CN109385414B - Purification method of bromelain - Google Patents
Purification method of bromelain Download PDFInfo
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- CN109385414B CN109385414B CN201710675070.0A CN201710675070A CN109385414B CN 109385414 B CN109385414 B CN 109385414B CN 201710675070 A CN201710675070 A CN 201710675070A CN 109385414 B CN109385414 B CN 109385414B
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- 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 purification method of bromelain, which comprises the steps of (1) adjusting the pH value of pineapple juice to 2.5-3.5 to obtain pineapple juice sample loading liquid; (2) passing the pineapple juice sample solution through an activated cation exchange resin column at the flow rate of 2.5-4.5BV/h, collecting effluent, and adjusting the pH of the effluent to 6.5-7.5 to obtain a first effluent; (3) passing the first effluent through an activated anion exchange resin column at a flow rate of 2.5-4.5BV/h, and collecting the effluent; adjusting the pH of the effluent to 3.0-4.0 to obtain a purified pineapple juice treatment solution. The method can effectively reduce the content of metal ions, particularly the content of iron ions, in the bromelain product, effectively improve the qualification rate of the bromelain product and ensure that the quality of the bromelain product is stable and controllable.
Description
Technical Field
The invention relates to an impurity removal and purification treatment method of bromelain and a preparation method of bromelain products.
Background
Bromelain, also known as bromelain or pineapple ferment, is a natural plant protease which is mainly extracted from pineapples, stems, leaves and skins and is obtained by steps of purification and the like. Bromelain is a complex mixture of protein digestive enzymes, is an enzyme system consisting of a plurality of enzymes with different molecular weights and molecular structures, mainly consists of cysteine protease, and also contains phosphatase, peroxidase, cellulase, other glycosidases and the like.
Modern researches show that the bromelain has important medicinal value and has an immunostimulating effect; can be effective in treating inflammation and edema in various tissues; treating diarrhea; can also be used for burn decrustation, and optionally removing dead skin; inhibiting tumor cell growth and reducing toxin binding activity. In addition to the pharmaceutical field, bromelain is also used in the fields of foods, cosmetics and the like.
The existing production process of bromelain mainly comprises an ammonium sulfate precipitation method, an ultrafiltration method, a tannin precipitation method and the like. In the ammonium sulfate precipitation method, salt ions competitively bind with water molecules on the surface of protein in a high-concentration ammonium sulfate solution to destroy a hydration film on the surface of the protein, so that the solubility is reduced, and the protein is aggregated under the hydrophobic action to form a precipitate. The ammonium sulfate precipitation method has serious environmental pollution because of the need of using a large amount of ammonium sulfate, and simultaneously, the produced protease may bring ammonium sulfate, so the method has low protein yield, unstable process and time consumption. The ultrafiltration method is characterized in that under certain pressure and flow, an asymmetric microporous structure and a semipermeable membrane medium are utilized, pressure difference on two sides of the membrane is used as a driving force, filtration is carried out in a cross flow mode, a solvent and small molecular substances pass through, and macromolecular proteins are retained by the filter membrane, so that the purposes of separation and purification are achieved. The ultrafiltration method requires large equipment investment, the viscosity of the pineapple juice is gradually increased in the ultrafiltration concentration process, and the ultrafiltration membrane is easy to block, so the production period is long, the cost is high, and the extraction yield is low. The tannin precipitation method is to precipitate proteins by utilizing the binding of tannic acid to the amino terminus of amino acids in the proteins.
In actual production, the quality difference of the bromelain prepared from raw materials with different sources and batches by using the same production method is large, the problem of unstable quality of the bromelain is prominent, and the product yield is low.
Disclosure of Invention
In order to solve the above problems, the inventors have conducted extensive studies and have unexpectedly found that the content of metal ions, particularly iron ions, in the pineapple raw material has a large influence on the enzymatic activity of bromelain. When the content of iron ions in the pineapple raw material for extracting the bromelain is high, the produced bromelain is easy to be unqualified, which is an important reason for the unstable quality of the bromelain produced by the existing method.
The invention aims to provide a purification method of bromelain, which can effectively reduce the content of metal ions, particularly the content of iron salt in bromelain products, thereby improving the qualification rate of the bromelain products and ensuring the quality of the bromelain products to be stable and controllable. The invention further aims to provide a purification method of bromelain, and the enzyme activity of the bromelain obtained by the method is obviously improved.
The invention also aims to provide a preparation method of the bromelain product.
The purpose of the invention is realized by the following technical scheme:
a purification method of bromelain comprises the following steps:
(1) adjusting the pH value of the pineapple juice to 2.5-3.5 to obtain pineapple juice sample liquid;
(2) passing the pineapple juice sample solution through an activated cation exchange resin column at the flow rate of 2.5-4.5BV/h, collecting effluent, and adjusting the pH of the effluent to 6.5-7.5 to obtain a first effluent;
(3) passing the first effluent through an activated anion exchange resin column at a flow rate of 2.5-4.5BV/h, and collecting the effluent; adjusting the pH of the effluent to 3.0-4.0 to obtain a purified pineapple juice treatment solution.
In the present invention, preferably, the pineapple juice in the step (1) is a pretreated pineapple juice; the pretreatment comprises the steps of mixing the pineapple juice with a preservative and an antioxidant, stirring, carrying out solid-liquid separation operation, and carrying out electrodialysis, thereby obtaining the pretreated pineapple juice.
In the invention, preferably, in the step (1), the pH of the pineapple normal juice is 3.5-5.5, the preservative is sodium benzoate, the antioxidant is sodium metabisulfite, and the weight ratio of the pineapple juice to the sodium benzoate to the sodium metabisulfite is 1000:0.05-0.2: 0.05-0.2; the solid-liquid separation operation comprises centrifugation at a centrifugation temperature of 0-6 ℃ and filtration with a microporous membrane.
In the present invention, preferably, in the step (2), the activated cation exchange resin column is prepared by:
(A) swelling cation exchange resin with purified water, and packing to obtain cation exchange resin column;
(B) passing 0.5-4mol/L hydrochloric acid solution through the column at a flow rate of 0.5-3BV/h and a consumption of 1-6BV, and washing with purified water until the effluent is nearly neutral;
(C) then 0.5-4mol/L sodium hydroxide solution is adopted to pass through the column, the flow rate of the sodium hydroxide solution is 0.5-3BV/h, the using amount of the sodium hydroxide solution is 1-6BV, and the column is washed by purified water until the effluent liquid is nearly neutral, thus obtaining the activated cation exchange resin column.
In the present invention, it is preferable that in the step (a), the cation exchange resin column is a cation exchange resin column having a sulfonic acid group.
In the present invention, preferably, in the step (3), the activated anion exchange resin column is prepared by the following steps:
(A') swelling the anion exchange resin with purified water, and packing the anion exchange resin into a column to obtain an anion exchange resin column;
(B') passing the sodium hydroxide solution through a column by using 0.5-4mol/L of sodium hydroxide solution, wherein the flow rate of the sodium hydroxide solution is 0.5-3BV/h, the using amount of the sodium hydroxide solution is 1-6BV, and then washing the effluent by using purified water until the effluent is nearly neutral;
(C') passing the activated anion exchange resin column through a 0.5-4mol/L hydrochloric acid solution with the flow rate of 0.5-3BV/h and the consumption of 1-6BV, and washing the activated anion exchange resin column with purified water until the effluent is nearly neutral.
In the present invention, preferably, in the step (a'), the anion exchange resin column is an anion exchange resin column having a quaternary ammonium group.
In the present invention, preferably, the purification method further comprises the steps of:
(4) and adding calcium chloride into the purified pineapple juice treatment liquid to ensure that the concentration of the calcium chloride is 2-5mmol/L to obtain the pineapple juice treatment liquid containing the calcium chloride.
In the present invention, preferably, the impurity removal and purification treatment method further includes the following steps:
(5) and (4) filtering the purified pineapple juice treatment liquid obtained in the step (3) or the calcium chloride-containing pineapple juice treatment liquid obtained in the step (4) by using a microporous filter membrane to obtain the sterilized pineapple juice treatment liquid.
The invention also provides a preparation method of the bromelain product, which comprises the steps of carrying out enzyme precipitation treatment on the purified pineapple juice treatment liquid, the pineapple juice treatment liquid containing calcium chloride or the pineapple juice treatment liquid subjected to impurity removal and purification treatment to precipitate bromelain; and drying the bromelain to obtain a bromelain product.
The impurity removal and purification treatment method can effectively remove iron ions, well protects the enzyme activity of bromelain in the impurity removal process, and can obtain bromelain products with stable quality. In a preferred embodiment of the present invention, the enzyme activity is further improved. In addition, the method has the advantages of simple process flow, less equipment investment, low resource consumption, determined parameters of each process, convenient control and suitability for industrial production.
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 the column volume.
The purification method of bromelain removes metal ions in the bromelain by using a resin impurity removal mode, and particularly can effectively remove iron ions. The impurity removal and purification treatment method can also comprise the step of pretreatment of pineapple juice. The purification process of the present invention may further comprise the step of activating the cation exchange resin and the anion exchange resin.
The preparation method of the bromelain product comprises the steps of carrying out enzyme analysis treatment and drying treatment on the bromelain treatment liquid after purification treatment.
< pretreatment of pineapple juice >
In the pretreatment step of the pineapple juice, the pineapple juice is the pineapple raw juice squeezed from the fruit, stem, leaf or peel of the pineapple. From the viewpoint of waste utilization and energy conservation, the pineapple juice is preferably pineapple raw juice squeezed from pineapple stems, leaves or peels, and more preferably pineapple raw juice squeezed from pineapple peels. The pressing method is not limited, and for example, a normal-temperature pressing method or a cold pressing method may be used.
In the invention, the pretreatment of the pineapple juice comprises the step of mixing the pineapple juice with a preservative and an antioxidant. The preservative is selected from one or more of sodium benzoate, potassium sorbate, methyl hydroxybenzoate, ethyl hydroxybenzoate, propyl hydroxybenzoate and butyl hydroxybenzoate; the antioxidant is selected from one or more of sodium metabisulfite, sodium sulfite, sodium bisulfite and sodium thiosulfate. According to one embodiment of the invention, the preservative is sodium benzoate, the antioxidant is sodium metabisulfite, and the weight ratio of the pineapple normal juice, sodium benzoate and sodium metabisulfite is 1000:0.05-0.2:0.05-0.2, preferably 1000:0.08-0.15:0.08-0.15, more preferably 1000:0.08-0.12: 0.08-0.12. According to a preferred embodiment of the invention, the weight ratio of pineapple normal juice, sodium benzoate and sodium metabisulphite is 1000:0.1: 0.1. By adopting the proportion of the invention, rancidity can be effectively prevented, and the activity of bromelain is kept. Preferably, the pH of the pineapple raw juice is adjusted to 3.5-5.5, preferably 4.0-5.5, before mixing the pineapple raw juice with the preservative and the antioxidant. Under the pH value, the bromelain has better activity when the pineapple raw juice is mixed with the preservative and the antioxidant. If the pH of the pineapple raw juice itself is within the above range, the pH does not need to be adjusted.
In the invention, the pretreatment of the pineapple juice further comprises the step of carrying out solid-liquid separation after mixing the pineapple juice with a preservative and an antioxidant. The solid-liquid separation method is selected from filtration and centrifugation, so as to obtain the pineapple juice after pretreatment. Preferably, the solid-liquid separation is carried out at low temperature, preferably at 0-10 ℃, more preferably 0-6 ℃, still more preferably at 4 ℃. The solid-liquid separation operation is carried out at the temperature, which is beneficial to keeping better activity of the bromelain. Preferably, the solid-liquid separation operation is centrifugation, and the centrifugation temperature is 0-6 ℃. Preferably, the centrifugation rate is 12000-20000rpm, preferably 14000-18000rmp, more preferably 15000-17000rmp, and still more preferably 16000rmp, to obtain the centrifugate. In the present invention, the pretreatment may further include a step of filtering the centrifugate obtained by the centrifugation with a microporous membrane, thereby obtaining a filtered pineapple juice. Preferably, the pore size of the microfiltration membrane is 0.22 μm. Then, the treatment is carried out by adopting an electrodialysis method, wherein the voltage of the electrodialysis is constant voltage of 50V. According to one embodiment of the invention, the method of electrodialysis is: adding tap water into the polar chamber, adding filtered pineapple juice with the same volume as the tap water into the weak chamber, and adding purified water with the same volume into the thick chamber; the electrodialysis operation is carried out at room temperature at a flow rate of 3-4 times/h of the filtered pineapple juice volume, and the operation time is 60-80 min. The pineapple juice treated by the electrodialysis instrument is further treated in the next step.
< pretreatment of ion exchange resin >
In the present invention, the pretreatment of the ion exchange resin includes an activation treatment of a cation exchange resin and an activation treatment of an anion exchange resin.
In the activation treatment of the cation exchange resin, the cation exchange resin is a cation exchange resin with sulfonic acid groups. Preferably, the cation exchange resin is selected from 732 type cation exchange resin, 734 type cation exchange resin, and D001 type cation exchange resin, and more preferably 732 type cation exchange resin. In the present invention, the 732 type cation exchange resin is significantly superior in effect to other types of cation exchange resins. Specifically, the activation treatment of the cation exchange resin comprises the following steps: (A) washing cation exchange resin with purified water, swelling, and packing to obtain cation exchange resin column; (B) passing 0.5-4mol/L hydrochloric acid solution through the column at a flow rate of 0.5-3BV/h and a consumption of 1-6BV, and washing with purified water until the effluent is nearly neutral; (C) then 0.5-4mol/L sodium hydroxide solution is adopted to pass through the column, the flow rate of the sodium hydroxide solution is 0.5-3BV/h, the using amount of the sodium hydroxide solution is 1-6BV, and the column is washed by purified water until the effluent liquid is nearly neutral, thus obtaining the activated cation exchange resin column.
In the present invention, the aspect ratio of the cation exchange resin column in the step (A) is preferably 1 to 8:1, more preferably 2 to 6:1, still more preferably 3 to 5:1, and still more preferably 3 to 4: 1. According to a preferred embodiment of the present invention, the cation exchange resin column has a height to diameter ratio of 4: 1.
In the present invention, preferably, the concentration of the hydrochloric acid solution in the step (B) is 1 to 3mol/L, more preferably 1 to 2 mol/L; the flow rate of the hydrochloric acid solution is 1-2.5BV/h, more preferably 1-2 BV/h; the amount of the hydrochloric acid solution is 2-5BV, more preferably 2-4 BV. According to a preferred embodiment of the present invention, the concentration of the hydrochloric acid solution in the step (B) is 1 to 2mol/L, the flow rate of the hydrochloric acid solution is 1 to 2BV/h, and the amount of the hydrochloric acid solution is 2 to 4 BV.
In the present invention, preferably, the concentration of the sodium hydroxide solution in the step (C) is 1 to 3mol/L, more preferably 1 to 2 mol/L; the flow rate of the sodium hydroxide solution is 1-2.5BV/h, more preferably 1-2 BV/h; the amount of sodium hydroxide solution is 2-5BV, more preferably 2-4 BV. According to a preferred embodiment of the present invention, the concentration of the sodium hydroxide solution in step (C) is 1 to 2mol/L, the flow rate of the sodium hydroxide solution is 1 to 2BV/h, and the amount of the sodium hydroxide solution is 2 to 4 BV.
In the activation treatment of the anion exchange resin, the anion exchange resin 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. In the present invention, the 717 type of anion exchange resin has a significantly better effect than other types of anion exchange resins. Specifically, the activation treatment of the anion exchange resin comprises the following steps: (A') washing the anion exchange resin with purified water, swelling, and packing to obtain an anion exchange resin column; (B') passing 0.5-4mol/L sodium hydroxide solution through the column, wherein the flow rate of the sodium hydroxide is 0.5-3BV/h, the using amount of the sodium hydroxide solution is 1-6BV, and then washing with purified water until the effluent liquid is nearly neutral; (C') passing the activated anion exchange resin column through a 0.5-4mol/L hydrochloric acid solution with the flow rate of 0.5-3BV/h and the consumption of 1-6BV, and washing the activated anion exchange resin column with purified water until the effluent is nearly neutral.
In the present invention, preferably, the aspect ratio of the anion exchange resin column in step (A') is 1 to 8:1, more preferably 2 to 6:1, still more preferably 3 to 5: 1.
In the present invention, preferably, the concentration of the sodium hydroxide solution in the step (B') is 1 to 3mol/L, more preferably 1 to 2 mol/L; the flow rate of the sodium hydroxide solution is 1-2.5BV/h, more preferably 1-2 BV/h; the amount of sodium hydroxide solution is 2-5BV, more preferably 2-4 BV. According to a preferred embodiment of the present invention, the concentration of the sodium hydroxide solution in step (B') is 1 to 2mol/L, the flow rate of the sodium hydroxide solution is 1 to 2BV/h, and the amount of the sodium hydroxide solution is 2 to 4 BV.
In the present invention, preferably, the concentration of the hydrochloric acid solution in the step (C') is 1 to 3mol/L, more preferably 1 to 2 mol/L; the flow rate of the hydrochloric acid solution is 1-2.5BV/h, more preferably 1-2 BV/h; the amount of the hydrochloric acid solution is 2-5BV, more preferably 2-4 BV. According to a preferred embodiment of the present invention, the concentration of the hydrochloric acid solution in the step (C') is 1 to 2mol/L, the flow rate of the hydrochloric acid solution is 1 to 2BV/h, and the amount of the hydrochloric acid solution is 2 to 4 BV.
< purification treatment >
In the invention, the impurity removal and purification treatment comprises the following steps:
(1) adjusting the pH value of the pineapple juice to 2.5-3.5 to obtain pineapple juice sample liquid;
(2) passing the pineapple juice sample solution through an activated cation exchange resin column at the flow rate of 2.5-4.5BV/h, collecting effluent, and adjusting the pH of the effluent to 6.5-7.5 to obtain a first effluent;
(3) passing the first effluent through an activated anion exchange resin column at a flow rate of 2.5-4.5BV/h, and collecting the effluent; adjusting the pH of the effluent to 3.0-4.0 to obtain a purified pineapple juice treatment solution.
In the present invention, in the step (1), the pineapple juice may be juice obtained by squeezing pineapple fruits, stems, leaves or peels directly, but is preferably pineapple juice subjected to the above-mentioned pretreatment. Preferably, the pH of the pineapple juice is adjusted by using a hydrochloric acid solution. Preferably, the concentration of the hydrochloric acid solution is 25 to 36 wt%, more preferably 30 to 36 wt%, and even more preferably 36 wt%, and with such a concentration of the hydrochloric acid solution, the pH can be adjusted rapidly without affecting the activity of bromelain. Preferably, the pineapple juice mixture is adjusted to a pH of 2.8-3.2, more preferably to a pH of 3.0, using a hydrochloric acid solution.
In the present invention, the cation exchange resin selected in step (2) is as described above and will not be described herein. Preferably, the activated cation exchange resin column is an activated type 732 cation exchange resin. Preferably, the flow rate is 3-4 BV/h. Preferably, the pH of the pineapple juice is adjusted with a sodium hydroxide solution. The concentration of the sodium hydroxide solution is 30-50 wt%, more preferably 35-45 wt%, and even more preferably 40 wt%, and by adopting the concentration of the sodium hydroxide solution, the pH can be quickly adjusted without influencing the activity of the bromelain. Preferably, the pineapple juice mixture is adjusted to ph6.8-7.2, more preferably ph7.0, with sodium hydroxide solution to obtain a first effluent. In the step (2), the sample loading amount of the pineapple juice sample loading liquid is not more than the sample loading amount of the activated cation 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. Preferably, the pineapple juice loading solution is loaded in an amount of 9 to 14 times, more preferably 10 to 12 times, the volume of the activated cation exchange resin column.
In the present invention, the anion exchange resin in step (3) is selected as described above, and will not be described herein again. Preferably, the activated anion exchange resin column is an activated 717 type anion exchange resin. Preferably, the flow rate is 3-4 BV/h. Preferably, the effluent is adjusted to a pH of 3.3-3.7, more preferably to a pH of 3.5, resulting in a purified pineapple juice treatment. In the step (3), the sample loading amount of the first effluent liquid is not more than the sample loading amount of the activated 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. Preferably, the first effluent is loaded in an amount of 9 to 14 times, more preferably 10 to 12 times the volume of the activated anion exchange resin column.
In the present invention, preferably, the purification treatment method further includes the steps of:
(4) and adding calcium chloride into the purified pineapple juice treatment liquid to ensure that the concentration of the calcium chloride is 2-5mmol/L to obtain the pineapple juice treatment liquid containing the calcium chloride.
In the present invention, the calcium chloride in step (4) may be added in the form of a solid, or may be added in the form of a calcium chloride solution, and is preferably added in the form of a calcium chloride solution. In the present invention, preferably, the purified pineapple juice treatment solution is added to a calcium chloride solution so that the concentration of calcium chloride is 2 to 4mmol/L, more preferably 2.5 to 3.5mmol/L, and still more preferably 3 mmol/L.
In the present invention, preferably, the impurity removal and purification treatment method further includes the following steps:
(5) and (4) filtering the purified pineapple juice treatment liquid obtained in the step (3) or the calcium chloride-containing pineapple juice treatment liquid obtained in the step (4) by using a microporous filter membrane to obtain the pineapple juice treatment liquid subjected to impurity removal and purification treatment. With such a procedure, microorganisms can be effectively removed. Preferably, the pore size of the microfiltration membrane is 0.22 μm.
By adopting the purification treatment method, the iron ions can be effectively removed, the enzyme activity of the bromelain is well protected in the impurity removal process, and the process flow is simple. In addition, calcium chloride is added into the purified pineapple juice treatment liquid, the concentration of the calcium chloride is controlled within the range of the invention, and the enzyme activity of the bromelain is effectively improved. After the method is adopted for removing impurities, the qualification rate of the quality of the finally obtained bromelain product reaches more than 98 percent, and the bromelain product with stable quality can be obtained from pineapple raw materials with different sources.
< enzyme analysis and drying treatment >
In the present invention, the pineapple proteinase can be isolated by subjecting the pineapple juice treated solution after impurity removal to an enzyme-isolating treatment by various methods, for example, ammonium sulfate precipitation, ultrafiltration, tannin precipitation, and the like, preferably tannin precipitation.
According to a preferred embodiment of the present invention, the enzyme analysis treatment method comprises: mixing the pineapple juice treatment solution filtered by the microporous membrane with tannic acid according to the weight ratio of 1000:1-3, stirring, standing at 4-15 ℃, and precipitating bromelain.
In the invention, preferably, the weight ratio of the pineapple juice treatment liquid subjected to impurity removal treatment to tannic acid is 1000: 2; the standing time is 2-3 h. According to one embodiment of the invention, the pineapple juice treatment solution after impurity removal treatment is mixed with tannic acid according to the weight ratio of 1000:2, stirred, kept stand at 4-15 ℃ for 2-3h, enzyme paste is settled, and supernatant is removed to obtain bromelain enzyme paste; and (2) centrifuging the enzyme paste at the centrifugal temperature of 0-10 ℃ to obtain bromelain enzyme paste, and drying the bromelain enzyme paste to obtain a bromelain product. Preferably, the centrifugation temperature is 0-6 ℃, more preferably 4 ℃; the centrifugation rate of the centrifugation treatment is 12000-20000rpm, preferably 14000-18000rmp, more preferably 15000-17000rmp, and still more preferably 16000 rmp. The drying method is, for example, vacuum drying or vacuum freeze drying, and preferably freeze drying.
The present invention will be described in detail with reference to specific examples, but the present invention is not limited to these examples.
Preparation examples 1-732 activation of cation exchange resin
(1) Washing 732 type cation exchange resin with purified water, swelling, and packing to obtain 732 type cation exchange resin column with height/diameter ratio of 4: 1;
(2) passing 2mol/L hydrochloric acid solution through a column, wherein the flow rate of the hydrochloric acid solution is 1.5BV/h, the using amount of the hydrochloric acid solution is 3BV, and then washing with purified water until the effluent liquid is nearly neutral;
(3) and then, passing a 2mol/L sodium hydroxide solution through the column, wherein the flow rate of the sodium hydroxide solution is 1.5BV/h, and the using amount of the sodium hydroxide solution is 3BV, and washing with purified water until the effluent liquid is nearly neutral to obtain the activated 732 type cation exchange resin column.
Preparation of anion exchange resin of types 2 to 717
(1) 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;
(2) passing a 2mol/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 then washing with purified water until the effluent liquid is nearly neutral;
(3) then 2mol/L hydrochloric acid solution is adopted to pass through the column, the flow rate of the hydrochloric acid solution is 1.5BV/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 717 type anion exchange resin column was obtained.
Preparation example 3 pretreatment of pineapple juice
The pretreatment method of the pineapple juice comprises the following steps: squeezing pineapple peel to obtain pineapple juice with pH of 4.1; mixing the pineapple juice with sodium benzoate and sodium metabisulfite according to the weight ratio of 1000:0.1:0.1, stirring, standing, centrifuging at 16000rpm and 4 ℃ by using a tubular centrifuge, filtering the filtrate by using a 1-micron microporous filter membrane, performing constant voltage of 50V by using an electrodialysis instrument at the flow rate of 4 times of the volume/h of the pineapple juice, and operating for 70min to obtain the pretreated pineapple juice.
EXAMPLE 1 purification of pineapple juice
(1) Adjusting the pH value of the pretreated pineapple juice of the preparation example 3 to 3.0 by adopting 36 wt% hydrochloric acid to obtain a pineapple juice sample loading liquid;
(2) passing the pineapple juice sample solution through an activated 732 type cation exchange resin column of preparation example 1 at a flow rate of 3.5BV/h, collecting effluent, and periodically detecting the pH and conductivity of the effluent to ensure that the activated 732 type cation exchange resin column is not overloaded; adjusting the effluent to pH7.0 to obtain a first effluent;
(3) passing the first effluent through the activated type 717 anion exchange resin column of preparative example 2 at a flow rate of 3.5BV/h, collecting the effluent, and periodically detecting the pH and conductivity of the effluent to ensure that the activated type 717 anion exchange resin column is not overloaded; adjusting the pH of the effluent to 3.5 to obtain a purified pineapple juice treatment solution;
(4) adding the purified pineapple juice treatment liquid into a calcium chloride solution to enable the concentration of calcium chloride to be 3mmol/L, so as to obtain a pineapple juice treatment liquid containing calcium chloride;
and (3) passing the pineapple juice treatment solution containing calcium chloride through a microporous filter membrane with the aperture of 0.22 mu m to obtain the pineapple juice treatment solution after impurity removal and purification treatment.
EXAMPLE 2 purification of pineapple juice
(1) Adjusting the pH value of the pretreated pineapple juice of the preparation example 3 to 3.0 by adopting 36 wt% of hydrochloric acid to obtain a pineapple juice sample loading liquid;
(2) passing the pineapple juice sample solution through an activated 732 type cation exchange resin column of preparation example 1 at a flow rate of 3.5BV/h, collecting effluent, and periodically detecting the pH and conductivity of the effluent to ensure that the activated 732 type cation exchange resin column is not overloaded; adjusting the effluent to pH7.0 to obtain a first effluent;
(3) passing the first effluent through the activated type 717 anion exchange resin column of preparative example 2 at a flow rate of 3.5BV/h, collecting the effluent, and periodically detecting the pH and conductivity of the effluent to ensure that the activated type 717 anion exchange resin column is not overloaded; adjusting the pH of the effluent to 3.5 to obtain a purified pineapple juice treatment solution;
and (3) passing the purified pineapple juice treatment solution through a microporous filter membrane with the aperture of 0.22 mu m to obtain the pineapple juice treatment solution after impurity removal treatment.
EXAMPLE 3 preparation of Bromelain product
Mixing the pineapple juice treatment solution obtained by the treatment of the example 1 with tannic acid according to the weight ratio of 1000:2, stirring, standing at 4-15 ℃ for 2.5h, settling enzyme paste, and removing supernatant to obtain bromelain enzyme paste; centrifuging the enzyme paste at a centrifugation temperature of 4 ℃ to obtain bromelain enzyme paste; and (3) carrying out vacuum freeze drying on the bromelain enzyme paste to obtain a bromelain product.
Example 4 preparation of Bromelain product
Mixing the pineapple juice treatment solution obtained by the treatment of the example 2 with tannic acid according to the weight ratio of 1000:2, stirring, standing at 4-15 ℃ for 2.5h, settling enzyme paste, and removing supernatant to obtain bromelain enzyme paste; centrifuging the enzyme paste at a centrifugation temperature of 4 ℃ to obtain bromelain enzyme paste; and (3) carrying out vacuum freeze drying on the bromelain enzyme paste to obtain a bromelain product.
The bromelain products of examples 3 and 4 were measured for metal ion content and bromelain enzyme activity, and the results are shown in table 1.
TABLE 1 Bromelain product-related parameters
In addition, the method for preparing the bromelain product from the pineapple raw materials with different sources and batches has the advantages that the quality of the obtained bromelain is very stable, and the unqualified phenomenon is avoided.
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 (5)
1. A method for purifying bromelain, comprising the steps of:
(1) adjusting the pH value of the pineapple juice to 2.5-3.5 to obtain pineapple juice sample liquid;
(2) passing the pineapple juice sample solution through an activated cation exchange resin column at the flow rate of 2.5-4.5BV/h, collecting effluent, and adjusting the pH of the effluent to 6.5-7.5 to obtain a first effluent; the cation exchange resin column is a 732 type cation exchange resin column;
(3) passing the first effluent through an activated anion exchange resin column at a flow rate of 2.5-4.5BV/h, and collecting the effluent; adjusting the pH of the effluent to 3.0-4.0 to obtain a purified pineapple juice treatment solution; the anion exchange resin column is a 717 type anion exchange resin column;
wherein, in the step (2), the preparation method of the activated cation exchange resin column comprises the following steps:
(A) swelling cation exchange resin with purified water, and packing to obtain cation exchange resin column;
(B) passing 0.5-4mol/L hydrochloric acid solution through the column at a flow rate of 0.5-3BV/h and a consumption of 1-6BV, and washing with purified water until the effluent is nearly neutral;
(C) then 0.5-4mol/L sodium hydroxide solution is adopted to pass through the column, the flow rate of the sodium hydroxide solution is 0.5-3BV/h, the using amount of the sodium hydroxide solution is 1-6BV, and purified water is used for washing until the effluent liquid is nearly neutral, so as to obtain activated cation exchange resin;
in the step (3), the preparation method of the activated anion exchange resin column comprises the following steps:
(A') swelling the anion exchange resin with purified water, and packing the anion exchange resin into a column to obtain an anion exchange resin column;
(B') passing the sodium hydroxide solution through a column by using 0.5-4mol/L of sodium hydroxide solution, wherein the flow rate of the sodium hydroxide solution is 0.5-3BV/h, the using amount of the sodium hydroxide solution is 1-6BV, and then washing the effluent by using purified water until the effluent is nearly neutral;
(C') passing the solution through a column by using 0.5-4mol/L hydrochloric acid solution, wherein the flow rate of the hydrochloric acid solution is 0.5-3BV/h, the using amount of the hydrochloric acid solution is 1-6BV, and washing the solution by using purified water until the effluent liquid is nearly neutral to obtain an activated anion exchange resin column;
the pineapple juice in the step (1) is pretreated pineapple juice; the pretreatment comprises the steps of mixing the pineapple juice with a preservative and an antioxidant, stirring, and carrying out solid-liquid separation operation and electrodialysis, so as to obtain the pretreated pineapple juice.
2. The method of claim 1, wherein the pH of the pineapple raw juice is 3.5-5.5, 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 1000:0.05-0.2: 0.05-0.2; the solid-liquid separation operation comprises centrifugation at a centrifugation temperature of 0-6 ℃ and filtration with a microporous membrane.
3. The method according to claim 1, characterized in that the method further comprises the steps of:
(4) and adding calcium chloride into the purified pineapple juice treatment liquid to ensure that the concentration of the calcium chloride is 2-5mmol/L to obtain the pineapple juice treatment liquid containing the calcium chloride.
4. The method according to claim 3, characterized in that the method further comprises the steps of:
(5) and (4) filtering the purified pineapple juice treatment liquid obtained in the step (3) or the calcium chloride-containing pineapple juice treatment liquid obtained in the step (4) by using a microporous filter membrane to obtain the sterilized pineapple juice treatment liquid.
5. A method for producing a bromelain product, which comprises subjecting the pineapple juice treated solution of any one of claims 1 to 4 to an enzyme-separating treatment to separate out bromelain; and drying the bromelain to obtain a bromelain product.
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| CN112044478A (en) * | 2020-09-02 | 2020-12-08 | 广东力恩普健康产业科技有限公司 | Method for removing harmful metal ions in pineapple peel juice |
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