined. The specific organic solvents used are detailed in Table 1. Each experimental and control group was repeated three times; Table 1 shows the average values ​​of the three repeated experiments.

[0030] Table 1: Comparison of extraction effects of different extractants (TCDCA recovery rate = mass of TCDCA in experimental group / mass of TCDCA in control group × 100%; TCDCA mass = chicken bile powder × TCDCA content; chicken bile powder and TCDCA content are the average values ​​of three experiments)

[0031]

[0032] The above experimental data demonstrate that using diethyl ether as the extraction solvent yields the best extraction effect for chicken bile, resulting in higher content of active ingredients and the highest recovery rate. Ethyl acetate, petroleum ether, diethyl ether, chloroform, dichloromethane, tert-butyl methyl ether, and n-hexane are all conventional organic solvents in existing technologies, capable of removing lipids from materials through the principle of "like dissolves like." In this technical solution, the extraction process not only needs to fully remove lipids but also needs to minimize the loss of bile acids (especially TCDCA). Therefore, no existing technologies have reported which solvents can achieve the above-mentioned effects. Regarding the TCDCA content in the obtained chicken bile powder, using diethyl ether as the extraction solvent resulted in a TCDCA content as high as 50.25%, which is 7%-10% higher than other experimental groups and the control group, a significant increase that the inventors did not anticipate before testing. Therefore, using diethyl ether can maintain a good balance between lipid removal and bile acid retention, ensuring a TCDCA recovery rate of over 93%.

[0033] Experimental Example 2: Comparison of Extraction Effects with Different Amounts of Ether

[0034] Prepare four 100ml portions of chicken bile, concentrate each to 25ml, and add 200ml, 100ml, 50ml, and 25ml of ether respectively. Stir for 1 hour, transfer each portion to a separatory funnel, let stand for 1 hour, and release the lower aqueous phase. Dry the mixture into powder in the same way as the unextracted sample, weigh it, and determine the content. The control group used was the control group of Experiment 1. The experimental results are shown in Table 2.

[0035] Table 2: Experimental results comparing the extraction effects of different amounts of diethyl ether

[0036]

[0037] The experimental data above show that the more ether used, the higher the content of TCDCA in chicken bile powder can be, but it will also reduce the yield of chicken bile powder (quality of chicken bile powder). Considering cost factors, TCDCA purity and TCDCA recovery rate, the original volume of bile is used for extraction in production, that is, the volume ratio of ether to chicken bile is 1:1.

[0038] Experimental Example 3: The Effect of Pre-extraction Concentration on the Efficacy of Ether Extraction

[0039] Prepare four 100ml portions of chicken bile, concentrate them to 100ml (unconcentrated), 50ml, 25ml, and 15ml respectively, add 100ml of diethyl ether to each, stir for 1 hour, transfer to separatory funnels and let stand for 1 hour, release the lower aqueous phase, dry into powder, weigh, and determine the content. The experimental results are shown in Table 3.

[0040] Table 3: Experimental results showing the effect of pre-extraction concentration on the extraction efficiency of diethyl ether.

[0041] Concentrated volume (compared to the original bile volume) Weighing (g) TCDCA content TCDCA(g) 100％ 7.5 46.56％ 3.49 50％ 7.2 49.67％ 3.58 25％ 7.0 50.23％ 3.52 15％ 6.3 50.82％ 3.20

[0042] The experimental data above show that concentrating the extract to 1 / 4 of its original volume before extraction can balance the cost of ether, the extraction effect, and the recovery rate. Concentrating it to 15% of the original bile volume will make the concentrated paste too viscous, causing too much sticking to the walls and resulting in too much loss.

[0043] Experiment Example 4: Exploration of the Process Sequence for Chicken Bile Paste Preparation

[0044] Method 1: Concentrate 100 mL of chicken bile to 25 mL, then add 100 mL of diethyl ether, stir for 1 hour, and let stand for 1 hour in a separatory funnel. Take the lower aqueous phase and concentrate under reduced pressure to 20 mL. Then add 100 mL of anhydrous ethanol, stir well, and let stand at 10°C for 12 hours. Filter to remove the solid phase, and then concentrate the supernatant under reduced pressure to obtain 20 mL of chicken bile extract. Following the method in Example 1, dry the chicken bile extract into chicken bile powder and test its TCDCA content. This method is a scale-down of the process in Example 1 (parameter settings not mentioned in this method are the same as in Example 1), and is an experimental test before formal large-scale production.

[0045] Method 2: Concentrate 100 mL of chicken bile to 20 mL, then add 100 mL of anhydrous ethanol, mix well, and let stand at 10°C for 12 hours. Filter to remove the solid phase, then concentrate the supernatant under reduced pressure to obtain 25 mL of material. Add 100 mL of diethyl ether to the 25 mL of material, stir for 1 hour, and let stand in a separatory funnel for 1 hour. Take the lower aqueous phase and concentrate under reduced pressure to 20 mL to obtain chicken bile extract. Following the method in Example 1, dry the chicken bile extract into chicken bile powder and determine its TCDCA content.

[0046] Method 3: Basically the same as Method 1, but the method of concentration under reduced pressure after ether extraction has been adjusted. The specific process is as follows:

[0047] 100 mL of chicken bile was concentrated to 25 mL (concentration under reduced pressure, temperature 80 °C, vacuum degree -0.09 MPa), then 100 mL of diethyl ether was added, and the mixture was stirred for 1 hour before being allowed to stand in a separatory funnel for 1 hour. The lower aqueous phase was collected and concentrated under reduced pressure to 20 mL (temperature at room temperature, approximately 25 °C, vacuum degree -0.09 MPa). Then, 100 mL of anhydrous ethanol was added and stirred thoroughly. The mixture was allowed to stand at 10 °C for 12 hours, and the solid phase was removed by filtration. The supernatant was then concentrated under reduced pressure (temperature at room temperature, approximately 25 °C, vacuum degree -0.09 MPa) to obtain 20 mL of chicken bile extract. Following the method in Experimental Example 1, the chicken bile extract was dried into chicken bile powder, and the TCDCA content was determined.

[0048] Table 4: Results of the exploratory experiment on the preparation process sequence of chicken bile paste (using the control in Experiment Example 1 as a reference).

[0049] Experimental Groups Extraction solvent Chicken bile powder (g) TCDCA content TCDCA(g) TCDCA recovery rate Comparison none 9.8 39.08％ 3.83 —— Method 1 Diethyl ether + ethanol 6.3 55.36％ 3.49 91.12％ Method 2 ethanol + diethyl ether 6.8 42.21％ 2.87 74.93％ Method 3 Diethyl ether + ethanol 6.9 51.01％ 3.52 91.90％

[0050] The experimental data above show that, compared with experimental group 3 of Experiment 1 in this experiment, adding ethanol precipitation after ether extraction can further increase the TCDCA content in chicken bile powder, resulting in higher quality chicken bile powder. Furthermore, the order of ether and ethanol treatment has a significant impact on the process effect. The inventors attempted to first use ethanol precipitation to remove impurities such as proteins from the bile, and then use ether to extract lipids. This method is a common operation in the prior art (ethanol precipitation first, i.e., method 2). However, this operation not only reduces the yield of chicken bile powder but also reduces the TCDCA content, leading to a significant decrease in TCDCA recovery rate. In addition, the reduced pressure concentration temperature after ether extraction and the ethanol precipitation step is crucial; if the temperature is too low, the organic solvent may not be fully removed. The main issue is that residual ether affects the subsequent ethanol precipitation operation.

[0051] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing high-quality chicken bile paste, characterized in that, It is used to enrich bile acids in chicken bile extract; chicken bile is concentrated under reduced pressure at a temperature of 80℃ and a vacuum degree of -0.09MPa to obtain a first concentrated material, the volume of which is one-quarter of that of chicken bile. The first concentrated material was extracted with diethyl ether at a volume ratio of 1:1 to chicken bile, and the aqueous phase was collected after phase separation. The aqueous phase was concentrated under reduced pressure at a temperature of 40°C and a vacuum of -0.09 MPa to obtain a second concentrated material, the volume of which was one-fifth that of chicken bile. The second concentrated material was mixed with anhydrous ethanol, with a volume ratio of anhydrous ethanol to chicken bile of 1:

1. The mixture was allowed to stand at 10°C for 10-12 hours, and then filtered to obtain the supernatant. The supernatant was concentrated under reduced pressure at a temperature of 40°C and a vacuum of -0.09MPa to obtain chicken bile paste, the volume of which was one-fifth that of chicken bile. The chicken bile is obtained by the following method: take a frozen chicken gallbladder, cut it into 3-5 mm thin slices; dissolve it at a temperature of 4-10℃, then filter to obtain the clear liquid, thus obtaining chicken bile.

2. The chicken bile paste prepared by the method for preparing a high-quality chicken bile paste according to claim 1.

3. The chicken bile paste according to claim 2, characterized in that, Its TCDCA content is >50%.

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