Preparation process of bovine prostate dry powder

Abstract

The invention discloses a preparation process of bovine prostate dry powder. The preparation process comprises the following steps: pretreating raw materials; preparing homogenate; freezing and thawing treatment; filtering and separating; carrying out alkaline protease enzymolysis; performing enzymolysis with acid protease; performing activation treatment; performing primary acetone precipitation; carrying out secondary and tertiary acetone precipitation; drying and crushing; the moistening is balanced; the invention relates to the technical field of bioengineering, through a three-stage system of freezing and thawing, double-stage enzymolysis and activation, cell structures are damaged by freezing and thawing, proteins are fully decomposed by double-stage enzymolysis, release of active substances is enhanced in the activation step, and the retention rate of core components such as alkaline phosphatase is effectively increased; after water-soluble impurities are removed step by step through acetone gradient precipitation and a moistening phase is balanced and optimized in a phase state, fat-soluble impurities are further removed through ethanol repeated precipitation, the product purity is improved, and the residual amount of an organic solvent is effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a preparation process for bovine prostate powder. Background Technology

[0002] Bovine prostate is rich in bioactive components such as alkaline phosphatase, polypeptides, and steroids, and has significant application value in the pharmaceutical and health product fields. Currently, only a few companies worldwide can produce active bovine prostate powder, and existing preparation processes suffer from significant technical bottlenecks: First, incomplete cell disruption leads to low release rates of active ingredients, and the single enzymatic hydrolysis method results in insufficient protein breakdown; second, the purification steps are simple, resulting in high residual levels of organic solvents (such as acetone and ethanol) and low impurity removal rates, affecting product safety and making it difficult to meet the quality standards for industrial production. To address these issues, there is an urgent need for a bovine prostate powder preparation process that can efficiently extract active ingredients, deeply purify and remove impurities, and is stable and controllable. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a preparation process for bovine prostate powder, which solves the problems of high loss rate of active ingredients, large amount of organic solvent residue, and insufficient product purity in existing extraction processes.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a preparation process for bovine prostate powder, comprising the following steps: Raw material pretreatment: Take fresh or frozen bovine prostate, trim and remove fat fascia, wash with water and pure water and set aside; Homogenization preparation: After trimming and washing, the bovine prostate is minced and then homogenized with pure water for later use; Freeze-thaw treatment: The prostate homogenate is frozen and then thawed for later use; Filtration and separation: The frozen-thawed prostate extract was filtered, the precipitate was used for enzymatic hydrolysis, and the filtrate was reserved; Alkaline protease hydrolysis: Take the freeze-thawed precipitate, adjust the pH to 8.5-10.0 with NaOH, add alkaline protease, stir evenly, hydrolyze for 10-12 hours, and inactivate at 70-80℃ to obtain alkaline protease hydrolysate. Acidic protease hydrolysis: Take the alkaline protease hydrolysate, adjust the pH to 3.0-5.0 with HCl, add the acidic protease, stir evenly, hydrolyze for 10-12 hours, and inactivate at 70-80℃ to obtain the acidic protease hydrolysate. Activation process: Combine the filtered filtrate with the acidic protease hydrolysate for activation; First acetone precipitation: After activation, add acetone to the material at a W / V ratio of 1:8-12, stir, let stand for 24-48 hours, filter to obtain filter cake, and recover acetone from the filtrate. Secondary and tertiary acetone precipitation: For the first acetone precipitation of the filter cake, add acetone at a W / V ratio of 1:4-7, stir evenly, and carry out the second and third acetone precipitation. Drying and pulverizing: The acetone precipitate is filtered to remove acetone. The filter cake is vacuum dried and then pulverized by a pulverizer and passed through a 100-mesh vibrating sieve to obtain prostate acetone dry powder. The acetone is recovered from the filtrate. Moistening phase equilibrium: Add the dry acetone powder of prostate to acetone and stir until the phase is balanced; Ethanol precipitation: Add ethanol to the dry powder after phase equilibrium, stir evenly, let stand and filter, and then filter by suction. Separate impurities from the filter cake, recover ethanol from the filtrate, repeat the operation, and then dry the filter cake to obtain the finished product.

[0005] Preferably, the process also includes the following step: inspecting the packaging: After the ethanol precipitation step, the finished product is inspected according to the items specified in the "Inspection Procedure for Bovine Prostate Powder" and then packaged and stored.

[0006] Preferably, in the homogenization preparation step, pure water is added at a ratio of 1:1-5 between the minced material and pure water, and the material is minced using a meat grinder and homogenized using a colloid mill.

[0007] Preferably, in the freeze-thaw process, the prostate homogenate is sealed in a tray with plastic wrap and frozen at -20°C for 36-48 hours before being thawed for later use.

[0008] Preferably, the alkaline protease is one or a mixture of two or more of the following enzymes: 2709 alkaline protease, serine protease, subtilisin protease, and papain.

[0009] Preferably, the acidic protease is one or a mixture of two or more of papain, protease M, Aspergillus niger protease, and yeast acidic protease.

[0010] Preferably, the activation time in the activation process is 5-12 hours.

[0011] Preferably, in the wetted phase balancing step, the injected acetone accounts for 5-10% of the dry powder weight, and is stirred using a V-type or double-cone mixer.

[0012] Preferably, a plate and frame filter is used for filtration in the filtration and separation step, and both the alkaline protease hydrolysis step and the acidic protease hydrolysis step are carried out in a stainless steel stirred jacketed reactor, and the pH is checked and adjusted by a pH meter and precision pH test paper.

[0013] Preferably, in the acetone precipitation step, acetone is recovered by distillation column, and in the ethanol precipitation step, ethanol is recovered by glass chromatography column filtration and distillation column.

[0014] Beneficial effects This invention provides a process for preparing bovine prostate powder, which has the following beneficial effects: Through a three-stage system of "freeze-thaw-dual enzymatic hydrolysis-activation", freeze-thaw disrupts cell structure, dual enzymatic hydrolysis fully decomposes proteins, and the activation step enhances the release of active substances, effectively improving the retention rate of core components such as alkaline phosphatase. Acetone gradient precipitation removes water-soluble impurities step by step. After the phase is optimized by wet phase equilibrium, repeated ethanol precipitation further removes fat-soluble impurities, improving product purity and effectively reducing residual organic solvents, meeting pharmaceutical grade standards. The process utilizes equipment such as plate and frame filtration, stainless steel reactors, and distillation recovery to adapt to large-scale production. Acetone and ethanol are recovered through a distillation column, reducing solvent consumption and environmental treatment costs, effectively lowering production energy consumption, and is easy to use. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the process of the present invention.

[0016] Figure 2 This is a screenshot of the first page of the test report for this invention.

[0017] Figure 3 This is a screenshot of the second page of the test report for this invention. Detailed Implementation

[0018] 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.

[0019] Please see Figure 1 This invention provides a technical solution: a preparation process for bovine prostate powder, comprising the following steps: Raw material pretreatment: Take fresh or frozen bovine prostate, trim and remove fat fascia, wash with water and pure water and set aside; Homogenization preparation: After trimming and washing, the bovine prostate is minced and then homogenized with pure water for later use; Freeze-thaw treatment: The prostate homogenate is frozen and then thawed for later use; Filtration and separation: The frozen-thawed prostate extract was filtered, the precipitate was used for enzymatic hydrolysis, and the filtrate was reserved; Alkaline protease hydrolysis: Take the freeze-thawed precipitate, adjust the pH to 8.5-10.0 with NaOH, add alkaline protease, stir evenly, hydrolyze for 10-12 hours, and inactivate at 70-80℃ to obtain alkaline protease hydrolysate. Acidic protease hydrolysis: Take the alkaline protease hydrolysate, adjust the pH to 3.0-5.0 with HCl, add the acidic protease, stir evenly, hydrolyze for 10-12 hours, and inactivate at 70-80℃ to obtain the acidic protease hydrolysate. Activation process: Combine the filtered filtrate with the acidic protease hydrolysate for activation; First acetone precipitation: After activation, add acetone to the material at a W / V ratio of 1:8-12, stir, let stand for 24-48 hours, filter to obtain filter cake, and recover acetone from the filtrate. Secondary and tertiary acetone precipitation: For the first acetone precipitation of the filter cake, add acetone at a W / V ratio of 1:4-7, stir evenly, and carry out the second and third acetone precipitation. Drying and pulverizing: The acetone precipitate is filtered to remove acetone. The filter cake is vacuum dried and then pulverized by a pulverizer and passed through a 100-mesh vibrating sieve to obtain prostate acetone dry powder. The acetone is recovered from the filtrate. Moistening phase equilibrium: Add the dry acetone powder of prostate to acetone and stir until the phase is balanced; Ethanol precipitation: Add ethanol to the dry powder after phase equilibrium, stir evenly, let stand and filter, and then filter by suction. Separate impurities from the filter cake, recover ethanol from the filtrate, repeat the operation, and then dry the filter cake to obtain the finished product.

[0020] This embodiment further includes the following step: inspecting the packaging: After the ethanol precipitation step, the finished product is inspected according to the items specified in the "Inspection Procedure for Bovine Prostate Powder" and then packaged and stored.

[0021] In this embodiment, the homogenization preparation step is further configured such that pure water is added at a ratio of 1:1-5 between the minced material and pure water, and the material is minced using a meat grinder and homogenized using a colloid mill.

[0022] In this embodiment, the prostate homogenate is sealed in a plastic wrap and frozen at -20°C for 36-48 hours before being thawed for later use.

[0023] In this embodiment, the alkaline protease is selected from one or more of the following enzymes: 2709 alkaline protease, serine protease, subtilisin protease, and papain.

[0024] In this embodiment, the acidic protease is selected from one or more of papain, protease M, Aspergillus niger protease, and yeast acidic protease.

[0025] In this embodiment, the activation time in the activation process is further set to 5-12 hours.

[0026] In this embodiment, the acetone sprayed in the wetted phase balancing step accounts for 5-10% of the dry powder weight, and is stirred using a V-type or double-cone mixer.

[0027] In this embodiment, the filtration and separation step is performed using a plate and frame filter, and both the alkaline protease hydrolysis step and the acidic protease hydrolysis step are carried out in a stainless steel stirred jacketed reactor, with the pH being checked and adjusted using a pH meter and precision pH test paper.

[0028] In this embodiment, acetone is recovered by distillation in the acetone precipitation step, and ethanol is recovered by glass chromatography column filtration in the ethanol precipitation step.

[0029] Those skilled in the art can connect the components in this case sequentially. The specific connection and operation sequence should refer to the working principle described below. The detailed connection methods are well-known technologies in the field. The working principle and process are mainly described below.

[0030] Example 1: A preparation process for bovine prostate powder, comprising the following steps: 1. 300 kg of frozen bovine prostate, trimmed and cleaned of fat and fascia, washed with water and purified water before use; 2. After trimming and washing, the bovine prostate is first minced using a meat grinder, then pure water is added at a ratio of 1:3 to the minced material, and the mixture is homogenized using a colloid mill for later use. 3. Homogenize the prostate and place it on a tray, seal it with plastic wrap, freeze it at -20℃ for 40 hours, and then thaw it for later use. 4. Filtration: The prostate freeze-thawed slow precipitate is filtered using a plate and frame filter press. The precipitate after filtration is then subjected to enzymatic hydrolysis, and the filtrate is reserved. 5. Alkaline protease hydrolysis: The precipitate was slowed down by freeze-thaw, and the pH was adjusted to 9.0 with NaOH. A 1:1 mixture of 2709 alkaline protease and serine protease was added, stirred evenly, and hydrolyzed for 12 hours. The mixture was then inactivated at 80°C to obtain the alkaline hydrolysate. 6. Acidic protease hydrolysis: The alkaline protease hydrolysate was adjusted to pH 4.0 with HCl, and a 1:1 mixture of papain and yeast acidic protease was added. The mixture was stirred evenly and hydrolyzed for 12 hours. The hydrolysate was then inactivated at 80°C to obtain the acidic hydrolysate. 7. Activation: After freeze-thaw thawing, the filtrate is combined with the acidic enzymatic hydrolysate and activated for 12 hours. 8. Primary acetone precipitation: After activation, acetone is added to the material at a ratio of 1:10 (w / v), stirred, and allowed to stand for 36 hours. The filter cake is obtained by filtration, and the acetone is recovered from the filtrate. 9. Secondary and tertiary acetone precipitation: After the first acetone precipitation of the filter cake, add acetone at a W / V ratio of 1:5 and stir evenly to carry out the second and third acetone precipitation. 10. Drying: The acetone precipitate is filtered to remove acetone, and the filter cake is vacuum dried to obtain prostate acetone powder. The acetone is recovered from the filtrate. 11. Grinding: After vacuum drying, the acetone precipitate is pulverized by a pulverizer and passed through a 100-mesh vibrating sieve to obtain prostate acetone powder. After the acetone precipitate is inspected and qualified according to the items under the inspection procedure for bovine prostate acetone powder, it is packaged into the semi-finished product warehouse.

[0031] 12. Moisturizing and balancing: Bovine prostate acetone powder is loaded into a V-type or double-cone mixer, and 10% acetone is slowly sprayed in while stirring to moisturize and achieve phase balance. 13. Ethanol precipitation: After reaching phase equilibrium, acetone precipitates dry powder, ethanol is added at a ratio of 1:6 (w / v), stirred evenly, allowed to stand for 24 hours, filtered, and vacuum filtered. The ethanol in the filtrate is recovered. This step is repeated once. The filter cake with impurities removed is then vacuum dried to obtain the finished product. 14. Inspection: After the finished product passes the inspection according to the items specified in the Inspection Procedure for Bovine Prostate Powder, it is packaged and put into storage.

[0032] Example 2: A preparation process for bovine prostate powder, comprising the following steps: 1. 100 kg of frozen bovine prostate, trimmed and cleaned of fat and fascia, washed with water and purified water before use; 2. After trimming and washing, the bovine prostate is first minced using a meat grinder, then pure water is added at a 1:1 ratio to the minced material, and the mixture is homogenized using a colloid mill for later use. 3. Homogenize the prostate and place it on a tray, seal it with plastic wrap, freeze it at -20℃ for 36 hours, and then thaw it for later use. 4. Filtration: The prostate freeze-thawed slow precipitate is filtered using a plate and frame filter press. The precipitate after filtration is then subjected to enzymatic hydrolysis, and the filtrate is reserved. 5. Alkaline protease hydrolysis: freeze-thaw the precipitate, adjust the pH to 8.5 with NaOH, add a 1:1 mixture of alkaline protease, stir well, hydrolyze for 10 hours, and inactivate at 70℃ to obtain alkaline hydrolysate. 6. Acidic protease hydrolysis: Adjust the pH of the alkaline protease hydrolysate to 3.0 with HCl, add a 1:1 mixture of acidic protease, stir well, hydrolyze for 10 hours, and inactivate at 70℃ to obtain the acidic hydrolysate. 7. Activation: Combine the filtrate after freeze-thaw thawing with the acidic enzymatic hydrolysate and activate for 5 hours; 8. Primary acetone precipitation: After activation, acetone is added to the material at a ratio of 1:8 (w / v), stirred, and allowed to stand for 24 hours. The filter cake is obtained by filtration, and the acetone is recovered from the filtrate. 9. Secondary and tertiary acetone precipitation: After the first acetone precipitation of the filter cake, add acetone at a W / V ratio of 1:4 and stir well to carry out the second and third acetone precipitation. 10. Drying: The acetone precipitate is filtered to remove acetone, and the filter cake is vacuum dried to obtain prostate acetone powder. The acetone is recovered from the filtrate. 11. Grinding: After vacuum drying, the acetone precipitate is pulverized by a pulverizer and passed through a 100-mesh vibrating sieve to obtain prostate acetone powder. After the acetone precipitate is inspected and qualified according to the items under the inspection procedure for bovine prostate acetone powder, it is packaged into the semi-finished product warehouse.

[0033] 12. Moisturizing and balancing: Bovine prostate acetone powder is loaded into a V-type or double-cone mixer, and 5% acetone is slowly sprayed in while stirring to moisturize and saturate the mixture until phase equilibrium is achieved. 12. Ethanol precipitation: After reaching phase equilibrium, acetone precipitates dry powder, ethanol is added at a W / V ratio of 1:4, stirred evenly, allowed to stand for 24 hours, filtered, and vacuum filtered. The ethanol in the filtrate is recovered. This step is repeated once. The filter cake with impurities removed is then vacuum dried to obtain the finished product. 13. Inspection: After the finished product passes the inspection according to the items specified in the Inspection Procedure for Bovine Prostate Powder, it is packaged and put into storage.

[0034] Example 3: A preparation process for bovine prostate powder, comprising the following steps: 1. 300 kg of frozen bovine prostate, trimmed and cleaned of fat and fascia, washed with water and purified water before use; 2. After trimming and washing, the bovine prostate is first minced using a meat grinder, then pure water is added at a ratio of 1:5 to the minced material, and the mixture is homogenized using a colloid mill for later use. 3. Homogenize the prostate and place it on a tray, seal it with plastic wrap, and freeze it at -20℃ for 48 hours. Thaw it before use. 4. Filtration: The prostate freeze-thawed slow precipitate is filtered using a plate and frame filter press. The precipitate after filtration is subjected to enzymatic hydrolysis, and the filtrate is reserved. 5. Alkaline protease hydrolysis: The precipitate was slowed down by freeze-thaw, the pH was adjusted to 10.0 with NaOH, and a 1:1 mixture of alkaline protease was added. The mixture was stirred evenly and hydrolyzed for 12 hours. The enzyme was then inactivated at 80°C to obtain the alkaline hydrolysate. 6. Acidic protease hydrolysis: Adjust the pH of the alkaline protease hydrolysate to 5.0 with HCl, add a 1:1 mixture of acidic protease, stir well, hydrolyze for 12 hours, and inactivate at 80℃ to obtain the acidic hydrolysate. 7. Activation: After freeze-thaw thawing, the filtrate is combined with the acidic enzymatic hydrolysate and activated for 12 hours. 8. Primary acetone precipitation: After activation, acetone is added to the material at a ratio of 1:12 (w / v), stirred, and allowed to stand for 48 hours. The filter cake is obtained by filtration, and the acetone is recovered from the filtrate. 9. Secondary and tertiary acetone precipitation: After the first acetone precipitation of the filter cake, add acetone at a W / V ratio of 1:7 and stir well to carry out the second and third acetone precipitation. 10. Drying: The acetone precipitate is filtered to remove acetone, and the filter cake is vacuum dried to obtain prostate acetone powder. The acetone is recovered from the filtrate. 11. Grinding: After vacuum drying, the acetone precipitate is pulverized by a pulverizer and passed through a 100-mesh vibrating sieve to obtain prostate acetone powder. After the acetone precipitate is inspected and qualified according to the items under the inspection procedure for bovine prostate acetone powder, it is packaged into the semi-finished product warehouse.

[0035] 11. Moisturizing and balancing: Bovine prostate acetone powder is loaded into a V-type or double-cone mixer, and 10% acetone is slowly sprayed in while stirring to moisturize and achieve phase balance. 12. Ethanol precipitation: After reaching phase equilibrium, acetone precipitates dry powder. Ethanol is added at a ratio of W / V 1:8, stirred evenly, allowed to stand for 24 hours, filtered, and vacuum filtered. The ethanol is recovered from the filtrate. This step is repeated once. The filter cake with impurities removed is then vacuum dried to obtain the finished product. 13. Inspection: After the finished product passes the inspection according to the items specified in the Inspection Procedure for Bovine Prostate Powder, it is packaged and put into storage.

[0036] It should be noted that, in this document, relational terms such as "first" and "second" are used merely 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, the phrase "comprising an element defined as..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0037] 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 preparation process for bovine prostate powder, characterized in that, Includes the following steps: Raw material pretreatment: Take fresh or frozen bovine prostate, trim and remove fat fascia, wash with water and pure water and set aside; Homogenization preparation: After trimming and washing, the bovine prostate is minced and then homogenized with pure water for later use; Freeze-thaw treatment: The prostate homogenate is frozen and then thawed for later use; Filtration and separation: The frozen-thawed prostate extract was filtered, the precipitate was used for enzymatic hydrolysis, and the filtrate was reserved; Alkaline protease hydrolysis: Take the freeze-thawed precipitate, adjust the pH to 8.5-10.0 with NaOH, add alkaline protease, stir evenly, hydrolyze for 10-12 hours, and inactivate at 70-80℃ to obtain alkaline protease hydrolysate. Acidic protease hydrolysis: Take the alkaline protease hydrolysate, adjust the pH to 3.0-5.0 with HCl, add the acidic protease, stir evenly, hydrolyze for 10-12 hours, and inactivate at 70-80℃ to obtain the acidic protease hydrolysate. Activation process: Combine the filtered filtrate with the acidic protease hydrolysate for activation; First acetone precipitation: After activation, add acetone to the material at a W / V ratio of 1:8-12, stir, let stand for 24-48 hours, filter to obtain filter cake, and recover acetone from the filtrate. Secondary and tertiary acetone precipitation: For the first acetone precipitation of the filter cake, add acetone at a W / V ratio of 1:4-7, stir evenly, and carry out the second and third acetone precipitation. Drying and pulverizing: The acetone precipitate is filtered to remove acetone. The filter cake is vacuum dried and then pulverized by a pulverizer and passed through a 100-mesh vibrating sieve to obtain prostate acetone dry powder. The acetone is recovered from the filtrate. Moistening phase equilibrium: Add the dry acetone powder of prostate to acetone and stir until the phase is balanced; Ethanol precipitation: Add ethanol to the dry powder after phase equilibrium, stir evenly, let stand and filter, and then filter by suction. Separate impurities from the filter cake, recover ethanol from the filtrate, repeat the operation, and then dry the filter cake to obtain the finished product.

2. The preparation process of bovine prostate powder according to claim 1, characterized in that, The following steps are also included to inspect the packaging: After the ethanol precipitation step, the finished product is inspected according to the items specified in the "Inspection Procedure for Bovine Prostate Powder" and then packaged and stored.

3. The preparation process of bovine prostate powder according to claim 1, characterized in that, In the homogenization preparation step, pure water is added at a ratio of 1:1-5 between the minced material and pure water, and the material is minced using a meat grinder and homogenized using a colloid mill.

4. A pneumatic device for preventing water splashing in a thick plate quenching machine according to claim 1, characterized in that, In the freeze-thaw process, the prostate homogenate is sealed in a tray with plastic wrap and frozen at -20°C for 36-48 hours before being thawed for later use.

5. The preparation process of bovine prostate powder according to claim 1, characterized in that, The alkaline protease is one or a mixture of two or more of the following enzymes: 2709 alkaline protease, serine protease, subtilisin protease, and papain.

6. The preparation process of bovine prostate powder according to claim 1, characterized in that, The acidic protease is one or a mixture of two or more of the following enzymes: papain, protease M, Aspergillus niger protease, and yeast acidic protease.

7. The preparation process of bovine prostate powder according to claim 1, characterized in that, The activation time in the activation process is 5-12 hours.

8. The preparation process of bovine prostate powder according to claim 1, characterized in that, In the phase balancing step, the injected acetone accounts for 5-10% of the dry powder weight, and is stirred using a V-type or double-cone mixer.

9. The preparation process of bovine prostate powder according to claim 1, characterized in that, The filtration and separation step uses a plate and frame filter for filtration. The alkaline protease hydrolysis step and the acidic protease hydrolysis step are both carried out in a stainless steel stirred jacket reactor, and the pH is checked and adjusted by a pH meter and precision pH test paper.

10. The preparation process of bovine prostate powder according to claim 1, characterized in that, In the acetone precipitation step, acetone is recovered by distillation column, and in the ethanol precipitation step, ethanol is recovered by glass chromatography column filtration and distillation column.

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