Preparation process and method of pig prostate extract

Abstract

This invention relates to the field of bio-extraction technology, specifically disclosing a method for preparing porcine prostate extract. The method includes: homogenizing porcine prostate tissue, defatting it using ultrasound combined with activated carbon at 4-10°C, centrifuging to collect the precipitate to obtain defatted tissue; sequentially hydrolyzing the defatted tissue with trypsin and pepsin to obtain an enzymatic hydrolysate; sequentially passing the enzymatic hydrolysate through a combined membrane separation process using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a nanofiltration membrane with a molecular weight cutoff of 1 kDa, collecting the 1-10 kDa molecular weight cutoff; and drying to obtain the final product. This invention utilizes a physical degreasing process combining ultrasound and activated carbon, avoiding organic solvent residue and reducing residual fat to below 2%; the stepwise enzymatic hydrolysis simulates the in vivo digestive environment, achieving a protein hydrolysis degree of 26.5%; and the combined membrane separation precisely enriches 1-10 kDa active peptides, with the target peptide accounting for over 82%, while effectively removing large molecular weight allergenic proteins and endotoxins. The product has high purity and good biosafety, making it suitable for the pharmaceutical and health product fields.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction technology, specifically to a preparation process and method for a porcine prostate extract. Background Technology

[0002] Porcine prostate tissue is rich in active substances such as polypeptides, amino acids, and prostaglandin precursors, and has broad application prospects in the fields of medicine and health products. However, prostate tissue has a high fat content (about 8-12% of wet weight), and traditional water extraction methods cannot effectively remove fat, resulting in the final product being prone to oxidation and rancidity with a fishy odor. Currently, the commonly used degreasing process mostly adopts organic solvent extraction methods, such as dehydration and degreasing with hexane and ethanol, but there are risks of solvent residue and environmental pollution problems.

[0003] A search revealed that, for example, Chinese invention patent CN103255190A discloses a method for extracting small molecule polypeptides and amino acids from animals using a compound enzyme and employing ultrasonic enzymatic hydrolysis-assisted extraction technology. However, this method is mainly used for the efficient dissociation of small molecule polypeptides and does not specifically degrease the fat-rich prostate tissue. As a result, the problem of fat content in the product extract has not been effectively solved.

[0004] In addition, traditional processes generally suffer from problems such as a wide molecular weight distribution of extracted active components and the presence of sensitizing macromolecular proteins, making it difficult to guarantee the biosafety of the product. Existing research on the preparation methods of porcine prostate extracts mostly focuses on the chemical treatment after direct extraction, while systematic processes from defatting and enzymatic hydrolysis to selective purification of target peptides are rarely reported. Therefore, it is necessary to develop a method for preparing porcine prostate extracts that is thoroughly defatted, has high enzymatic hydrolysis efficiency, high purity of target peptides, and no organic solvent residues. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing porcine prostate extract, which has the advantages of thorough degreasing, high enzymatic hydrolysis efficiency, high purity of target peptides, and no organic solvent residue. This method solves the problems of high fat residue, low yield of active ingredients, and poor product purity in traditional processes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing porcine prostate extract, comprising the following steps: Step 1: Take fresh or frozen-thawed pig prostate tissue, remove surface fat and connective tissue, cut it into small pieces and place it in a tissue homogenizer, add 1-3 times the weight of purified water, and homogenize it for 10-30 minutes to obtain tissue homogenate. Step 2: Introduce the tissue homogenate obtained in Step 1 into the ultrasonic degreasing tank through the feed port, add 0.5-2% (w / w) of activated carbon through the activated carbon feed port, start the ultrasonic transducer group, and perform ultrasonic treatment at 4-10℃ by controlling the temperature through the cooling jacket. The power is 200-500W, and the treatment time is 20-60 minutes. Then open the discharge port and centrifuge the tissue to separate the precipitate and obtain the degreased prostate tissue. Step 3: Resuspend the defatted prostate tissue obtained in Step 2 in purified water and transfer it to the enzymatic hydrolysis reactor (11). Adjust the pH to 7.0-8.0 through the pH adjuster tank, add 0.1-0.5% (w / w) of trypsin through the trypsin feed port, and hydrolyze at 35-40℃ for 1-3 hours. Then adjust the pH to 3.0-5.0 through the pH adjuster tank, add 0.1-0.3% (w / w) of pepsin through the pepsin feed port, and continue hydrolyzing at 37-42℃ for 0.5-2 hours to obtain the hydrolysate. Step 4: Remove the residue from the enzymatic hydrolysate obtained in Step 3 by passing it through a plate and frame filter and collect the filtrate; pass the filtrate sequentially through an ultrafiltration membrane module with a molecular weight cutoff of 10 kDa and a nanofiltration membrane module with a molecular weight cutoff of 1 kDa, and collect the cutoff solution in the molecular weight range of 1-10 kDa. Step 5: Transfer the retentate obtained in Step 4 to a freeze dryer for vacuum freeze drying or spray drying to obtain porcine prostate extract powder.

[0007] Preferably, in step two, the frequency of ultrasonic treatment is 20-40kHz, the centrifugal speed of the centrifuge is 4000-8000 rpm, and the centrifugation time is 15-30 minutes.

[0008] Preferably, the activated carbon added in step two has a mesh size of 200-400, and its mass-to-volume ratio (w / v) with the tissue homogenate is 1:50 to 1:100. The activated carbon is added to the ultrasonic degreasing tank through the activated carbon feeding port, and circulating cooling water is circulated in the cooling jacket to maintain the temperature.

[0009] Preferably, in step three, the enzyme activity of trypsin is 2500-4000 USP U / mg, and the enzyme activity of pepsin is 1:12000-1:15000.

[0010] Preferably, in step three, before adjusting the pH for the second enzymatic hydrolysis, the pH of the enzymatic hydrolysate in the enzymatic hydrolysis reactor is adjusted from 7.0-8.0 to 3.0-5.0 using 1M hydrochloric acid through the pH adjuster storage tank (12), and 0.1-0.5M sodium chloride is added when adding pepsin through the pepsin feeding port to maintain ionic strength.

[0011] Preferably, the operating conditions for the combined membrane separation and purification in step four are as follows: the temperature is controlled below 25°C, the operating pressure is 0.2-0.6 MPa, and the membrane flux of the ultrafiltration membrane module and the nanofiltration membrane module is maintained at 20-50 L / (m²·h).

[0012] Preferably, in step four, before passing through the ultrafiltration membrane assembly (16), the filtrate is first centrifuged at a speed of 3000-5000 rpm for 10-20 minutes and pre-filtered using a microfiltration membrane with a pore size of 0.45 μm.

[0013] Preferably, the conditions for vacuum freeze drying in step five are as follows: in the freeze dryer, the pre-freezing temperature is -40°C to -30°C, maintained for 2-4 hours, and the vacuum degree during the sublimation drying stage is 10-30 Pa, the temperature is -20°C to -10°C, and the temperature during the desorption drying stage is 20-30°C.

[0014] Preferably, in the obtained porcine prostate extract, peptides with a content of less than 10 kDa account for more than 85% of the total protein, and the nucleic acid content (measured by absorbance at 260 nm) is less than 0.1%.

[0015] Preferably, the extract contains active peptides with a molecular weight between 1 and 10 kDa, and the total protein content of the extract is not less than 70%, the residual fat content is less than 5%, and the ash content is less than 8%.

[0016] Compared with the prior art, the technical solution of this application has the following beneficial effects: 1. The preparation method of the porcine prostate extract of the present invention adopts a physical combined degreasing technology of "activated carbon adsorption + ultrasonic treatment" in the degreasing step, avoiding the use of organic solvents. Under low temperature conditions of 4-10℃, the residual fat content can be reduced to below 1.8%, which is significantly better than the 7.2% of the traditional water extraction method. It effectively solves the problems of easy oxidation and rancidity and fishy smell of the final product. By adopting a stepwise enzymatic hydrolysis process of trypsin (pH 7.0-8.0) followed by pepsin (pH 3.0-5.0), the digestive environment in vivo is simulated, and the degree of protein hydrolysis reaches 26.5%, which is much higher than the 15% of single enzyme hydrolysis, significantly improving the release efficiency of active peptides.

[0017] 2. The preparation method of the porcine prostate extract of the present invention adopts a combined membrane separation and purification process of 10kDa ultrafiltration membrane and 1kDa nanofiltration membrane to accurately retain 1-10kDa target peptides. The proportion of target peptides in the final product can reach more than 82%. At the same time, it effectively removes sensitizing macromolecular proteins, endotoxins and inorganic salts, resulting in high product purity and good biosafety. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the process flow of the present invention; Figure 2 This is a flowchart of the ultrasonic-assisted degreasing process of the present invention; Figure 3 This is a graph showing the change in pH value inside the enzymatic hydrolysis reactor of the present invention over reaction time; Figure 4 This is a graph showing the temperature change in the enzymatic hydrolysis reactor of the present invention over time. Figure 5 This is a structural diagram of the combined membrane separation and purification system of the present invention.

[0019] In the diagram: 1. Raw material pretreatment unit; 2. Tissue grinder; 3. Homogenizer; 4. Feed inlet; 5. Ultrasonic degreasing tank; 6. Activated carbon feed inlet; 7. Ultrasonic transducer assembly; 8. Cooling jacket; 9. Discharge outlet; 10. Centrifuge; 11. Enzymatic hydrolysis reactor; 12. pH adjuster storage tank; 13. Trypsin feed inlet; 14. Pepsin feed inlet; 15. Plate and frame filter; 16. Ultrafiltration membrane module; 17. Nanofiltration membrane module; 18. Freeze dryer. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that in this invention, the terms "preparation process" and "preparation method" have exactly the same technical meaning and both refer to the technical solution described in this application. The use of both terms is merely a convention and is intended to describe the technical implementation process more comprehensively. It does not constitute semantic redundancy, nor does it create any substantial difference in the scope of protection. Example

[0022] (1) Raw material pretreatment: Take 500g of fresh pig prostate tissue from the day of slaughter, transport it to the workshop at 4℃, remove the surface fat, capsule and visible connective tissue, cut it into small pieces of about 1cm³, put the small pieces of tissue into tissue grinder 2, add 1000mL of 4℃ pre-cooled purified water, start homogenizer 3, homogenize at 10000rpm for 20 minutes to obtain tissue homogenate; (2) Ultrasonic-assisted degreasing: The tissue homogenate obtained in step one is transferred to the ultrasonic degreasing tank 5 through the feed port 4. 1% (w / w) of 300-mesh activated carbon is added through the activated carbon feed port 6. The ultrasonic transducer group 7 is started, the power is set to 350W and the frequency is 30kHz. At the same time, the cooling jacket 8 is turned on and 4℃ circulating cooling water is introduced to control the temperature of the material in the tank at 6±2℃. After ultrasonic treatment for 40 minutes, the discharge port 9 is opened and the material is transported to the centrifuge 10. It is centrifuged at 6000rpm for 20 minutes, the supernatant is discarded, and the precipitate is collected, which is the degreased prostate tissue. (3) Stepwise enzymatic hydrolysis: The defatted prostate tissue precipitate obtained in step two was resuspended in 1500 mL of purified water and transferred to the enzymatic hydrolysis reactor 11. 0.5 M sodium hydroxide solution was added dropwise through the pH adjuster tank 12 to slowly adjust the pH to 7.5 ± 0.1. Trypsin was added through the trypsin feed port 13 at a rate of 0.3% (w / w) of the wet weight of the precipitate. The mixture was stirred and hydrolyzed for 2 hours at 37℃ ± 0.5℃. Subsequently, 1 M hydrochloric acid was added dropwise through the pH adjuster tank 12 to adjust the pH of the reaction solution to 4.0 ± 0.1. Sodium chloride was added to a final concentration of 0.2 M to maintain ionic strength. Pepsin was added through the pepsin feed port 14 at a rate of 0.2% (w / w) of the wet weight of the precipitate. The mixture was stirred and hydrolyzed for 1 hour at 40℃ ± 0.5℃. After the enzymatic hydrolysis was completed, a sample was taken to determine the degree of protein hydrolysis. (4) Combined membrane separation and purification: The enzymatic hydrolysate was transferred to a tubular centrifuge and centrifuged at 4000 rpm for 15 minutes to remove unhydrolyzed residue and some denatured proteins. The supernatant was then clarified by passing it through a microfiltration membrane with a pore size of 0.45 μm and a plate and frame filter 15. The clarified filtrate first entered the ultrafiltration membrane module 16. The membrane material was polyethersulfone with a molecular weight cutoff of 10 kDa. The operating temperature was 22±1℃, the pressure was 0.4 MPa, and the membrane flux was maintained at 35 L / (m²·h). The permeate (molecular weight < 10 kDa) was collected, and the retentate was discarded. The permeate was then entered into the nanofiltration membrane module 17. The membrane material was also polyethersulfone with a molecular weight cutoff of 1 kDa. The operation was carried out under the same temperature and pressure conditions. The retentate (molecular weight 1-10 kDa) was collected, and the permeate was discarded. (5) Drying: Transfer the retentate obtained in step four to freeze dryer 18, maintain at -35°C for 3 hours for the first drying, vacuum degree 20Pa, gradually raise the temperature of the partition to -15°C for the second drying, raise the temperature of the partition to 25°C, maintain for 4 hours until the pressure is stable, and discharge the material to obtain pig prostate extract powder.

[0023]

[0024] The total protein content was determined using the Kjeldahl method, the residual fat content was determined using the Soxhlet extraction method, the ash content was determined using the muffle furnace ignition method, the nucleic acid content was determined by measuring the A260 absorbance using a UV spectrophotometer, the endotoxin content was determined using the Limulus amebocyte lysate (LAL) gel electrophoresis method, the molecular weight distribution and proportion of target peptides were determined using high performance liquid chromatography with size exclusion chromatography, and the degree of protein hydrolysis was determined by pre-column derivatization using OPA to determine and calculate the amino nitrogen content. Three batches of pilot-scale tests were conducted continuously according to the method described in Example 1. The inter-batch RSD of the main indicators was <5%, demonstrating that the method is stable and reliable.

[0025] Example

[0026] Following the method in Example 1, parallel experiments were conducted by changing only the parameters to boundary values ​​to verify the critical effectiveness of the parameter range in the claims of this invention.

[0027] (1) Boundary of ultrasonic degreasing parameters: Three 500g portions of porcine prostate tissue were taken, and the ultrasonic power was set to 200W, 350W and 500W respectively. The treatment time was 60 minutes, 40 minutes and 20 minutes respectively, corresponding to low-medium-high power combinations. All three groups used 1% activated carbon and 6℃ conditions. The results showed that the fat residue after degreasing in the three groups was 1.9%, 1.7% and 2.1% respectively, all below 2.5%, which was significantly better than the traditional process. An additional blank control experiment showed that when the power was below 200W, the fat residue was still as high as 4.5% after 60 minutes, which was not effective. When the power was above 500W, the local temperature rise was significantly higher than 10℃, which may cause protein denaturation. Therefore, the limitation of the power range of 200-500W in this invention has critical significance.

[0028] (2) Enzymatic hydrolysis time boundary: Three defatted tissue samples were taken. The first step of trypsin hydrolysis was set to 1 hour, 2 hours and 3 hours respectively, and the second step of pepsin hydrolysis was set to 0.5 hours, 1 hour and 2 hours respectively. The degree of hydrolysis was detected to be 22.3%, 27.1% and 28.4% respectively. The results showed that the 1-3 hour range was effective. The degree of hydrolysis was insufficient below 1 hour, and the degree of hydrolysis increased slowly and energy consumption increased significantly above 3 hours. Therefore, the 1-3 hour range was considered reasonable.

[0029] (3) Membrane separation molecular weight boundary: Add a control group: ① The ultrafiltration membrane is 5kDa (without nanofiltration step), and the target peptide accounts for only 52% of the product, with a high content of small molecule impurities. ② The ultrafiltration membrane is 30kDa + nanofiltration is 1kDa, and 10-30kDa impurities enter the product, and the target peptide accounts for 68%, which confirms that the 10kDa + 1kDa combination membrane system defined in this invention can optimally retain 1-10kDa active peptides.

[0030] Comparative Example 1: Traditional Water Extraction and Alcohol Precipitation Method Referring to the water extraction and alcohol precipitation process disclosed in the background technology, 500g of the same batch of porcine prostate tissue was taken, chopped, and added with twice the amount of purified water. The mixture was freeze-thawed three times at 4℃ and extracted twice at 80℃ for one hour each time. The extracts were combined, centrifuged, and the supernatant was collected. Twice the volume of 95% ethanol was added for alcohol precipitation overnight. The precipitate was collected by centrifugation and freeze-dried. The test results were as follows: total protein content 48.3%, residual fat 7.2%, nucleic acid content 0.42%, endotoxin 5.2 EU / mg, and the target peptide ratio was only 31.5%. The product was dark yellow with a distinct fishy odor, and the acid value increased significantly in the accelerated oxidation test.

[0031] Comparative Example 2: Single Enzyme Digestion Method After defatting 500g of tissue using steps one and two of this invention, it was enzymatically hydrolyzed with trypsin at pH 7.5 and 37°C for 3 hours. Subsequent membrane separation and drying were the same as in Example 1. The results showed that the degree of protein hydrolysis was only 15.6%, the target peptide accounted for 57.3% of the final product, and it contained a large number of large molecular protein fragments greater than 10kDa. In the in vitro simulated digestion test, this product could be further degraded after treatment with pepsin, but its predicted bioavailability for direct oral absorption was lower than that of the stepwise enzymatic hydrolysis product of this invention.

[0032]

[0033] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0034] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a porcine prostate extract, characterized in that, Includes the following steps: Step 1: Take fresh or frozen-thawed pig prostate tissue, remove surface fat and connective tissue, cut it into small pieces and place it in a tissue grinder (2), add 1-3 times the mass of purified water, and homogenize it for 10-30 minutes using a homogenizer (3) to obtain tissue homogenate. Step 2: The tissue homogenate obtained in Step 1 is introduced into the ultrasonic degreasing tank (5) through the feed port (4). 0.5-2% (w / w) of activated carbon is added through the activated carbon feed port (6). The ultrasonic transducer group (7) is started. The temperature is controlled by the cooling jacket (8) at 4-10℃. Ultrasonic treatment is performed with a power of 200-500W and a treatment time of 20-60 minutes. Then the discharge port (9) is opened and centrifuged by centrifuge (10). The precipitate is collected to obtain degreased prostate tissue. Step 3: Resuspend the defatted prostate tissue obtained in Step 2 in purified water and transfer it to the enzymatic hydrolysis reactor (11). Adjust the pH to 7.0-8.0 through the pH adjuster tank (12), add 0.1-0.5% (w / w) of trypsin through the trypsin feed port (13), and hydrolyze at 35-40℃ for 1-3 hours. Then adjust the pH to 3.0-5.0 through the pH adjuster tank (12), add 0.1-0.3% (w / w) of pepsin through the pepsin feed port (14), and continue hydrolyzing at 37-42℃ for 0.5-2 hours to obtain the hydrolysate. Step 4: Remove the residue from the enzymatic hydrolysate obtained in Step 3 by passing it through a plate and frame filter (15) and collect the filtrate; pass the filtrate through an ultrafiltration membrane module (16) with a molecular weight cutoff of 10 kDa and a nanofiltration membrane module (17) with a molecular weight cutoff of 1 kDa in sequence, and collect the cutoff liquid in the molecular weight range of 1-10 kDa. Step 5: Transfer the retentate obtained in Step 4 to a freeze dryer (18) for vacuum freeze drying or spray drying to obtain porcine prostate extract powder.

2. The method for preparing a porcine prostate extract according to claim 1, characterized in that, In step two, the frequency of ultrasonic treatment is 20-40kHz, the centrifugal speed of centrifuge (10) is 4000-8000 rpm, and the centrifugation time is 15-30 minutes.

3. The method for preparing a porcine prostate extract according to claim 1, characterized in that, The activated carbon added in step two has a mesh size of 200-400 and a mass-to-volume ratio (w / v) of 1:50 to 1:100 with respect to the tissue homogenate. The activated carbon is added to the ultrasonic degreasing tank (5) through the activated carbon feeding port (6), and circulating cooling water is introduced into the cooling jacket (8) to maintain the temperature.

4. The method for preparing a porcine prostate extract according to claim 1, characterized in that, In step three, the enzyme activity of trypsin is 2500-4000 USP U / mg, and the enzyme activity of pepsin is 1:12000-1:15000.

5. The method for preparing a porcine prostate extract according to claim 1, characterized in that, In step three, before adjusting the pH for the second enzymatic hydrolysis, the pH of the enzymatic hydrolysate in the enzymatic hydrolysis reactor (11) must be adjusted from 7.0-8.0 to 3.0-5.0 using 1M hydrochloric acid through the pH adjuster storage tank (12), and 0.1-0.5M sodium chloride is added when adding pepsin through the pepsin feeding port (14) to maintain the ionic strength.

6. The method for preparing a porcine prostate extract according to claim 1, characterized in that, The operating conditions for the combined membrane separation and purification in step four are as follows: the temperature is controlled below 25℃, the operating pressure is 0.2-0.6MPa, and the membrane flux of the ultrafiltration membrane module (16) and the nanofiltration membrane module (17) is maintained at 20-50L / (m²·h).

7. The method for preparing a porcine prostate extract according to claim 1, characterized in that, In step four, before passing through the ultrafiltration membrane assembly (16), the filtrate is first centrifuged at 3000-5000 rpm for 10-20 minutes and pre-filtered using a microfiltration membrane with a pore size of 0.45 μm.

8. The method for preparing a porcine prostate extract according to claim 1, characterized in that, The conditions for vacuum freeze drying in step five are as follows: in the freeze dryer (18), the pre-freezing temperature is -40℃ to -30℃, maintained for 2-4 hours, and the vacuum degree of the sublimation drying stage is 10-30Pa, the temperature is -20℃ to -10℃, and the temperature of the desorption drying stage is 20-30℃.

9. The method for preparing a porcine prostate extract according to claim 1, characterized in that, In the obtained porcine prostate extract, peptides with a content of less than 10 kDa accounted for more than 85% of the total protein, and the nucleic acid content (measured by absorbance at 260 nm) was less than 0.1%.

10. A porcine prostate extract prepared by the method according to any one of claims 1-9, characterized in that, The extract contains active peptides with a molecular weight between 1 and 10 kDa, and the total protein content of the extract is not less than 70%, the residual fat content is less than 5%, and the ash content is less than 8%.

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