CN109385414A - The purification process of bromelain - Google Patents
The purification process of bromelain Download PDFInfo
- Publication number
- CN109385414A CN109385414A CN201710675070.0A CN201710675070A CN109385414A CN 109385414 A CN109385414 A CN 109385414A CN 201710675070 A CN201710675070 A CN 201710675070A CN 109385414 A CN109385414 A CN 109385414A
- Authority
- CN
- China
- Prior art keywords
- exchange resin
- pineapple juice
- efflux
- bromelain
- pineapple
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Non-Alcoholic Beverages (AREA)
Abstract
The invention discloses a kind of purification process of bromelain, including pineapple juice is adjusted to pH 2.5-3.5 by (1), obtains pineapple juice sample solution；(2) by the cation exchange resin column of the pineapple juice sample solution overactivation, flow velocity 2.5-4.5BV/h collects efflux, and the efflux is adjusted to pH 6.5-7.5, obtains the first efflux；(3) by the anion-exchange resin column of the first efflux overactivation, flow velocity 2.5-4.5BV/h collects efflux；The efflux is adjusted to pH 3.0-4.0, the pineapple juice treatment fluid purified.Method of the invention can be effectively reduced the content of the metal ion content in bromelain enzyme product, especially iron ion, effectively improve the qualification rate of bromelain enzyme product, keep its stable and controllable for quality.
Description
Technical field
The present invention relates to a kind of edulcoration purification processing methods of bromelain, further relate to the preparation of bromelain enzyme product
Method.
Background technique
Bromelain abbreviation bromelain, also known as bromelin or Bromelain are mainly extracted from pineapple sarcocarp, stem, leaf, skin
Out, and by a kind of natural plant protein enzyme obtained from purifying.Bromelain is a kind of protein digestibility
The complex mixts of enzyme, the enzyme system being made of the enzyme of a variety of different molecular weights and molecular structure, mainly by cysteine proteinase
Composition, additionally containing phosphate, peroxidase, cellulase, other glycosidases etc..
Modern research shows that bromelain has important medical value, with immunostimulation；It can be each
Inflammation and oedema are effectively treated in kind tissue；Treat diarrhea；It can also be used in decrustation after burn, be optionally removed dead skin；Inhibit swollen
Tumor cell growth reduces toxin and combines activity.In addition to medicinal applications, bromelain is also applied to the fields such as food, cosmetics.
Existing bromelain production technology mainly has ammonium sulfate precipitation method, ultrafiltration, tannin precipitation method etc..Wherein,
Ammonium sulfate precipitation method is in the ammonium sulfate of high concentration, and the hydrone on salt ion competitively binding protein surface destroys
The hydration shell of protein surface, solubility reduce, and protein assembles formation precipitating under hydrophobic effect.Ammonium sulfate precipitation method is due to needing
A large amount of ammonium sulfate is used, environmental pollution is serious, while may bring ammonium sulfate into the protease produced, and albumen yield is low,
Technique is unstable and time-consuming.Ultrafiltration be under certain pressure and flow, using asymmetric microcellular structure and semi-transparent film medium, according to
Pressure difference by film two sides is filtered as motive force with cross-current flow, passes through solvent and small-molecule substance, macromolecular egg
White matter is by filter membrane detention, to achieve the purpose that isolate and purify.Equipment investment needed for ultrafiltration is big, and pineapple juice had been concentrated by ultrafiltration
Viscosity is gradually increased in journey, and ultrafiltration membrane easily blocks, therefore the production cycle is long, at high cost, and extract yield is low.The tannin precipitation method are
It can make protein precipitation in conjunction with the aminoterminal of amino acid in albumen using tannic acid.
It is found in actual production, raw material bromelain made from identical production method of separate sources, different batches
Enzyme difference in quality is larger, and for bromelain unstable quality problem than more prominent, product qualification rate is relatively low.
Summary of the invention
To solve the above problems, inventor has conducted extensive research, it has unexpectedly been found that metal ion, spy in pineapple raw material
It is not that the content of iron ion has larger impact to the enzyme activity of bromelain.When the pineapple raw material for extracting bromelain
When the content of middle iron ion is higher, it is easy to appear the bromelain of production situation off quality, this is to lead to existing side
The major reason of method production bromelain unstable quality.
The purpose of the present invention is to provide a kind of purification process of bromelain, this method can be effectively reduced pineapple egg
The content of metal ion content in white enzyme product, especially molysite makes it to improve the qualification rate of bromelain enzyme product
It is stable and controllable for quality.It is a further object to provide a kind of purification process of bromelain, this method is obtained
The enzyme activity of bromelain significantly improve.
Another object of the present invention is to provide a kind of preparation methods of bromelain enzyme product.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of purification process of bromelain, includes the following steps:
(1) pineapple juice is adjusted to pH 2.5-3.5, obtains pineapple juice sample solution；
(2) by the cation exchange resin column of the pineapple juice sample solution overactivation, flow velocity 2.5-4.5BV/h is collected
Efflux, and the efflux is adjusted to pH 6.5-7.5, obtain the first efflux；
(3) by the anion-exchange resin column of the first efflux overactivation, flow velocity 2.5-4.5BV/h collects stream
Liquid out；The efflux is adjusted to pH 3.0-4.0, the pineapple juice treatment fluid purified.
In the present invention, it is preferable that pineapple juice described in step (1) is the pineapple juice after pre-treatment；The pre-treatment
Including mixing pineapple Normal juice with preservative, antioxidant, stir, carry out separation of solid and liquid operation, by electrodialysis, thus obtain through
Pineapple juice after crossing pre-treatment.
In the present invention, it is preferable that in step (1), the pH of the pineapple Normal juice is 3.5-5.5, and the preservative is benzene first
Sour sodium, the antioxidant is Sodium Metabisulfite, and the weight ratio of the pineapple juice, sodium benzoate and Sodium Metabisulfite is
1000:0.05-0.2:0.05-0.2；The separation of solid and liquid operation, which is included under 0-6 DEG C of centrifuging temperature, to be centrifuged, and is then used
Filtering with microporous membrane.
In the present invention, it is preferable that in step (2), the cation exchange resin column of the activation the preparation method comprises the following steps:
(A) by cation exchange resin purified water swollen, column is filled, cation exchange resin column is obtained；
(B) column is crossed using 0.5-4mol/L hydrochloric acid solution, the flow velocity of hydrochloric acid solution is 0.5-3BV/h, the use of hydrochloric acid solution
Amount is 1-6BV, then is washed to efflux weakly acidic pH with purifying；
(C) column is crossed using 0.5-4mol/L sodium hydroxide solution again, the flow velocity of sodium hydroxide solution is 0.5-3BV/h, hydrogen
The dosage of sodium hydroxide solution be 1-6BV, then with purify be washed to efflux weakly acidic pH, the cation exchange resin activated
Column.
In the present invention, it is preferable that in step (A), the cation exchange resin column is to hand over sulfonic cation
Change resin column.
In the present invention, it is preferable that in step (3), the anion-exchange resin column of the activation the preparation method comprises the following steps:
Anion exchange resin purified water swollen is filled column, obtains anion-exchange resin column by (A ')；
(B ') crosses column using 0.5-4mol/L sodium hydroxide solution, and the flow velocity of sodium hydroxide solution is 0.5-3BV/h, hydrogen-oxygen
The dosage for changing sodium solution is 1-6BV, then is washed to efflux weakly acidic pH with purifying；
(C ') crosses column using 0.5-4mol/L hydrochloric acid solution again, and the flow velocity of hydrochloric acid solution is 0.5-3BV/h, hydrochloric acid solution
Dosage is 1-6BV, then is washed to efflux weakly acidic pH with purifying, the anion-exchange resin column activated.
In the present invention, it is preferable that in step (A '), the anion-exchange resin column is that the anion with quaternary ammonium group is handed over
Change resin column.
In the present invention, it is preferable that the purification process further includes following steps:
(4) calcium chloride is added in the pineapple juice treatment fluid of the purifying and makes the concentration 2-5mmol/L of calcium chloride, obtained
The pineapple juice treatment fluid of chloride containing calcium.
In the present invention, it is preferable that the edulcoration purification processing method further includes following steps:
(5) pineapple juice for the chloride containing calcium for obtaining the pineapple juice treatment fluid for the purifying that step (3) obtains or step (4)
Treatment fluid carries out filtering with microporous membrane, the pineapple juice treatment fluid after obtaining bacteria removing.
The present invention also provides a kind of preparation methods of bromelain enzyme product comprising handles the pineapple juice of above-mentioned purifying
Liquid, the pineapple juice treatment fluid of chloride containing calcium or edulcoration purification treated pineapple juice treatment fluid carries out analysis enzymatic treatment, are precipitated pineapple
Protease；The bromelain is dried again, obtains bromelain enzyme product.
Edulcoration purification processing method of the invention, can effectively remove iron ion, while dedoping step protects well
The enzyme activity of bromelain can obtain the stable bromelain enzyme product of quality.The preferred embodiment of the present invention, enzyme activity into
One step improves.In addition, present invention process process is simple, equipment investment is few, low in resources consumption, and each process parameter determines, convenient for control
System is suitble to industrialized production.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, but protection scope of the present invention is not limited to
This.
In the present invention, " BV " indicates column volume.
The purification process of bromelain of the invention removes metal ion therein in such a way that resin cleans, especially
Iron ion can effectively be removed.The edulcoration purification processing method can also include the steps that pineapple juice pre-treatment.The present invention
Purification process can also include cation exchange resin and anion exchange resin activation step.
The preparation method of bromelain enzyme product of the invention includes by the bromelain treatment fluid Jing Guo purification process
The step of carrying out analysis enzymatic treatment and being dried.
The pre-treatment > of < pineapple juice
In the present invention, in the pre-treatment step of pineapple juice, the pineapple juice refers to presses in the fruit from pineapple, stem, leaf or skin
The pineapple Normal juice squeezed.From waste utilization and energy saving angle, it is preferable that the pineapple juice refers to from pineapple stem, leaf
Or the pineapple Normal juice of acquirement is squeezed in skin, the pineapple Normal juice of acquirement is more preferably squeezed from pineapple peel.The pressing method is not
Limit, such as can be room temperature squeezing method or cold-press.
In the present invention, the pre-treatment of the pineapple juice includes mixing the pineapple Normal juice with preservative, antioxidant
Step.The preservative is in sodium benzoate, potassium sorbate, methyl hydroxybenzoate, ethyl hydroxy benzoate, Nipasol, butyl hydroxybenzoate
It is one or more；The antioxidant is selected from one of Sodium Metabisulfite, sodium sulfite, sodium hydrogensulfite, sodium thiosulfate
Or it is a variety of.According to embodiment of the present invention, the preservative is sodium benzoate, and the antioxidant is to lay particular stress on sulfurous acid
Sodium, 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
It is further preferably 1000:0.08-0.12:0.08-0.12 for 1000:0.08-0.15:0.08-0.15.According to the present invention one it is excellent
The embodiment of choosing, the weight ratio of the pineapple Normal juice, sodium benzoate and Sodium Metabisulfite are 1000:0.1:0.1.Using this
The proportion of invention can effectively prevent activity that is rancid, and keeping bromelain.Preferably, when pineapple Normal juice and preservative,
Before antioxidant mixing, pineapple Normal juice is adjusted into pH to 3.5-5.5, preferably 4.0-5.5.Under such pH value, Neng Goubao
When card pineapple Normal juice is mixed with preservative and antioxidant, bromelain has preferable activity.It should be noted that if pineapple
The pH of Normal juice itself i.e. within the above range, then no longer needs to adjust pH.
In the present invention, the pre-treatment of the pineapple juice further include after the pineapple Normal juice is mixed with preservative, antioxidant,
The step of being separated by solid-liquid separation.The method of the separation of solid and liquid is selected from filtering, centrifugation, to obtain the pineapple juice after pre-treatment.
Preferably, the operation of the separation of solid and liquid carries out at low temperature, and preferably at 0-10 DEG C, more preferably 0-6 DEG C, be further preferably 4 DEG C
Lower progress.Separation of solid and liquid operation is carried out at said temperatures, is conducive to bromelain and is kept preferable activity.Preferably, institute
Separation of solid and liquid operation is stated as centrifugation, centrifuging temperature is 0-6 DEG C.Preferably, centrifugation rate 12000-20000rpm, preferably
14000-18000rmp, more preferably 15000-17000rmp are further preferably 16000rmp, obtain centrifugate.In the present invention, institute
Stating pre-treatment can also include the steps that for the resulting centrifugate of centrifugally operated being filtered with miillpore filter, thus obtain by
The pineapple juice of filtering.Preferably, the aperture of the miillpore filter is 0.22 μm.Then it is handled using electroosmose process, electric osmose
The voltage of analysis is constant voltage 50V.According to embodiment of the present invention, the electrodialytic method are as follows: be added into pole room
The filtered pineapple juice with tap water same volume is added into light room, the pure of same volume is added into dense room for tap water
Change water；Electrodialysis operation carries out the 3-4 times/h that flow control is filtered pineapple juice volume at room temperature, and runing time is
60-80min.The processing of next step is further continued for through the processed pineapple juice of electric osmose analyzer.
The pre-treatment > of < ion exchange resin
In the present invention, the pre-treatment of the ion exchange resin includes the activation processing and anion of cation exchange resin
The activation processing of exchanger resin.
In the activation processing of the cation exchange resin, the cation exchange resin is with sulfonic cation
Exchanger resin.Preferably, the cation exchange resin is selected from 732 type cation exchange resins, 734 type cation exchange trees
Rouge, D001 type cation exchange resin, more preferably 732 type cation exchange resins.In the present invention, 732 type cations are handed over
The effect for changing resin is substantially better than the cation exchange resin of other models.Specifically, the activation of the cation exchange resin
Processing includes the following steps: that (A) cleans cation exchange resin with purified water, swollen, fills column, obtains cation exchange resin
Column；(B) column is crossed using 0.5-4mol/L hydrochloric acid solution, the flow velocity of hydrochloric acid solution is 0.5-3BV/h, and the dosage of hydrochloric acid solution is 1-
6BV, then efflux weakly acidic pH is washed to purifying；(C) column is crossed using 0.5-4mol/L sodium hydroxide solution again, sodium hydroxide is molten
The flow velocity of liquid is 0.5-3BV/h, and the dosage of sodium hydroxide solution is 1-6BV, then is washed to efflux weakly acidic pH with purifying, is obtained
The cation exchange resin column of activation.
In the present invention, it is preferable that the ratio of height to diameter 1-8:1, more preferably 2-6 of step (A) described cation exchange resin column:
1, it is further preferably 3-5:1, and preferably 3-4:1.According to a preferred embodiment of the present invention, the cation exchange resin
The ratio of height to diameter 4:1 of column.
In the present invention, it is preferable that the concentration of step (B) described hydrochloric acid solution is 1-3mol/L, more preferably 1-2mol/L；
The flow velocity of hydrochloric acid solution is 1-2.5BV/h, more preferably 1-2BV/h；The dosage of hydrochloric acid solution is 2-5BV, more preferably 2-
4BV.According to a preferred embodiment of the present invention, step (B) concentration of hydrochloric acid solution is 1-2mol/L, hydrochloric acid solution
Flow velocity is 1-2BV/h, and the dosage of hydrochloric acid solution is 2-4BV.
In the present invention, it is preferable that the concentration of step (C) described sodium hydroxide solution is 1-3mol/L, more preferably 1-
2mol/L；The flow velocity of sodium hydroxide solution is 1-2.5BV/h, more preferably 1-2BV/h；The dosage of sodium hydroxide solution is 2-
5BV, more preferably 2-4BV.According to a preferred embodiment of the present invention, step (C) described concentration of sodium hydroxide solution is
1-2mol/L, the flow velocity of sodium hydroxide solution are 1-2BV/h, and the dosage of sodium hydroxide solution is 2-4BV.
In the activation processing of the anion exchange resin, the anion exchange resin is the anion with quaternary ammonium group
Exchanger resin.Preferably, the anion exchange resin is selected from 717 type anion exchange resin, 711 type anion exchange trees
Rouge, D201 type anion exchange resin, more preferably 717 type anion exchange resin.In the present invention, 717 type anion are handed over
The effect for changing resin is substantially better than the anion exchange resin of other models.Specifically, the activation of the anion exchange resin
Processing includes the following steps: that (A ') clean, swollen, dress column obtain anion exchange tree by anion exchange resin purified water
Rouge column；(B ') crosses column using 0.5-4mol/L sodium hydroxide solution, and the flow velocity of sodium hydroxide is 0.5-3BV/h, and sodium hydroxide is molten
The dosage of liquid is 1-6BV, then is washed to efflux weakly acidic pH with purifying；(C ') crosses column using 0.5-4mol/L hydrochloric acid solution again,
The flow velocity of hydrochloric acid solution is 0.5-3BV/h, and the dosage of hydrochloric acid solution is 1-6BV, then is washed to efflux weakly acidic pH with purifying, is obtained
To the anion-exchange resin column of activation.
In the present invention, it is preferable that the ratio of height to diameter 1-8:1, more preferably 2- of step (A ') described anion-exchange resin column
6:1 is further preferably 3-5:1.
In the present invention, it is preferable that the concentration of step (B ') described sodium hydroxide solution is 1-3mol/L, more preferably 1-
2mol/L；The flow velocity of sodium hydroxide solution is 1-2.5BV/h, more preferably 1-2BV/h；The dosage of sodium hydroxide solution is 2-
5BV, more preferably 2-4BV.According to a preferred embodiment of the present invention, step (B ') described concentration of sodium hydroxide solution is
1-2mol/L, the flow velocity of sodium hydroxide solution are 1-2BV/h, and the dosage of sodium hydroxide solution is 2-4BV.
In the present invention, it is preferable that the concentration of step (C ') described hydrochloric acid solution is 1-3mol/L, more preferably 1-2mol/
L；The flow velocity of hydrochloric acid solution is 1-2.5BV/h, more preferably 1-2BV/h；The dosage of hydrochloric acid solution is 2-5BV, more preferably 2-
4BV.According to a preferred embodiment of the present invention, step (the C ') concentration of hydrochloric acid solution is 1-2mol/L, hydrochloric acid solution
Flow velocity be 1-2BV/h, the dosage of hydrochloric acid solution is 2-4BV.
< purification process >
In the present invention, the edulcoration purification processing includes the following steps:
(1) pineapple juice is adjusted to pH 2.5-3.5, obtains pineapple juice sample solution；
(2) by the cation exchange resin column of the pineapple juice sample solution overactivation, flow velocity 2.5-4.5BV/h is collected
Efflux, and the efflux is adjusted to pH 6.5-7.5, obtain the first efflux；
(3) by the anion-exchange resin column of the first efflux overactivation, flow velocity 2.5-4.5BV/h collects stream
Liquid out；The efflux is adjusted to pH 3.0-4.0, the pineapple juice treatment fluid purified.
In the present invention, in step (1), the pineapple juice can be the squeezing acquirement directly from pineapple sarcocarp, stem, leaf or skin
Juice, but the pineapple juice preferably after above-mentioned pre-treatment.Preferably, pH is adjusted to pineapple juice using hydrochloric acid solution.
Preferably, the concentration of the hydrochloric acid solution is 25-36wt%, more preferably 30-36wt%, is further preferably 36wt%, using this
The concentration of hydrochloric acid solution of sample can either quickly adjust pH, and be unlikely to influence the activity of bromelain.Preferably, using salt
The pineapple juice mixed liquor is adjusted to pH2.8-3.2 by acid solution, is further preferably pH 3.0.
In the present invention, the selection of step (2) described cation exchange resin is as described above, which is not described herein again.Preferably,
The cation exchange resin column of the activation is 732 type cation exchange resins of activation.Preferably, the flow velocity is 3-4BV/
h.Preferably, pH is adjusted to pineapple juice using sodium hydroxide solution.The concentration of the sodium hydroxide solution is 30-
50wt%, more preferably 35-45wt% are further preferably 40wt%, can either be quick using such concentration of sodium hydroxide solution
PH is adjusted, and is unlikely to influence the activity of bromelain.Preferably, using sodium hydroxide solution by the pineapple juice mixed liquor
It is adjusted to pH6.8-7.2, is further preferably pH7.0, obtains the first efflux.In step (2), the loading of the pineapple juice sample solution
Measure the volume containing the sample being subject to no more than the cation exchange resin column of the activation；Such as it can be by being flowed out described in periodic detection
The pH and conductivity of liquid, as the reference index for determining whether overload.Preferably, the applied sample amount of the pineapple juice sample solution is institute
9-14 times for stating the cation exchange resin column volume of activation, more preferably 10-12 times.
In the present invention, the selection of step (3) described anion exchange resin is as described above, which is not described herein again.Preferably,
The anion-exchange resin column of the activation is 717 type anion exchange resin of activation.Preferably, the flow velocity is 3-4BV/
h.Preferably, the efflux is adjusted to pH 3.3-3.7, more preferably pH3.5, the pineapple juice treatment fluid purified.Step
Suddenly in (3), the applied sample amount of first efflux be subject to no more than the activation anion-exchange resin column volume containing the sample；
Such as it can be by the pH and conductivity of efflux described in periodic detection, as the reference index for determining whether overload.Preferably,
The applied sample amount of first efflux is 9-14 times of the anion-exchange resin column volume of the activation, more preferably 10-12
Times.
In the present invention, it is preferable that the purification treating method further includes following steps:
(4) calcium chloride is added in the pineapple juice treatment fluid of the purifying and makes the concentration 2-5mmol/L of calcium chloride, obtained
The pineapple juice treatment fluid of chloride containing calcium.
In the present invention, step (4) described calcium chloride can be added in solid form, can also be added in the form of calcium chloride solution
Enter, and is added preferably in the form of calcium chloride solution.In the present invention, it is preferable that the pineapple juice treatment fluid of the purifying is added
Calcium chloride solution and the concentration 2-4mmol/L for making calcium chloride, more preferably 2.5-3.5mmol/L are further preferably 3mmol/L.
In the present invention, it is preferable that the edulcoration purification processing method further includes following steps:
(5) pineapple juice for the chloride containing calcium for obtaining the pineapple juice treatment fluid for the purifying that step (3) obtains or step (4)
Treatment fluid carries out filtering with microporous membrane, obtains edulcoration purification treated pineapple juice treatment fluid.Can be had using such step
Effect removes microorganism.Preferably, the aperture of the miillpore filter is 0.22 μm.
Using purification treating method of the invention, iron ion can be effectively removed, while dedoping step protects well
The enzyme activity of bromelain, and process flow is simple.In addition, calcium chloride is added in the pineapple juice treatment fluid of purifying, and by chlorine
The concentration for changing calcium controls within the scope of the present invention, and the enzyme activity of bromelain effectively improves.Using method of the invention into
After row removal of impurities, finally obtained bromelain Qualified Products Rate is up to 98% or more, and the pineapple raw material of separate sources
It can get the stable bromelain enzyme product of quality.
< analyses enzyme and is dried >
In the present invention, can using a variety of methods, to removal of impurities, treated that pineapple juice treatment fluid carries out analysis enzymatic treatment, such as
Ammonium sulfate precipitation method, ultrafiltration, tannin precipitation method etc., preferably the tannin precipitation method, so that bromelain be precipitated.
A preferred embodiment according to the present invention, the analysis method of enzymatically treating are as follows: after filtering with microporous membrane
Pineapple juice treatment fluid is that 1000:1-3 is mixed according to weight ratio with tannic acid, stirs, stands at 4~15 DEG C, pineapple is precipitated
Protease.
In the present invention, it is preferable that the weight ratio of removal of impurities treated the pineapple juice treatment fluid and tannic acid is 1000:2；Institute
Stating time of repose is 2-3h.According to embodiment of the present invention, removal of impurities treated pineapple juice treatment fluid is pressed with tannic acid
It is that 1000:2 is mixed according to weight ratio, stirs, 2-3h is stood at 4~15 DEG C, and enzyme paste sedimentation removes supernatant, obtains spinach
Trailing plants protease enzyme paste；Enzyme paste is subjected to centrifugal treating under 0-10 DEG C of centrifuging temperature, to obtain bromelain enzyme
Then the bromelain enzyme cream is dried, obtains bromelain enzyme product by cream.Preferably, the centrifuging temperature is
0-6 DEG C, more preferably 4 DEG C；The centrifugation rate of the centrifugal treating is 12000-20000rpm, preferably 14000-
18000rmp, more preferably 15000-17000rmp are further preferably 16000rmp.The mode of the drying is, for example, to be dried in vacuo
Or vacuum freeze drying, and preferably freeze-drying.
Below by way of specific embodiment, the present invention is described in detail, but these embodiments are not limited to this hair
It is bright.
The activation of preparation example 1-732 type cation exchange resin
(1) 732 type cation exchange resins are sufficiently cleaned with purified water, swollen fills column, obtains the friendship of 732 type cations
Change resin column, ratio of height to diameter 4:1；
(2) column is crossed using 2mol/L hydrochloric acid solution, the flow velocity of hydrochloric acid solution is 1.5BV/h, and the dosage of hydrochloric acid solution is
3BV, then efflux weakly acidic pH is washed to purifying；
(3) column is crossed using 2mol/L sodium hydroxide solution again, the flow velocity of sodium hydroxide solution is 1.5BV/h, sodium hydroxide
The dosage of solution be 3BV, then with purify be washed to efflux weakly acidic pH, the 732 type cation exchange resin columns activated.
The activation of preparation example 2-717 type anion exchange resin
(1) 717 type anion exchange resin are sufficiently cleaned with purified water, swollen fills column, obtains the friendship of 717 type anion
Change resin column, ratio of height to diameter 4:1；
(2) column is crossed using 2mol/L sodium hydroxide solution, the flow velocity of sodium hydroxide solution is 1.5BV/h, and sodium hydroxide is molten
The dosage of liquid is 3BV, then is washed to efflux weakly acidic pH with purifying；
(3) column is crossed using 2mol/L hydrochloric acid solution again, the flow velocity of hydrochloric acid solution is 1.5BV/h, and the dosage of hydrochloric acid solution is
3BV, then efflux weakly acidic pH is washed to purifying；The 717 type anion-exchange resin columns activated.
The pre-treatment of preparation example 3- pineapple juice
The pre-treating method of pineapple juice: the pineapple juice obtained from pineapple peel squeezing, pH 4.1 are taken；By the pineapple juice
It is mixed with sodium benzoate, Sodium Metabisulfite according to weight ratio for 1000:0.1:0.1, stirs, stand, be then centrifuged with tubular type
Machine is centrifuged at revolving speed 16000rpm, 4 DEG C, and filtrate crosses 1 μm of miillpore filter, and using electric osmose analyzer 50V constant voltage, flow velocity is 4 times
Pineapple juice volume/h runs 70min, the pineapple juice after obtaining pre-treatment.
The purification process of embodiment 1- pineapple juice
(1) pineapple juice after the pre-treatment of preparation example 3 is adjusted to pH 3.0 using the hydrochloric acid of 36wt%, obtains pineapple juice
Sample solution；
(2) the pineapple juice sample solution is crossed to 732 type cation exchange resin columns of the activation of preparation example 1, flow velocity is
3.5BV/h collects efflux, passes through the pH and conductivity of efflux described in periodic detection, it is ensured that 732 type cations of activation are handed over
Resin column is changed not overload；The efflux is adjusted to pH 7.0 again, obtains the first efflux；
(3) first efflux is crossed to 717 type anion-exchange resin columns of the activation of preparation example 2, flow velocity is
3.5BV/h collects efflux, passes through the pH and conductivity of efflux described in periodic detection, it is ensured that 717 type anion of activation are handed over
Resin column is changed not overload；The efflux is adjusted to pH 3.5, the pineapple juice treatment fluid purified；
(4) calcium chloride solution is added in the pineapple juice treatment fluid of the purifying and makes the concentration 3mmol/L of calcium chloride, obtained
To the pineapple juice treatment fluid of chloride containing calcium；
It is 0.22 μm of miillpore filter that the pineapple juice treatment fluid of the chloride containing calcium, which is crossed aperture, after obtaining edulcoration purification processing
Pineapple juice treatment fluid.
The purification process of embodiment 2- pineapple juice
(1) pineapple juice after the pre-treatment of preparation example 3 is adjusted to pH3.0 using the hydrochloric acid of 36wt%, obtains pineapple juice
Sample solution；
(2) the pineapple juice sample solution is crossed to 732 type cation exchange resin columns of the activation of preparation example 1, flow velocity is
3.5BV/h collects efflux, passes through the pH and conductivity of efflux described in periodic detection, it is ensured that 732 type cations of activation are handed over
Resin column is changed not overload；The efflux is adjusted to pH 7.0 again, obtains the first efflux；
(3) first efflux is crossed to 717 type anion-exchange resin columns of the activation of preparation example 2, flow velocity is
3.5BV/h collects efflux, passes through the pH and conductivity of efflux described in periodic detection, it is ensured that 717 type anion of activation are handed over
Resin column is changed not overload；The efflux is adjusted to pH 3.5, the pineapple juice treatment fluid purified；
It is 0.22 μm of miillpore filter that the pineapple juice treatment fluid of the purifying, which is crossed aperture, obtains removal of impurities treated pineapple juice
Treatment fluid.
The preparation of embodiment 3- bromelain enzyme product
The pineapple juice treatment fluid that 1 method of embodiment is handled is mixed with tannic acid according to weight ratio for 1000:2,
Stirring, stands 2.5h at 4~15 DEG C, and enzyme paste sedimentation removes supernatant, obtains bromelain enzyme paste；The enzyme is pasted in 4
DEG C centrifuging temperature under carry out centrifugal treating, to obtain bromelain enzyme cream；The bromelain enzyme cream is carried out true
Vacuum freecing-dry obtains bromelain enzyme product.
The preparation of embodiment 4- bromelain enzyme product
The pineapple juice treatment fluid that 2 method of embodiment is handled is mixed with tannic acid according to weight ratio for 1000:2,
Stirring, stands 2.5h at 4~15 DEG C, and enzyme paste sedimentation removes supernatant, obtains bromelain enzyme paste；The enzyme is pasted in 4
DEG C centrifuging temperature under carry out centrifugal treating, to obtain bromelain enzyme cream；The bromelain enzyme cream is carried out true
Vacuum freecing-dry obtains bromelain enzyme product.
The metal ion content of the bromelain enzyme product of embodiment 3 and embodiment 4, bromelain enzyme activity are surveyed
It is fixed, it the results are shown in Table 1.
1 bromelain enzyme product relevant parameter of table
In addition, preparing bromelain using method of the invention by the pineapple raw material to separate sources, different batches
Product, gained bromelain quality is sufficiently stable, does not occur unqualified phenomenon.
Present invention is not limited to the embodiments described above, without departing from the essence of the present invention, this field skill
Any deformation, improvement, the replacement that art personnel are contemplated that each fall within the scope of the present invention.
Claims (10)
1. a kind of purification process of bromelain, which is characterized in that described method includes following steps:
(1) pineapple juice is adjusted to pH 2.5-3.5, obtains pineapple juice sample solution；
(2) by the cation exchange resin column of the pineapple juice sample solution overactivation, flow velocity 2.5-4.5BV/h collects outflow
Liquid, and the efflux is adjusted to pH 6.5-7.5, obtain the first efflux；
(3) by the anion-exchange resin column of the first efflux overactivation, flow velocity 2.5-4.5BV/h collects efflux；
The efflux is adjusted to pH 3.0-4.0, the pineapple juice treatment fluid purified.
2. the method according to claim 1, wherein pineapple juice described in step (1) is after pre-treatment
Pineapple juice；The pre-treatment includes mixing the pineapple Normal juice with preservative, antioxidant, and stirring carries out separation of solid and liquid operation
And electrodialysis, to obtain the pineapple juice after pre-treatment.
3. according to the method described in claim 2, it is characterized in that, the pH of the pineapple Normal juice is 3.5-5.5, the preservative
For sodium benzoate, the antioxidant is Sodium Metabisulfite, and the weight of the pineapple juice, sodium benzoate and Sodium Metabisulfite
Than for 1000:0.05-0.2:0.05-0.2；The separation of solid and liquid operation, which is included under 0-6 DEG C of centrifuging temperature, to be centrifuged, so
After use filtering with microporous membrane.
4. the method according to claim 1, wherein in step (2), the cation exchange resin column of the activation
The preparation method comprises the following steps:
(A) by cation exchange resin purified water swollen, column is filled, cation exchange resin column is obtained；
(B) column is crossed using 0.5-4mol/L hydrochloric acid solution, the flow velocity of hydrochloric acid solution is 0.5-3BV/h, and the dosage of hydrochloric acid solution is
1-6BV, then efflux weakly acidic pH is washed to purifying；
(C) column is crossed using 0.5-4mol/L sodium hydroxide solution again, the flow velocity of sodium hydroxide solution is 0.5-3BV/h, hydroxide
The dosage of sodium solution be 1-6BV, then with purify be washed to efflux weakly acidic pH, the cation exchange resin column activated.
5. according to the method described in claim 4, it is characterized in that, in step (A), the cation exchange resin column be with
Sulfonic cation exchange resin column.
6. the method according to claim 1, wherein in step (3), the anion-exchange resin column of the activation
The preparation method comprises the following steps:
Anion exchange resin purified water swollen is filled column, obtains anion-exchange resin column by (A ')；
(B ') crosses column using 0.5-4mol/L sodium hydroxide solution, and the flow velocity of sodium hydroxide solution is 0.5-3BV/h, sodium hydroxide
The dosage of solution is 1-6BV, then is washed to efflux weakly acidic pH with purifying；
(C ') crosses column using 0.5-4mol/L hydrochloric acid solution again, and the flow velocity of hydrochloric acid solution is 0.5-3BV/h, the dosage of hydrochloric acid solution
For 1-6BV, then with purifying it is washed to efflux weakly acidic pH, the anion-exchange resin column activated.
7. according to the method described in claim 6, it is characterized in that, in step (A '), the tree of the anion-exchange resin column
Rouge is the anion-exchange resin column with quaternary ammonium group.
8. the method according to claim 1, wherein the method also includes following steps:
(4) calcium chloride is added in the pineapple juice treatment fluid of the purifying and makes the concentration 2-5mmol/L of calcium chloride, obtained containing chlorine
Change the pineapple juice treatment fluid of calcium.
9. according to the method described in claim 8, it is characterized in that, the method also includes following steps:
(5) the pineapple juice processing for the chloride containing calcium for obtaining the pineapple juice treatment fluid for the purifying that step (3) obtains or step (4)
Liquid carries out filtering with microporous membrane, the pineapple juice treatment fluid after obtaining bacteria removing.
10. a kind of preparation method of bromelain enzyme product comprising handle the described in any item pineapple juice of claim 1-9
Liquid carries out analysis enzymatic treatment, and bromelain is precipitated；The bromelain is dried again, obtains bromelain production
Product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710675070.0A CN109385414B (en) | 2017-08-09 | 2017-08-09 | Purification method of bromelain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710675070.0A CN109385414B (en) | 2017-08-09 | 2017-08-09 | Purification method of bromelain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109385414A true CN109385414A (en) | 2019-02-26 |
| CN109385414B CN109385414B (en) | 2020-10-30 |
Family
ID=65414802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710675070.0A Active CN109385414B (en) | 2017-08-09 | 2017-08-09 | Purification method of bromelain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109385414B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996183A (en) * | 2020-09-02 | 2020-11-27 | 广东力恩普健康产业科技有限公司 | Preparation method of bromelain |
| 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 |
| 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361333A (en) * | 2013-07-29 | 2013-10-23 | 广西民族大学 | Preparation method of bromelain leaching liquor and stabilization protective agent used therein |
| CN104531643A (en) * | 2015-01-21 | 2015-04-22 | 扬州大学 | Method for separating and purifying of bromelain from pineapple peels |
-
2017
- 2017-08-09 CN CN201710675070.0A patent/CN109385414B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361333A (en) * | 2013-07-29 | 2013-10-23 | 广西民族大学 | Preparation method of bromelain leaching liquor and stabilization protective agent used therein |
| CN104531643A (en) * | 2015-01-21 | 2015-04-22 | 扬州大学 | Method for separating and purifying of bromelain from pineapple peels |
Non-Patent Citations (4)
| Title |
|---|
| OTA S等: "Reinvestigation of fractionation and some properties of the proteolytically active components of stem and fruit bromelains", 《J BIOCHEM.》 * |
| 朱明英等: "菠萝蛋白酶的提取和分离", 《云南热作科技》 * |
| 王燕等: "菠萝蛋白酶工业化提取工艺的改良及酶学性质研究", 《食品工业科技》 * |
| 赵力超 等: "多糖和添加剂对菠萝茎蛋白酶活力的影响", 《食品科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996183A (en) * | 2020-09-02 | 2020-11-27 | 广东力恩普健康产业科技有限公司 | Preparation method of bromelain |
| 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109385414B (en) | 2020-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102907558A (en) | Processing method of sea cucumber polypeptide | |
| CN104961823B (en) | A kind of method of purification suitable for large scale preparation food-grade ovalbumin | |
| CN104788511B (en) | A kind of method of persimmon tannin from persimmon more than preparation small molecule tannin content | |
| CN105925368B (en) | A method of walnut oil is prepared by water law demulsification walnut oil body | |
| CN102115690A (en) | Method for comprehensively utilizing rice bran | |
| CN109385414A (en) | The purification process of bromelain | |
| CN103992402A (en) | Preparation method of high-purity phycocyanin | |
| CN102406048B (en) | Method for preparing sea cucumber glycoprotein by using sea cucumber blanching solution | |
| CN109021096A (en) | A kind of separation and Extraction purifying process of spirulina polysaccharide and phycocyanin | |
| CN103073652A (en) | Method for extracting polysaccharide of spirulina platensis | |
| CN105167064A (en) | Method of processing clear wolfberry juice by comprehensive utilization of wolfberry residues | |
| CN106749743A (en) | A kind of celestial grass Polyose extraction system and extraction process | |
| CN101869169B (en) | Method for preparing fish oligopeptide from gurry by combining fermentation and membrane technology | |
| CN101319208A (en) | Method for preparation of bromelin | |
| CN102276849A (en) | Method for extracting gutta-percha from folium cortex eucommiae | |
| CN106318919A (en) | Method for preparing wild rosa SOD complex enzyme | |
| CN104611316A (en) | Method for extracting bromelin | |
| CN106754834A (en) | A kind of preparation technology of high activity papain | |
| CN114369564B (en) | Preparation method and application of hovenia dulcis-derived exosome | |
| CN106488927A (en) | Method for extracting soluble protein from microalgae biomass | |
| CN107011457A (en) | A kind of method that extraction prepares SNSP and small molecule nutrient molecule in waste water from sweet potato | |
| CN105837703A (en) | Method for extracting spirulina polysaccharide | |
| CN205409475U (en) | Solid sugarcane juice processingequipment is strained to multistage mixed membrane | |
| CN102220296B (en) | Method for extracting garlic superoxide dismutase from garlic sheet processing waste water | |
| CN107628944A (en) | The method and system of low-ester pectin and calcium citrate is extracted from passion fruit shell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: No.39, Jinyang Road, Nanning City, Guangxi Zhuang Autonomous Region, 530032 Patentee after: GUANGXI NANNING BAIHUI PHARMACEUTICAL GROUP Co.,Ltd. Address before: 530005 No. 2, South Central Yao Road, XiXiangTang District, Nanning, the Guangxi Zhuang Autonomous Region Patentee before: GUANGXI NANNING BAIHUI PHARMACEUTICAL GROUP Co.,Ltd. |