CN111876401A - Method for extracting bromelain from pineapple stems and leaves - Google Patents
Method for extracting bromelain from pineapple stems and leaves Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22032—Stem bromelain (3.4.22.32)
Abstract
The invention discloses a method for extracting and obtaining bromelain from pineapple stem leaves, which completely removes impurities in the extracted bromelain by two-time extraction, greatly improves the quality of the bromelain, and prepares an extraction microsphere in the extraction process, the extraction microsphere is characterized in that carboxyl on carboxyl cellulose reacts with amino on amino-terminated silicon oil under the action of 4-dimethylamino pyridine to form amide, the amino-terminated silicon oil is fixed on the surface of the cellulose, the cellulose has good adsorption capacity and can adsorb the bromelain, the amino-terminated silicon oil is further fixed on the surface of the cellulose, the amino-terminated silicon oil is a surfactant, the adsorption effect on the bromelain is further improved, the cellulose is prepared into the microsphere, so that the extraction microsphere can be repeatedly used, greatly improves the extraction efficiency of the bromelain.
Description
Technical Field
The invention belongs to the technical field of enzyme extraction, and relates to a method for extracting bromelain from pineapple stems and leaves.
Background
The pineapple has high nutritive value, contains rich nutritive substances and functional substances, is more processed products, is greatly popular in multiple fields of food, medicine, health care products, living goods and the like, and the bromelain is one of the functional components with higher application value in the pineapple, can quickly decompose protein, tenderize meat, dissolve fibrin and blood clots blocked in tissues, improve local blood circulation and eliminate inflammation and edema, and is one of the main protease sources in the current market.
In the existing method for extracting bromelain from pineapple stem leaves, a large amount of bromelain still remains in the pineapple stem leaves in the process of extracting the bromelain, and the extracted bromelain contains partial impurities, so that the quality of the extracted bromelain is low.
Disclosure of Invention
The invention aims to provide a method for extracting bromelain from pineapple stems and leaves.
The technical problems to be solved by the invention are as follows:
in the existing method for extracting bromelain from pineapple stem leaves, a large amount of bromelain still remains in the pineapple stem leaves in the process of extracting the bromelain, and the extracted bromelain contains partial impurities, so that the quality of the extracted bromelain is low.
The purpose of the invention can be realized by the following technical scheme:
a method for extracting bromelain from pineapple stems and leaves comprises the following steps:
step S1: washing pineapple stem and leaf for 2-4 times, drying after washing for 3-5min each time, preserving heat for 10-15min at the temperature of 3-5 ℃, crushing, adding phosphate buffer, standing for 10-15min at the pH value of 5-6, filtering, centrifuging the filtrate for 15-20min at the rotation speed of 4000 plus materials at 5000r/min, taking supernatant to obtain base liquid, and preserving heat for later use at the temperature of 3-5 ℃;
step S2: adding the base liquid and the extraction microspheres prepared in the step S1 into a stirring kettle, stirring for 20-30min at the rotation speed of 150-;
step S3: regulating the eluent prepared in the step S2 with hydrochloric acid solution until the pH value is 5, adding ammonium sulfate, stirring at the rotation speed of 60-80r/min until the ammonium sulfate is completely dissolved, keeping the temperature at 3-5 ℃ for 12-15h, centrifuging at the rotation speed of 4000-5000r/min for 5-10min, and removing the supernatant to obtain crude enzyme;
step S4: adding the crude enzyme and the phosphate buffer solution into a reaction kettle, stirring at the rotation speed of 60-80r/min and the temperature of 3-5 ℃ until the crude enzyme is completely dissolved, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cut-off of 100, performing ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight cut-off of 20, and performing reduced pressure drying at the temperature of 0 ℃ to obtain the bromelain.
Further, the using amount ratio of the pineapple stem and leaf and the phosphate buffer solution in the step S1 is 1g:2-2.5mL, the concentration of the phosphate buffer solution is 0.05-0.1mol/L, the using amount ratio of the base solution and the extraction microsphere in the step S2 is 10mL:2-3g, the mass fraction of the sodium hydroxide solution is 15-18%, the mass fraction of the hydrochloric acid solution in the step S2 is 12-15%, and the using amount of ammonium sulfate is 20-25% of the using amount of the eluent.
Further, the extraction microspheres are prepared by the following steps:
step A1: crushing moso bamboo, sieving the crushed moso bamboo through a sieve of 80-100 meshes to obtain moso bamboo powder, drying the moso bamboo powder, adding the dried moso bamboo powder into a nitric acid solution, stirring the mixture for 5-10min at the rotation speed of 500r/min, carrying out heating reflux for 3-5h at the temperature of 85-90 ℃, filtering to remove the nitric acid solution, washing a filter cake with deionized water until the pH value of a washing solution is 7, adding the filter cake into a sodium hydroxide solution, carrying out heating reflux for 1-2h at the temperature of 105-110 ℃, filtering to remove the sodium hydroxide solution, adding the filter cake into hydrogen peroxide with the pH value of 9-10, soaking the hydrogen peroxide for 20-30min, filtering to remove the hydrogen peroxide, and drying the filter cake to obtain cellulose;
step A2: adding the cellulose prepared in the step A1 and deionized water into a reaction kettle, stirring for 10-15min at the rotating speed of 500-800r/min, adding sodium bromide, stirring for 3-5min, adding 2,2,6, 6-tetramethylpiperidine oxide solution, stirring for 5-10min, adding sodium hypochlorite until the reaction solution is stirred continuously and the pH value of the reaction solution is kept at 10, stirring for 4-5h, adding ethanol, centrifuging at 3000-4000r/min for 3-5min, removing supernatant, dispersing substrate with deionized water, adding hydrochloric acid solution until the pH value of the reaction solution is 5-6, reacting for 30-60min, centrifuging again to remove supernatant, cleaning the substrate with deionized water until the cleaning solution is neutral, and drying to obtain carboxylated cellulose;
step A3: adding octamethylcyclotetrasiloxane and sodium hydroxide into a reaction kettle, stirring for 10-15min under the condition that the rotation speed is 300-150℃, introducing nitrogen for protection, keeping the temperature for 6-8h under the condition that the temperature is 140-150℃, adding aminopropyltriethoxysilane and dimethyl sulfoxide, stirring for 3-5h under the condition that the rotation speed is 150-200℃ and the temperature is 90-100 ℃, adding carboxylated cellulose prepared in the step A2 and 4-dimethylaminopyridine after carrying out equivalent neutralization by using a hydrochloric acid solution, stirring for 8-10h under the conditions that the rotation speed is 100-150℃ and the temperature is 40-50 ℃, and filtering to remove filtrate to prepare carrier cellulose;
step A4: adding the carrier cellulose prepared in the step A3 and the chloride ion liquid into a reaction kettle, stirring at the rotation speed of 200-300r/min and the temperature of 80-85 ℃ until the carrier cellulose is completely dissolved to prepare a cellulose solution, adding gelatin and deionized water into the reaction kettle, stirring until the gelatin is completely dissolved, adding span 80, stirring at the rotation speed of 300-500r/min until the mixture is uniformly mixed, adding the cellulose solution at the temperature of 80-85 ℃ and continuously stirring for 30-40min, centrifuging to remove the supernatant, soaking the substrate in the deionized water for 20-25h, and drying to prepare the extraction microspheres.
Further, the using amount ratio of the moso bamboo powder and the nitric acid solution in the step A1 is 1g:8-10mL, the mass fraction of the nitric acid solution is 5-10%, the using amount ratio of the filter cake and the sodium hydroxide solution is 1g:8-10mL, and the mass fraction of the sodium hydroxide solution is 1.5-3%.
Further, the dosage ratio of the cellulose, the deionized water and the 2,2,6, 6-tetramethylpiperidine oxide solution in the step A2 is 1-1.5g:100mL:100mL, the dosage of the sodium bromide is 30-35% of the mass of the cellulose, the 2,2,6, 6-tetramethylpiperidine oxide solution is prepared by mixing 2,2,6, 6-tetramethylpiperidine oxide and deionized water in the dosage ratio of 2.5-3mg:10mL, the dosage of the sodium hypochlorite is 4-5 times of the mass of the cellulose, and the dosage of the ethanol is 1-3% of the total volume of the reaction solution.
Further, the dosage mass ratio of the octamethylcyclotetrasiloxane, the sodium hydroxide, the aminopropyl triethoxysilane and the dimethyl sulfoxide in the step A3 is 100:1-1.5:80:2.5-3, the dosage of the carboxylated cellulose is 2-3 times of that of the octamethylcyclotetrasiloxane, and the dosage of the 4-dimethylaminopyridine is 5-10% of that of the carboxylated cellulose.
Further, the mass ratio of the carrier cellulose to the chloride ion liquid in the step A4 is 1:2-2.5, the dosage ratio of the gelatin, the deionized water and the span 80 is 3g:20mL:0.5mL, and the dosage of the cellulose solution is 60-80% of the dosage of the gelatin.
The invention has the beneficial effects that: the invention prepares an extraction microsphere in the preparation process of bromelain, which takes moso bamboo as a raw material, the moso bamboo is crushed, the crushed moso bamboo is treated by nitric acid solution, the nitric acid solution can destroy lignin in the moso bamboo to form nitrolignin, the nitrolignin and hemicellulose generated are dissolved by sodium hydroxide treatment, then the cellulose is obtained by reflux drying, the cellulose is oxidized under the action of 2,6, 6-tetramethylpiperidine oxide solution, the hydroxyl on the surface of the cellulose is converted into carboxyl, then carboxyl cellulose is prepared, octamethylcyclotetrasiloxane is reacted with aminopropyltriethoxysilane under the action of sodium hydroxide to prepare silicone oil containing amino end, and then the carboxyl cellulose and the silicone oil containing the amino end are reacted under the action of 4-dimethylaminopyridine, carboxyl on the carboxyl cellulose reacts with amino on the amino-terminal silicone oil to form amide, and then the amino-terminal silicone oil is fixed on the surface of the cellulose, the cellulose has good adsorption capacity and can adsorb bromelain, the amino-terminal silicone oil is further fixed on the surface of the cellulose, the amino-terminal silicone oil is a surfactant, the adsorption effect on the bromelain is further improved, the cellulose is made into microspheres, the extracted microspheres can be reused, the extraction efficiency of the bromelain is greatly improved, impurities in the extracted bromelain are completely removed by two-time extraction, and the quality of the bromelain is greatly improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for extracting bromelain from pineapple stems and leaves comprises the following steps:
step S1: washing pineapple stem and leaf for 2 times, washing for 3min each time, drying, preserving heat for 10min at the temperature of 3 ℃, crushing, adding a phosphate buffer solution, standing for 10min at the pH value of 5, filtering, centrifuging the filtrate for 15min at the rotation speed of 4000r/min, taking the supernatant to obtain a base solution, and preserving heat for later use at the temperature of 3 ℃;
step S2: adding the base solution and the extraction microspheres prepared in the step S1 into a stirring kettle, stirring for 20min at the rotation speed of 150r/min and the temperature of 3 ℃, heating to 8 ℃, continuously stirring for 30min, filtering to remove filtrate, adding the filter cake and the sodium hydroxide solution into the reaction kettle until the pH value is 7, adding sodium chloride, stirring for 30min at the rotation speed of 100r/min, and filtering to remove filtrate to obtain eluent;
step S3: adjusting the eluent prepared in the step S2 with hydrochloric acid solution, adding ammonium sulfate after the pH value is 5, stirring until the ammonium sulfate is completely dissolved under the condition that the rotation speed is 60r/min, preserving the temperature for 12h under the condition that the temperature is 3 ℃, centrifuging for 5min under the condition that the rotation speed is 4000r/min, and removing the supernatant to obtain crude enzyme;
step S4: adding the crude enzyme and the phosphate buffer solution into a reaction kettle, stirring at the rotation speed of 60r/min and the temperature of 3 ℃ until the crude enzyme is completely dissolved, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100, performing ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 20, and performing reduced pressure drying at the temperature of 0 ℃ to obtain the bromelain.
The extraction microsphere is prepared by the following steps:
step A1: crushing moso bamboos, sieving the moso bamboos by a 80-mesh sieve to obtain moso bamboo powder, drying the moso bamboo powder, adding the moso bamboo powder into a nitric acid solution, stirring the mixture for 50min at the rotation speed of 300r/min, heating and refluxing the mixture for 3h at the temperature of 85 ℃, filtering the mixture to remove the nitric acid solution, washing a filter cake by deionized water until the pH value of a washing solution is 7, adding the filter cake into a sodium hydroxide solution, heating and refluxing the mixture for 1h at the temperature of 105 ℃, filtering the mixture to remove the sodium hydroxide solution, adding the filter cake into hydrogen peroxide with the pH value of 9, soaking the mixture for 20min, filtering the mixture to remove the hydrogen peroxide, and drying the filter cake to obtain cellulose;
step A2: adding the cellulose prepared in the step A1 and deionized water into a reaction kettle, stirring for 10min at the rotation speed of 500r/min, adding sodium bromide, continuously stirring for 3min, adding a2, 2,6, 6-tetramethylpiperidine oxide solution, continuously stirring for 5min, adding sodium hypochlorite until the reaction solution is continuously stirred and the pH value of the reaction solution is kept at 10, stirring for 4h, adding ethanol, centrifuging for 3min at the rotation speed of 3000r/min, removing supernatant, dispersing a substrate with the deionized water, adding a hydrochloric acid solution until the pH value of the reaction solution is 5, reacting for 30min, centrifuging again to remove the supernatant, cleaning the substrate with the deionized water until the cleaning solution is neutral, and drying to prepare carboxylated cellulose;
step A3: adding octamethylcyclotetrasiloxane and sodium hydroxide into a reaction kettle, stirring for 10min at the rotation speed of 300r/min, introducing nitrogen for protection, keeping the temperature at 140 ℃ for 6h, adding aminopropyltriethoxysilane and dimethyl sulfoxide, stirring for 3h at the rotation speed of 150r/min and the temperature of 90 ℃, performing equivalent neutralization with a hydrochloric acid solution, adding the carboxylated cellulose prepared in the step A2 and 4-dimethylaminopyridine, stirring for 8h at the rotation speed of 100r/min and the temperature of 40 ℃, and filtering to remove filtrate to obtain carrier cellulose;
step A4: adding the carrier cellulose prepared in the step A3 and the chloride ion liquid into a reaction kettle, stirring at the rotation speed of 200r/min and the temperature of 80 ℃ until the carrier cellulose is completely dissolved to prepare a cellulose solution, adding gelatin and deionized water into the reaction kettle, stirring until the gelatin is completely dissolved, adding span 80, stirring at the rotation speed of 300r/min until the mixture is uniformly mixed, adding the cellulose solution at the temperature of 80 ℃ and continuously stirring for 30min, centrifuging to remove supernatant, soaking a substrate in deionized water for 20h, and drying to prepare the extraction microspheres.
Example 2
A method for extracting bromelain from pineapple stems and leaves comprises the following steps:
step S1: washing pineapple stem and leaf for 3 times, drying after washing for 4min each time, preserving heat for 12min at the temperature of 4 ℃, crushing, adding phosphate buffer, standing for 12min at the pH value of 5, filtering, centrifuging the filtrate for 17min at the rotation speed of 4000r/min, taking supernatant to obtain base solution, and preserving heat for later use at the temperature of 4 ℃;
step S2: adding the base solution and the extraction microspheres prepared in the step S1 into a stirring kettle, stirring for 22min at the rotation speed of 150r/min and the temperature of 4 ℃, heating to 9 ℃, continuously stirring for 40min, filtering to remove filtrate, adding the filter cake and the sodium hydroxide solution into the reaction kettle until the pH value is 7, adding sodium chloride, stirring for 32min at the rotation speed of 130r/min, and filtering to remove filtrate to obtain eluent;
step S3: adjusting the eluent prepared in the step S2 with hydrochloric acid solution, adding ammonium sulfate after the pH value is 5, stirring until the ammonium sulfate is completely dissolved under the condition that the rotation speed is 70r/min, preserving the temperature for 13h under the condition that the temperature is 4 ℃, centrifuging for 7min under the condition that the rotation speed is 4000r/min, and removing the supernatant to obtain crude enzyme;
step S4: adding the crude enzyme and the phosphate buffer solution into a reaction kettle, stirring at the rotation speed of 70r/min and the temperature of 4 ℃ until the crude enzyme is completely dissolved, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100, performing ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 20, and performing reduced pressure drying at the temperature of 0 ℃ to obtain the bromelain.
The extraction microsphere is prepared by the following steps:
step A1: crushing moso bamboos, sieving the moso bamboos by a 80-mesh sieve to obtain moso bamboo powder, drying the moso bamboo powder, adding the moso bamboo powder into a nitric acid solution, stirring the mixture for 6min at the rotation speed of 300r/min, heating and refluxing the mixture for 4h at the temperature of 87 ℃, filtering the mixture to remove the nitric acid solution, washing a filter cake by deionized water until the pH value of a washing solution is 7, adding the filter cake into a sodium hydroxide solution, heating and refluxing the mixture for 1.5h at the temperature of 107 ℃, filtering the mixture to remove the sodium hydroxide solution, adding the filter cake into hydrogen peroxide with the pH value of 9, soaking the mixture for 22min, filtering the mixture to remove the hydrogen peroxide, and drying the filter cake to obtain cellulose;
step A2: adding the cellulose prepared in the step A1 and deionized water into a reaction kettle, stirring for 10min at the rotation speed of 500r/min, adding sodium bromide, continuously stirring for 3min, adding a2, 2,6, 6-tetramethylpiperidine oxide solution, continuously stirring for 5min, adding sodium hypochlorite until the reaction solution is continuously stirred and the pH value of the reaction solution is kept at 10, stirring for 4h, adding ethanol, centrifuging for 4min at the rotation speed of 3000r/min, removing supernatant, dispersing a substrate with the deionized water, adding a hydrochloric acid solution until the pH value of the reaction solution is 5, reacting for 40min, centrifuging again to remove supernatant, cleaning the substrate with the deionized water until the cleaning solution is neutral, and drying to obtain carboxylated cellulose;
step A3: adding octamethylcyclotetrasiloxane and sodium hydroxide into a reaction kettle, stirring for 12min at the rotation speed of 400r/min, introducing nitrogen for protection, keeping the temperature at 142 ℃ for 7h, adding aminopropyltriethoxysilane and dimethyl sulfoxide, stirring for 4h at the rotation speed of 150r/min and the temperature of 92 ℃, performing equivalent neutralization by using a hydrochloric acid solution, adding carboxylated cellulose prepared in the step A2 and 4-dimethylaminopyridine, stirring for 9h at the rotation speed of 100r/min and the temperature of 42 ℃, and filtering to remove filtrate to prepare carrier cellulose;
step A4: adding the carrier cellulose prepared in the step A3 and the chloride ion liquid into a reaction kettle, stirring at the rotation speed of 200r/min and the temperature of 80 ℃ until the carrier cellulose is completely dissolved to prepare a cellulose solution, adding gelatin and deionized water into the reaction kettle, stirring until the gelatin is completely dissolved, adding span 80, stirring at the rotation speed of 400r/min until the mixture is uniformly mixed, adding the cellulose solution at the temperature of 82 ℃ and continuously stirring for 32min, centrifuging to remove supernatant, soaking a substrate in deionized water for 22h, and drying to prepare the extraction microspheres.
Example 3
A method for extracting bromelain from pineapple stems and leaves comprises the following steps:
step S1: washing pineapple stem and leaf for 3 times, drying after washing for 4min each time, preserving heat for 14min at the temperature of 4 ℃, crushing, adding phosphate buffer, standing for 14min at the pH value of 6, filtering, centrifuging the filtrate for 15-20min at the rotation speed of 5000r/min, taking supernatant to obtain base solution, and preserving heat for later use at the temperature of 4 ℃;
step S2: adding the base solution and the extraction microspheres prepared in the step S1 into a stirring kettle, stirring for 25min at the rotation speed of 200r/min and the temperature of 4 ℃, heating to 9 ℃, continuously stirring for 50min, filtering to remove filtrate, adding the filter cake and the sodium hydroxide solution into the reaction kettle until the pH value is 7, adding sodium chloride, stirring for 35min at the rotation speed of 140r/min, and filtering to remove filtrate to obtain eluent;
step S3: adjusting the eluent prepared in the step S2 with hydrochloric acid solution, adding ammonium sulfate after the pH value is 5, stirring until the ammonium sulfate is completely dissolved under the condition that the rotation speed is 70r/min, preserving the temperature for 14h under the condition that the temperature is 4 ℃, centrifuging for 8min under the condition that the rotation speed is 5000r/min, and removing the supernatant to obtain crude enzyme;
step S4: adding the crude enzyme and the phosphate buffer solution into a reaction kettle, stirring at the rotation speed of 70r/min and the temperature of 4 ℃ until the crude enzyme is completely dissolved, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100, performing ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 20, and performing reduced pressure drying at the temperature of 0 ℃ to obtain the bromelain.
The extraction microsphere is prepared by the following steps:
step A1: crushing moso bamboos, sieving the moso bamboos by a 100-mesh sieve to obtain moso bamboo powder, drying the moso bamboo powder, adding the moso bamboo powder into a nitric acid solution, stirring the mixture for 8min at the rotation speed of 500r/min, heating and refluxing the mixture for 4h at the temperature of 90 ℃, filtering the mixture to remove the nitric acid solution, washing a filter cake by deionized water until the pH value of a washing solution is 7, adding the filter cake into a sodium hydroxide solution, heating and refluxing the mixture for 2h at the temperature of 108 ℃, filtering the mixture to remove the sodium hydroxide solution, adding the filter cake into hydrogen peroxide with the pH value of 10, soaking the mixture for 25min, filtering the mixture to remove the hydrogen peroxide, and drying the filter cake to obtain cellulose;
step A2: adding the cellulose prepared in the step A1 and deionized water into a reaction kettle, stirring for 15min at the rotation speed of 800r/min, adding sodium bromide, continuously stirring for 5min, adding a2, 2,6, 6-tetramethylpiperidine oxide solution, continuously stirring for 10min, adding sodium hypochlorite until the reaction solution is continuously stirred and the pH value of the reaction solution is kept at 10, stirring for 5h, adding ethanol, centrifuging for 5min at the rotation speed of 4000r/min, removing supernatant, dispersing a substrate with deionized water, adding a hydrochloric acid solution until the pH value of the reaction solution is 6, reacting for 50min, centrifuging again to remove supernatant, cleaning the substrate with deionized water until the cleaning solution is neutral, and drying to obtain carboxylated cellulose;
step A3: adding octamethylcyclotetrasiloxane and sodium hydroxide into a reaction kettle, stirring for 14min at the rotation speed of 500r/min, introducing nitrogen for protection, keeping the temperature at 145 ℃ for 8h, adding aminopropyltriethoxysilane and dimethyl sulfoxide, stirring for 5h at the rotation speed of 180r/min and the temperature of 95 ℃, performing equivalent neutralization with a hydrochloric acid solution, adding the carboxylated cellulose prepared in the step A2 and 4-dimethylaminopyridine, stirring for 10h at the rotation speed of 120r/min and the temperature of 45 ℃, and filtering to remove filtrate to obtain carrier cellulose;
step A4: adding the carrier cellulose prepared in the step A3 and the chloride ion liquid into a reaction kettle, stirring at the rotation speed of 300r/min and the temperature of 85 ℃ until the carrier cellulose is completely dissolved to prepare a cellulose solution, adding gelatin and deionized water into the reaction kettle, stirring until the gelatin is completely dissolved, adding span 80, stirring at the rotation speed of 500r/min until the mixture is uniformly mixed, adding the cellulose solution at the temperature of 85 ℃ and continuously stirring for 35min, centrifuging to remove supernatant, soaking a substrate in deionized water for 23h, and drying to prepare the extraction microspheres.
Example 4
A method for extracting bromelain from pineapple stems and leaves comprises the following steps:
step S1: washing pineapple stem and leaf for 4 times, washing for 5min each time, drying, preserving heat for 15min at the temperature of 5 ℃, crushing, adding a phosphate buffer solution, standing for 15min at the pH value of 6, filtering, centrifuging the filtrate for 20min at the rotation speed of 5000r/min, taking the supernatant to obtain a base solution, and preserving heat for later use at the temperature of 5 ℃;
step S2: adding the base solution and the extraction microspheres prepared in the step S1 into a stirring kettle, stirring for 30min at the rotation speed of 200r/min and the temperature of 5 ℃, heating to 10 ℃, continuously stirring for 60min, filtering to remove filtrate, adding the filter cake and the sodium hydroxide solution into the reaction kettle until the pH value is 7, adding sodium chloride, stirring for 40min at the rotation speed of 150r/min, and filtering to remove filtrate to obtain eluent;
step S3: adjusting the eluent prepared in the step S2 with hydrochloric acid solution, adding ammonium sulfate after the pH value is 5, stirring until the ammonium sulfate is completely dissolved under the condition of the rotation speed of 80r/min, preserving the temperature for 15h under the condition of the temperature of 5 ℃, centrifuging for 10min under the condition of the rotation speed of 5000r/min, and removing the supernatant to obtain crude enzyme;
step S4: adding the crude enzyme and the phosphate buffer solution into a reaction kettle, stirring at the rotation speed of 80r/min and the temperature of 5 ℃ until the crude enzyme is completely dissolved, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 100, performing ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 20, and performing reduced pressure drying at the temperature of 0 ℃ to obtain the bromelain.
The extraction microsphere is prepared by the following steps:
step A1: crushing moso bamboos, sieving the moso bamboos by a 100-mesh sieve to obtain moso bamboo powder, drying the moso bamboo powder, adding the moso bamboo powder into a nitric acid solution, stirring the mixture for 10min at the rotation speed of 500r/min, heating and refluxing the mixture for 5h at the temperature of 90 ℃, filtering the mixture to remove the nitric acid solution, washing a filter cake by deionized water until the pH value of a washing solution is 7, adding the filter cake into a sodium hydroxide solution, heating and refluxing the mixture for 2h at the temperature of 110 ℃, filtering the mixture to remove the sodium hydroxide solution, adding the filter cake into hydrogen peroxide with the pH value of 10, soaking the mixture for 30min, filtering the mixture to remove the hydrogen peroxide, and drying the filter cake to obtain cellulose;
step A2: adding the cellulose prepared in the step A1 and deionized water into a reaction kettle, stirring for 15min at the rotation speed of 800r/min, adding sodium bromide, continuously stirring for 5min, adding a2, 2,6, 6-tetramethylpiperidine oxide solution, continuously stirring for 10min, adding sodium hypochlorite until the reaction solution is continuously stirred and the pH value of the reaction solution is kept at 10, stirring for 5h, adding ethanol, centrifuging for 5min at the rotation speed of 4000r/min, removing supernatant, dispersing a substrate with deionized water, adding a hydrochloric acid solution until the pH value of the reaction solution is 6, reacting for 60min, centrifuging again to remove supernatant, cleaning the substrate with deionized water until the cleaning solution is neutral, and drying to obtain carboxylated cellulose;
step A3: adding octamethylcyclotetrasiloxane and sodium hydroxide into a reaction kettle, stirring for 15min at the rotation speed of 500r/min, introducing nitrogen for protection, keeping the temperature at 150 ℃ for 8h, adding aminopropyltriethoxysilane and dimethyl sulfoxide, stirring for 5h at the rotation speed of 200r/min and the temperature of 100 ℃, performing equivalent neutralization with a hydrochloric acid solution, adding the carboxylated cellulose prepared in the step A2 and 4-dimethylaminopyridine, stirring for 10h at the rotation speed of 150r/min and the temperature of 50 ℃, and filtering to remove filtrate to obtain carrier cellulose;
step A4: adding the carrier cellulose prepared in the step A3 and the chloride ion liquid into a reaction kettle, stirring at the rotation speed of 300r/min and the temperature of 85 ℃ until the carrier cellulose is completely dissolved to prepare a cellulose solution, adding gelatin and deionized water into the reaction kettle, stirring until the gelatin is completely dissolved, adding span 80, stirring at the rotation speed of 500r/min until the mixture is uniformly mixed, adding the cellulose solution at the temperature of 85 ℃ and continuously stirring for 40min, centrifuging to remove supernatant, soaking a substrate in deionized water for 25h, and drying to prepare the extraction microspheres.
Comparative example
The comparative example is a common extraction method of bromelain in the market.
The results of the test of the amount of extraction performed on the methods for extracting bromelain according to the above examples 1 to 4 and comparative example are shown in table 1 below;
TABLE 1
From table 1 above, it can be seen that the extraction amount of bromelain extracted by the method for extracting bromelain of examples 1 to 4 is 1.325 to 1.354mg/g, and the extraction amount of bromelain extracted by the method for extracting bromelain of comparative example is 0.932mg/g, indicating that the extraction amount of the method for extracting bromelain of the present invention is larger.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (7)
1. A method for extracting bromelain from pineapple stems and leaves is characterized in that: the method comprises the following steps:
step S1: washing pineapple stem and leaf for 2-4 times, drying after washing for 3-5min each time, preserving heat for 10-15min at the temperature of 3-5 ℃, crushing, adding phosphate buffer, standing for 10-15min at the pH value of 5-6, filtering, centrifuging the filtrate for 15-20min at the rotation speed of 4000 plus materials at 5000r/min, taking supernatant to obtain base liquid, and preserving heat for later use at the temperature of 3-5 ℃;
step S2: adding the base liquid and the extraction microspheres prepared in the step S1 into a stirring kettle, stirring for 20-30min at the rotation speed of 150-;
step S3: regulating the eluent prepared in the step S2 with hydrochloric acid solution until the pH value is 5, adding ammonium sulfate, stirring at the rotation speed of 60-80r/min until the ammonium sulfate is completely dissolved, keeping the temperature at 3-5 ℃ for 12-15h, centrifuging at the rotation speed of 4000-5000r/min for 5-10min, and removing the supernatant to obtain crude enzyme;
step S4: adding the crude enzyme and the phosphate buffer solution into a reaction kettle, stirring at the rotation speed of 60-80r/min and the temperature of 3-5 ℃ until the crude enzyme is completely dissolved, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cut-off of 100, performing ultrafiltration on the filtrate by using an ultrafiltration membrane with the molecular weight cut-off of 20, and performing reduced pressure drying at the temperature of 0 ℃ to obtain the bromelain.
2. The method for extracting and obtaining bromelain from pineapple stems and leaves as claimed in claim 1, wherein: the using amount ratio of the pineapple stem leaves and the phosphate buffer solution in the step S1 is 1g:2-2.5mL, the concentration of the phosphate buffer solution is 0.05-0.1mol/L, the using amount ratio of the base solution and the extraction microspheres in the step S2 is 10mL:2-3g, the mass fraction of the sodium hydroxide solution is 15-18%, the mass fraction of the hydrochloric acid solution in the step S2 is 12-15%, and the using amount of ammonium sulfate is 20-25% of the using amount of the eluent.
3. The method for extracting and obtaining bromelain from pineapple stems and leaves as claimed in claim 1, wherein: the extraction microsphere is prepared by the following steps:
step A1: crushing moso bamboo, sieving the crushed moso bamboo through a sieve of 80-100 meshes to obtain moso bamboo powder, drying the moso bamboo powder, adding the dried moso bamboo powder into a nitric acid solution, stirring the mixture for 5-10min at the rotation speed of 500r/min, carrying out heating reflux for 3-5h at the temperature of 85-90 ℃, filtering to remove the nitric acid solution, washing a filter cake with deionized water until the pH value of a washing solution is 7, adding the filter cake into a sodium hydroxide solution, carrying out heating reflux for 1-2h at the temperature of 105-110 ℃, filtering to remove the sodium hydroxide solution, adding the filter cake into hydrogen peroxide with the pH value of 9-10, soaking the hydrogen peroxide for 20-30min, filtering to remove the hydrogen peroxide, and drying the filter cake to obtain cellulose;
step A2: adding the cellulose prepared in the step A1 and deionized water into a reaction kettle, stirring for 10-15min at the rotating speed of 500-800r/min, adding sodium bromide, stirring for 3-5min, adding 2,2,6, 6-tetramethylpiperidine oxide solution, stirring for 5-10min, adding sodium hypochlorite until the reaction solution is stirred continuously and the pH value of the reaction solution is kept at 10, stirring for 4-5h, adding ethanol, centrifuging at 3000-4000r/min for 3-5min, removing supernatant, dispersing substrate with deionized water, adding hydrochloric acid solution until the pH value of the reaction solution is 5-6, reacting for 30-60min, centrifuging again to remove supernatant, cleaning the substrate with deionized water until the cleaning solution is neutral, and drying to obtain carboxylated cellulose;
step A3: adding octamethylcyclotetrasiloxane and sodium hydroxide into a reaction kettle, stirring for 10-15min under the condition that the rotation speed is 300-150℃, introducing nitrogen for protection, keeping the temperature for 6-8h under the condition that the temperature is 140-150℃, adding aminopropyltriethoxysilane and dimethyl sulfoxide, stirring for 3-5h under the condition that the rotation speed is 150-200℃ and the temperature is 90-100 ℃, adding carboxylated cellulose prepared in the step A2 and 4-dimethylaminopyridine after carrying out equivalent neutralization by using a hydrochloric acid solution, stirring for 8-10h under the conditions that the rotation speed is 100-150℃ and the temperature is 40-50 ℃, and filtering to remove filtrate to prepare carrier cellulose;
step A4: adding the carrier cellulose prepared in the step A3 and the chloride ion liquid into a reaction kettle, stirring at the rotation speed of 200-300r/min and the temperature of 80-85 ℃ until the carrier cellulose is completely dissolved to prepare a cellulose solution, adding gelatin and deionized water into the reaction kettle, stirring until the gelatin is completely dissolved, adding span 80, stirring at the rotation speed of 300-500r/min until the mixture is uniformly mixed, adding the cellulose solution at the temperature of 80-85 ℃ and continuously stirring for 30-40min, centrifuging to remove the supernatant, soaking the substrate in the deionized water for 20-25h, and drying to prepare the extraction microspheres.
4. The method for extracting and obtaining bromelain from pineapple stems and leaves as claimed in claim 3, wherein: the dosage ratio of the moso bamboo powder and the nitric acid solution in the step A1 is 1g:8-10mL, the mass fraction of the nitric acid solution is 5-10%, the dosage ratio of the filter cake and the sodium hydroxide solution is 1g:8-10mL, and the mass fraction of the sodium hydroxide solution is 1.5-3%.
5. The method for extracting and obtaining bromelain from pineapple stems and leaves as claimed in claim 3, wherein: the dosage ratio of the cellulose, the deionized water and the 2,2,6, 6-tetramethylpiperidine oxide solution in the step A2 is 1-1.5g:100mL:100mL, the dosage of the sodium bromide is 30-35% of the mass of the cellulose, the 2,2,6, 6-tetramethylpiperidine oxide solution is prepared by mixing 2,2,6, 6-tetramethylpiperidine oxide and deionized water with the dosage ratio of 2.5-3mg:10mL, the dosage of the sodium hypochlorite is 4-5 times of the mass of the cellulose, and the dosage of the ethanol is 1-3% of the total volume of the reaction solution.
6. The method for extracting and obtaining bromelain from pineapple stems and leaves as claimed in claim 3, wherein: the mass ratio of the octamethylcyclotetrasiloxane, the sodium hydroxide, the aminopropyltriethoxysilane and the dimethyl sulfoxide in the step A3 is 100:1-1.5:80:2.5-3, the mass of the carboxylated cellulose is 2-3 times of that of the octamethylcyclotetrasiloxane, and the mass of the 4-dimethylaminopyridine is 5-10% of that of the carboxylated cellulose.
7. The method for extracting and obtaining bromelain from pineapple stems and leaves as claimed in claim 3, wherein: the mass ratio of the carrier cellulose and the chloride ion liquid in the step A4 is 1:2-2.5, the dosage ratio of the gelatin, the deionized water and the span-80 is 3g:20mL:0.5mL, and the dosage of the cellulose solution is 60-80% of that of the gelatin.
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