CN111996183A - Preparation method of bromelain - Google Patents
Preparation method of bromelain Download PDFInfo
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- CN111996183A CN111996183A CN202010911902.6A CN202010911902A CN111996183A CN 111996183 A CN111996183 A CN 111996183A CN 202010911902 A CN202010911902 A CN 202010911902A CN 111996183 A CN111996183 A CN 111996183A
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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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- 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
- B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
- B01J41/04—Processes using organic exchangers
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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
- B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
- B01J41/08—Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
- B01J41/09—Organic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22033—Fruit bromelain (3.4.22.33), i.e. juice bromelain
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Abstract
The invention discloses a preparation method of bromelain, which comprises the following steps: (1) enabling the pineapple pretreatment juice to pass through an anion exchange resin column at the flow speed of 2-5 BV/h to obtain an effluent liquid; (2) filtering the effluent with microporous membrane to obtain filtrate; (3) mixing the filtrate with a precipitator, and standing for 1-5 h at 4-15 ℃ to obtain bromelain paste; (4) and (3) carrying out centrifugal separation on the bromelain paste at the temperature of 0-10 ℃ and the rotating speed of 10000-18000 rpm to obtain bromelain paste, and then carrying out freeze drying to obtain powdery bromelain. The extraction method of the invention ensures that the activity of the extracted bromelain is higher.
Description
Technical Field
The invention relates to a preparation method of bromelain.
Background
Bromelain allows the body's immune system to more readily recognize unhealthy cells and provides a more optimal protective regime. The bromelain is mainly extracted from pineapple fruits, stems, leaves and peels and is obtained by steps of purification and the like. The preparation method of the bromelain is very important, and is inappropriate, so that the activity of the obtained bromelain is low, and the application of the bromelain is greatly influenced.
CN104611316 discloses a method for extracting bromelain, which comprises the steps of dialyzing by ultrafiltration equipment after obtaining pineapple juice to reduce conductivity, adjusting pH and cooling to enable the bromelain to generate flocculent precipitate, separating enzyme paste, and freeze-drying to obtain the finished product of the bromelain. The method has long production period and low extraction yield. CN106032530A discloses a method for extracting bromelain from pineapple stem, which adopts tannic acid method, and the purity of bromelain still needs to be improved. CN109385414A discloses a purification method of bromelain, which adopts an ion exchange resin column to purify bromelain, and controls the content of metal ions in the purified bromelain within a certain range. The method comprises the steps of enabling pineapple juice to pass through a cation exchange resin column and an anion exchange resin column in sequence, so that the metal content can be further reduced well, but the activity of bromelain needs to be improved due to the fact that the pineapple juice needs to be subjected to pH value adjustment for many times during the period, and the pineapple juice passes through the anion exchange resin column and the cation exchange resin column.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a method for extracting bromelain, which can further retain the activity of bromelain. The invention adopts the following technical scheme to achieve the purpose.
The invention provides a preparation method of bromelain, which comprises the following steps:
(1) enabling the pineapple pretreatment juice to pass through an anion exchange resin column at the flow speed of 2-5 BV/h to obtain an effluent liquid;
(2) filtering the effluent with microporous membrane to obtain filtrate;
(3) mixing the filtrate with a precipitator, and standing for 1-5 h at 4-15 ℃ to obtain bromelain paste;
(4) and (3) carrying out centrifugal separation on the bromelain paste at the temperature of 0-10 ℃ and the rotating speed of 10000-18000 rpm to obtain bromelain paste, and then carrying out freeze drying to obtain powdery bromelain.
According to the preparation method provided by the invention, preferably, in the step (1), the pH value of the pineapple pretreatment juice is 4.5-6.5.
According to the preparation method of the invention, the preparation method preferably further comprises a pineapple juice pretreatment step: mixing the pineapple juice, the preservative and the antioxidant, and carrying out solid-liquid separation treatment and filtration to obtain the pineapple pretreatment juice.
According to the preparation method of the invention, preferably, in the pretreatment step of the pineapple juice, the preservative is sodium benzoate, the antioxidant is sodium metabisulfite, and the weight ratio of the pineapple juice, the sodium benzoate and the sodium metabisulfite is 1000: 0.05-0.18.
According to the preparation method of the invention, preferably, in the pretreatment step of the pineapple juice, the solid-liquid separation treatment comprises centrifugal separation at the temperature of 0-10 ℃ and the rotation speed of 10000-18000 rpm; the filtration adopts a microporous filter membrane with the aperture of 0.5-2 microns.
According to the preparation method of the present invention, preferably, in the step (1), the anion exchange resin column is obtained by activating treatment using the following steps:
(a) swelling anion exchange resin with purified water, and then loading the anion exchange resin into a column;
(b) passing a 1-2 mol/L sodium hydroxide solution through a column, and then cleaning with purified water until an effluent liquid is nearly neutral;
(c) and (3) passing a 1-2 mol/L hydrochloric acid solution through a column, and then washing with purified water until an effluent liquid is nearly neutral to obtain the anion exchange resin column.
According to the production method of the present invention, preferably, in the step (a), the anion exchange resin is an anion exchange resin having a quaternary ammonium group.
According to the preparation method of the invention, preferably, in the step (b), the flow rate of the sodium hydroxide solution is 1-2 BV/h, and the dosage of the sodium hydroxide solution is 2-4 BV; in the step (c), the flow rate of the hydrochloric acid solution is 1-2 BV/h, and the dosage of the hydrochloric acid solution is 2-4 BV.
According to the preparation method of the invention, in the step (3), the ratio of the volume of the filtrate in mL to the weight of the precipitant in g is preferably 1000: 2-5.
According to the preparation method of the present invention, it is preferable that the activity of bromelain is more than 800 units/mg.
The preparation method of the bromelain only passes through the anion exchange resin column, thereby reducing the influence on the activity of the bromelain and enabling the activity of the extracted bromelain to be higher.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
In the present invention, BV represents a column volume. The extraction method comprises the steps of pineapple juice pretreatment, ion exchange resin column activation and bromelain preparation. The order of the pineapple juice pretreatment step and the ion exchange resin column activation step is not particularly limited. As will be explained in detail below.
< pineapple juice pretreatment step >
In the present invention, the pineapple juice is obtained by squeezing the fruit, stem, leaf or skin of pineapple, and the squeezing method is not limited. The pineapple juice pretreatment step is to mix pineapple juice, preservative and antioxidant, and obtain the pineapple pretreatment juice through solid-liquid separation treatment and filtration.
The pineapple juice pretreatment step comprises the step of mixing the pineapple juice with a preservative and an antioxidant. The manner and order of mixing are not particularly limited. For example, the preservative and the antioxidant are added to the pineapple juice simultaneously, and the mixture is stirred while being added, thereby forming the pineapple pretreatment juice. Freezing and storing the pineapple pretreatment juice. The weight ratio of the pineapple juice to the preservative to the antioxidant is 1000: 0.05-0.18, preferably 1000: 0.08-0.15, and more preferably 1000: 0.10-0.12. According to one embodiment of the invention, the preservative is sodium benzoate and the antioxidant is sodium metabisulfite. The weight ratio of the pineapple juice to the sodium benzoate to the sodium metabisulfite is 1000: 0.05-0.18, preferably 1000: 0.08-0.15, and more preferably 1000: 0.10-0.12. According to a preferred embodiment of the invention, the weight ratio of pineapple juice, sodium benzoate and sodium metabisulphite is 1000:0.1: 0.1. By adopting the proportion of the invention, the activity of bromelain can be effectively maintained.
The pineapple juice pretreatment step can also comprise a step of carrying out solid-liquid separation after the pineapple juice is mixed with the preservative and the antioxidant. The solid-liquid separation method is selected from filtration or centrifugal separation. The solid-liquid separation is performed at a low temperature, preferably 0 to 10 ℃, more preferably 0 to 6 ℃, and still more preferably 4 ℃. The solid-liquid separation operation at the temperature is beneficial to keeping better activity of the bromelain. The solid-liquid separation preferably adopts centrifugal separation, and the centrifugal temperature is 0-6 ℃. The centrifugal rate is 10000-18000 rpm, preferably 14000-18000 rmp, more preferably 15000-17000 rmp, and still more preferably 16000 rmp.
The pineapple juice pretreatment can also comprise a step of filtering a product obtained by solid-liquid separation by using a microporous filter membrane. The aperture of the microporous filter membrane is 0.5-5 μm, preferably 1-2 μm.
According to one embodiment of the invention, in the pretreatment step of the pineapple juice, the preservative is sodium benzoate, the antioxidant is sodium metabisulfite, and the weight ratio of the pineapple juice, the sodium benzoate and the sodium metabisulfite is 1000: 0.05-0.18; the solid-liquid separation treatment comprises the step of carrying out centrifugal separation at the temperature of 0-10 ℃ and the rotating speed of 10000-18000 rpm; the filtration adopts a microporous filter membrane with the aperture of 0.5-2 microns.
< step of activating ion exchange resin column >
The anion exchange resin column used in the bromelain extraction step is an activated anion exchange resin column.
The anion exchange resin column is obtained by adopting the following steps of activation treatment: (a) swelling anion exchange resin with purified water, and then loading the anion exchange resin into a column; (b) passing a 1-2 mol/L sodium hydroxide solution through a column, and then cleaning with purified water until an effluent liquid is nearly neutral; (c) and (3) passing a 1-2 mol/L hydrochloric acid solution through a column, and then washing with purified water until an effluent liquid is nearly neutral to obtain the anion exchange resin column. According to one embodiment of the present invention, the activation treatment of the anion exchange resin column comprises the steps of: (a) washing anion exchange resin with purified water, swelling, and packing to obtain anion exchange resin column; (b) passing a 1-2 mol/L sodium hydroxide solution through the column, wherein the flow rate of the sodium hydroxide is 1-2 BV/h, the using amount of the sodium hydroxide solution is 2-4 BV, and washing with purified water until the effluent liquid is nearly neutral; (c) and then, passing a 1-2 mol/L hydrochloric acid solution through the column, wherein the flow rate of the hydrochloric acid solution is 1-2 BV/h, the using amount of the hydrochloric acid solution is 2-4 BV, and washing with purified water until the effluent liquid is nearly neutral to obtain the activated anion exchange resin column.
The anion exchange resin of the present invention is an anion exchange resin with quaternary ammonium groups. Preferably, the anion exchange resin is selected from the group consisting of 717 type anion exchange resin, 711 type anion exchange resin, D201 type anion exchange resin, more preferably 717 type anion exchange resin. The invention finds that compared with 711 type anion exchange resin, 717 type anion exchange resin is adopted to reduce the metal content of bromelain and better retain higher activity of bromelain. In the present invention, the 717 type of anion exchange resin has a significantly better effect than other types of anion exchange resins. Preferably, when the pineapple pretreatment juice passes through the activated anion exchange resin column, the sample loading amount is 9-14 times, more preferably 10-12 times of the volume of the anion exchange resin column.
The height-diameter ratio of the anion exchange resin column in the step (a) can be 1-8: 1, preferably 2-6: 1, more preferably 3-5: 1, and further preferably 3-4: 1. According to a preferred embodiment of the invention, the anion exchange resin column has an aspect ratio of 4: 1.
The concentration of the sodium hydroxide solution in the step (b) can be 1-2 mol/L, preferably 1.2-1.8 mol/L, and more preferably 1.3-1.5 mol/L. The flow rate of the sodium hydroxide solution can be 1-2 BV/h, preferably 1.2-1.8 BV/h, and more preferably 1.3-1.5 BV/h. The amount of the sodium hydroxide solution may be 2 to 4BV, preferably 2.5 to 3.5BV, more preferably 2.8 to 3 BV. According to a preferred embodiment of the present invention, the concentration of the sodium hydroxide solution in step (b) is 1.5mol/L, the flow rate of the sodium hydroxide solution is 1.5BV/h, and the amount of the sodium hydroxide solution is 3 BV.
The concentration of the hydrochloric acid solution in the step (c) can be 1-2 mol/L, preferably 1.2-1.8 mol/L, and more preferably 1.5 mol/L. The flow rate of the hydrochloric acid solution can be 1-2 BV/h, preferably 1.2-1.8 BV/h, and more preferably 1.5 BV/h; the amount of the hydrochloric acid solution may be 2-4 BV, preferably 2.5-3.5 BV, more preferably 2.8-3 BV. According to a preferred embodiment of the present invention, the hydrochloric acid solution of step (c) has a concentration of 1.5mol/L, a flow rate of 1.5BV/h and an amount of 3 BV.
< Bromelain extraction step >
In the invention, the extraction step comprises the following steps:
(1) enabling the pineapple pretreatment juice to pass through an anion exchange resin column at the flow speed of 2-5 BV/h to obtain an effluent liquid;
(2) filtering the effluent with microporous membrane to obtain filtrate;
(3) mixing the filtrate with a precipitator, and standing for 1-5 h at 4-15 ℃ to obtain bromelain paste;
(4) and (3) carrying out centrifugal separation on the bromelain paste at the temperature of 0-10 ℃ and the rotating speed of 10000-18000 rpm to obtain bromelain paste, and then carrying out freeze drying to obtain powdery bromelain. The preparation method can be used for preparing bromelain activity with activity more than 800 units/mg.
In the step (1), the pineapple pretreatment juice is obtained by the method, and details are not repeated. The pH value of the pineapple pretreatment juice is adjusted to 5.5-6.5, preferably 5-6, and more preferably 5.2-4.8 before passing through an anion exchange resin column. The pH of the pineapple pretreatment juice can be adjusted by using a sodium hydroxide solution. The concentration of the sodium hydroxide solution can be 30-50 wt%, preferably 35-45 wt%, and more preferably 38-40 wt%, and by adopting the concentration of the sodium hydroxide solution, the pH can be quickly adjusted without affecting the activity of the bromelain. The invention discovers that the pineapple pretreatment juice with the pH value is used as the sample loading liquid for anion exchange, so that the influence on the activity of the bromelain is less when impurity ions are removed, and the higher activity of the bromelain is ensured compared with the traditional ion column impurity removal.
The pineapple pretreatment juice passes through an anion exchange resin column, and the flow rate is 2-5 BV/h, preferably 3-4 BV/h, and more preferably 3.5-3.7 BV/h. Collecting the effluent. During the anion exchange process, the loading amount is not more than the loading amount of the anion exchange resin column. For example, the pH and conductivity of the effluent may be periodically measured as a reference indicator to determine if it is overloaded.
And (3) in the step (2), filtering the effluent liquid by using a microporous membrane to obtain filtrate after impurity removal and purification treatment. This enables efficient removal of microorganisms. The pore diameter of the microporous filter membrane is 0.1-0.5 micron, preferably 0.15-0.22 micron.
And (3) mixing the filtrate with a precipitator, and standing for 1-5 hours at 4-15 ℃ to obtain the bromelain paste. The ratio of the volume of the filtrate in mL to the weight of the precipitant in g may be 1000:2 to 5, preferably 1000:2 to 4, and more preferably 1000:2 to 2.5. After being uniformly mixed, the mixture is kept stand at 4-15 ℃, preferably 5-10 ℃, and more preferably 6 ℃. The bromelain activity precipitated at this temperature was higher. The standing time can be 1-5 h, preferably 2-3 h. In the present invention, the precipitant is one selected from tannic acid, polyacrylic acid, or ethanol; preferably, the precipitating agent is selected from one of tannic acid or polyacrylic acid; more preferably, the precipitating agent is tannic acid. The invention finds that the precipitant is more beneficial to the retention of the activity of bromelain by adopting the precipitant, and particularly, the bromelain with higher activity can be obtained by adopting tannic acid.
And (4) centrifuging the bromelain paste at a centrifugal temperature of 0-10 ℃ to obtain bromelain paste, and drying the bromelain paste to obtain a bromelain product. The centrifugation temperature may be 0 to 10 ℃, preferably 0 to 6 ℃, and more preferably 1 to 4 ℃. The centrifugation rate can be 12000-20000 rpm, preferably 14000-18000 rmp, more preferably 15000-17000 rmp, and more preferably 16000 rmp. The drying mode can be vacuum drying or vacuum freeze drying, and is preferably freeze drying.
Example 1
< activation of type 717 anion exchange resin column >
(a) Fully washing the 717 type anion exchange resin with purified water, swelling, and packing to obtain a 717 type anion exchange resin column with the height-diameter ratio of 4: 1;
(b) passing a 1.5mol/L sodium hydroxide solution through the column, wherein the flow rate of the sodium hydroxide solution is 1.5BV/h, the using amount of the sodium hydroxide solution is 3BV, and washing with purified water until the effluent liquid is nearly neutral;
(c) then 1.5mol/L hydrochloric acid solution is adopted to pass through the column, the flow rate of the hydrochloric acid solution is 1.5BV/h, the using amount of the hydrochloric acid solution is 3BV, and purified water is used for washing until the effluent liquid is nearly neutral; an activated 717 type anion exchange resin column was obtained.
< pretreatment of pineapple juice >
Taking pineapple juice obtained by squeezing pineapple peel, and adjusting the pH value of the pineapple juice to 5.5 by using a sodium hydroxide solution with the concentration of 40 wt%; mixing pineapple juice with sodium benzoate and sodium metabisulfite at a weight ratio of 1000:0.1:0.1, stirring, standing, centrifuging at 16000rpm and 4 deg.C with a tubular centrifuge, and filtering with 1 μm microporous membrane to obtain pineapple pretreatment juice.
< extraction of Bromelain >
(1) The pineapple pretreatment juice is passed through an anion exchange resin column at the flow rate of 3.5BV/h to obtain an effluent liquid;
(2) filtering the effluent through a 0.22 mu m microporous filter membrane to obtain filtrate;
(3) mixing the filtrate with tannic acid, and standing at 6 deg.C for 3.5 hr to obtain bromelain paste.
(4) Mixing 1000mL of filtrate with 2g of tannic acid, stirring, standing at 6 ℃ for 2.5h, settling enzyme paste, and removing supernatant to obtain bromelain enzyme paste;
(5) centrifuging the enzyme paste at 16000rpm at 4 deg.C to obtain bromelain enzyme paste; and (4) carrying out vacuum freeze drying on the bromelain enzyme paste to obtain a bromelain product. The properties of the bromelain product are shown in table 1.
Example 2
< activation of type 717 anion exchange resin column >
(a) Washing 717 type anion exchange resin with purified water, swelling, and packing to obtain 711 type anion exchange resin column with height-diameter ratio of 4: 1;
(b) passing 1.5mol/L sodium hydroxide solution through the column, wherein the flow rate of the sodium hydroxide solution is 2BV/h, the using amount of the sodium hydroxide solution is 3BV, and washing with purified water until the effluent liquid is nearly neutral;
(c) then 1.5mol/L hydrochloric acid solution is adopted to pass through the column, the flow rate of the hydrochloric acid solution is 2BV/h, the consumption of the hydrochloric acid solution is 3BV, and purified water is used for washing until the effluent liquid is nearly neutral; an activated 711 type anion exchange resin column was obtained.
< pretreatment of pineapple juice >
Taking pineapple juice obtained by squeezing pineapple peel, and adjusting the pH value of the pineapple juice to be 6 by using a sodium hydroxide solution with the concentration of 40 wt%; mixing pineapple juice with sodium benzoate and sodium metabisulfite at a weight ratio of 1000:0.1:0.1, stirring, standing, centrifuging at 16000rpm and 4 deg.C with a tubular centrifuge, and filtering with 1 μm microporous membrane to obtain pineapple pretreatment juice.
< extraction of Bromelain >
(1) The pineapple pretreatment juice is passed through an anion exchange resin column at the flow rate of 3.5BV/h to obtain an effluent liquid;
(2) filtering the effluent through a 0.22 mu m microporous filter membrane to obtain filtrate;
(3) mixing the filtrate with tannic acid, and standing at 6 deg.C for 3.5 hr to obtain bromelain paste.
(4) Mixing 1000mL of filtrate with 2g of tannic acid, stirring, standing at 6 ℃ for 2.5h, settling enzyme paste, and removing supernatant to obtain bromelain enzyme paste;
(5) centrifuging the enzyme paste at 16000rpm at 4 deg.C to obtain bromelain enzyme paste; and (4) carrying out vacuum freeze drying on the bromelain enzyme paste to obtain a bromelain product. The properties of the bromelain product are shown in table 1.
Comparative example
(1) Adjusting the pineapple pretreatment juice of example 1 to a pH of 3.0 with 30 wt% hydrochloric acid to obtain a first sample solution;
(2) passing the first sample solution through an activated 732 type cation exchange resin column at the flow rate of 1.5BV/h, and collecting the effluent liquid;
(3) regulating the effluent to pH 7.0 through a 40 wt% hydrochloric acid solution to obtain a second sample solution;
(4) passing the second sample solution through an activated 717 type anion exchange resin column at the flow rate of 3.5BV/h, and collecting effluent liquid as purified pineapple juice;
(5) passing the purified pineapple juice through a microporous filter membrane with the aperture of 0.22 mu m to obtain filtrate after impurity removal and purification treatment;
(6) mixing 1000mL of filtrate with 2g of tannic acid, stirring, standing at 6 ℃ for 2.5h, settling enzyme paste, and removing supernatant to obtain bromelain enzyme paste;
(7) centrifuging the bromelain paste at the temperature of 4 ℃ and the rpm of 16000 to obtain bromelain paste; and (4) carrying out vacuum freeze drying on the bromelain enzyme paste to obtain a bromelain product. The properties of the bromelain product are shown in table 1.
TABLE 1
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (10)
1. The preparation method of the bromelain is characterized by comprising the following steps:
(1) enabling the pineapple pretreatment juice to pass through an anion exchange resin column at the flow speed of 2-5 BV/h to obtain an effluent liquid;
(2) filtering the effluent with microporous membrane to obtain filtrate;
(3) mixing the filtrate with a precipitator, and standing for 1-5 h at 4-15 ℃ to obtain bromelain paste;
(4) and (3) carrying out centrifugal separation on the bromelain paste at the temperature of 0-10 ℃ and the rotating speed of 10000-18000 rpm to obtain bromelain paste, and then carrying out freeze drying to obtain powdery bromelain.
2. The preparation method according to claim 1, wherein in the step (1), the pH value of the pineapple pretreatment juice is 4.5-6.5.
3. The method of claim 1, further comprising a step of pre-treating the pineapple juice: mixing the pineapple juice, the preservative and the antioxidant, and carrying out solid-liquid separation treatment and filtration to obtain the pineapple pretreatment juice.
4. The method according to claim 3, wherein in the step of pretreating pineapple juice, the preservative is sodium benzoate, the antioxidant is sodium metabisulfite, and the weight ratio of the pineapple juice, the sodium benzoate and the sodium metabisulfite is 1000: 0.05-0.18.
5. The method according to claim 4, wherein in the step of pretreating pineapple juice, the solid-liquid separation treatment comprises centrifugal separation at a temperature of 0 to 10 ℃ and a rotation speed of 10000 to 18000 rpm; the filtration adopts a microporous filter membrane with the aperture of 0.5-2 microns.
6. The method according to claim 1, wherein in the step (1), the anion exchange resin column is obtained by activating treatment using the following steps:
(a) swelling anion exchange resin with purified water, and then loading the anion exchange resin into a column;
(b) passing a 1-2 mol/L sodium hydroxide solution through a column, and then cleaning with purified water until an effluent liquid is nearly neutral;
(c) and (3) passing a 1-2 mol/L hydrochloric acid solution through a column, and then washing with purified water until an effluent liquid is nearly neutral to obtain the anion exchange resin column.
7. The method according to claim 6, wherein in the step (a), the anion exchange resin is an anion exchange resin having a quaternary ammonium group.
8. The method according to claim 7, wherein in the step (b), the flow rate of the sodium hydroxide solution is 1-2 BV/h, and the amount of the sodium hydroxide solution is 2-4 BV; in the step (c), the flow rate of the hydrochloric acid solution is 1-2 BV/h, and the dosage of the hydrochloric acid solution is 2-4 BV.
9. The method of claim 8, wherein: in the step (3), the ratio of the volume of the filtrate in mL to the weight of the precipitant in g is 1000: 2-5.
10. The method of any one of claims 1 to 9, wherein the activity of bromelain is greater than 800 units/mg.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215137A (en) * | 2021-06-11 | 2021-08-06 | 江苏格局生物医药科技有限公司 | Preparation method of enzyme for pharmaceutical industry |
| CN114381449A (en) * | 2022-01-28 | 2022-04-22 | 中国热带农业科学院海口实验站 | Purification method of bromelain extract |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109385414A (en) * | 2017-08-09 | 2019-02-26 | 广西南宁百会药业集团有限公司 | The purification process of bromelain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109385414A (en) * | 2017-08-09 | 2019-02-26 | 广西南宁百会药业集团有限公司 | The purification process of bromelain |
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| CN113215137A (en) * | 2021-06-11 | 2021-08-06 | 江苏格局生物医药科技有限公司 | Preparation method of enzyme for pharmaceutical industry |
| CN114381449A (en) * | 2022-01-28 | 2022-04-22 | 中国热带农业科学院海口实验站 | Purification method of bromelain extract |
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