CN107475226A - A kind of new Bromenzyme and preparation method thereof - Google Patents
A kind of new Bromenzyme and preparation method thereof Download PDFInfo
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- CN107475226A CN107475226A CN201710851725.5A CN201710851725A CN107475226A CN 107475226 A CN107475226 A CN 107475226A CN 201710851725 A CN201710851725 A CN 201710851725A CN 107475226 A CN107475226 A CN 107475226A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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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 kind of preparation method of new Bromenzyme, comprise the following steps：(1) pineapple juice is prepared；(2) preparing natural polyanionic polysaccharide solution；(3) pineapple juice, polyanionic polysaccharide solution to same pH are adjusted respectively, pineapple juice and polyanionic polysaccharide solution are sufficiently mixed, stirring, the bromelain in pineapple juice is set to interact to form bromelain polyanionic polysaccharide complex precipitate by electrostatic attraction with polyanionic polysaccharide；(4) bromelain polyanionic polysaccharide complex precipitate is collected by centrifugation, through being dried to obtain Bromenzyme.The present invention uses polyanionic polysaccharide and bromelain, the principle precipitated is produced by electrostatic adelphotaxy to reach the purpose of bromelain separation and preparation, said preparation can both keep stability of the bromelain during saving and processing, bromelain can also be overcome to the shortcomings that thermo-responsive, oxidizable, and there is good sustained release and controlled release properties.
Description
Technical field
The invention belongs to protease preparation technical field, and in particular to a kind of new Bromenzyme and its preparation side
Method.
Background technology
Bromelain is a kind of protease for extracting to obtain from pineapple peel and pineapple stem, and the production of bromelain can
To solve environmental pollution and problem of resource waste caused by a large amount of waste materials are dropped in pineapple industry, it can also make pineapple product high level
Change and utilize.Bromelain belongs to pawpaw enzyme system, is divided into stem bromelain according to the difference at separation and Extraction position
(EC3.4.22.32) and seed-timber stand (EC3.4.22.33), both molecular weight are respectively 33kDa and 28kDa, wait electricity
Point is respectively 9.5 and 4.6, is mostly commercially stem enzyme with bromelain.Bromelain is applied in pharmacy and food service industry
It is in extensive range.In field of medicaments, because bromelain has the effect such as antithrombotic, antioedematous, anti-inflammatory, it can be used for ulcer
With the removing of burn wound slough.In field of food industry, bromelain is widely used in the tenderization of meat, beer
Clarification, cheesemaking and protein hydrolysate etc..
At present, isolating and purifying the commercial run of bromelain mainly has kaolin absorption method, the tannin precipitation method and ultrafiltration
Concentration method etc..Kaolin absorption method products obtained therefrom enzymatic activity is higher, but production technology is complex, equipment and raw materials consumption
More, the enzyme activity rate of recovery is low, pollutes environment, and environmental protection investments are larger；The tannin precipitation method are simple to operate, but selectivity is poor, enzyme activity
The rate of recovery and enzymatic activity are relatively low, and tannin is a kind of noxious material, and environment can be polluted；Ultrafiltration concentration method is simple to operate,
Enzyme activity is lost and environmental pollution is less, but its selectivity is poor, and the foreign protein contained in bromelain enzyme preparation is more, it is difficult to
Meet requirement.
It is also a kind of hand commonly used in the prior art to prepare bromelain by immobilization technology to improve its stability
Section, immobilised enzymes have that heat endurance is high, storage stability is good, repellence is strong, not facile hydrolysis and to change compared with resolvase
The characteristics such as property agent better tolerance, thus immobilization technology obtains the important means utilized extensively and effectively as biology enzyme.
The conventional embedded material of immobilised enzymes has a lot, and wherein sodium alginate is most commonly seen.The glad grade of nobleness (" sodium alginate-
The research of chitosan co-immobilization pineapple stem protease ", noble glad etc., food industry science and technology, the 5th phase of volume 32,209-211
Page, 2011) pineapple stem protease is slowly injected into sodium alginate soln with pipettor, then with No. seven needle applicators
Sodium alginate-enzyme mixation is highly implanted containing CaCl with 5cm2Chitosan solution in form smooth microballoon, it is this with marine alga
Sour sodium, chitosan are that the pineapple stem protease that material co-immobilization prepares is reusable, its optimal reactive temperature and heat
The enzymatic properties such as stability are also improved.Open (" sodium alginate investment prepares immobilization bromelain ", the Zhang Chen such as treasure
Deng, biotechnology, the 6th phase of volume 7, the 31-34 pages) propose using sodium alginate as carrier immobilized bromelain, carry
The high optimal pH of bromelain and to alkaline environment and the stability of temperature.Document above using soluble sodium alginate with
Bivalent metal ion Ca2+The principle that reaction can form microballoon embeds to bromelain, can realize in a mild condition
Enzyme immobilizatio, help to maintain the bioactivity of enzyme.Whether occur in the immobilization technology, between enzyme and sodium alginate mutually
Effect does not influence on embedding；But the sodium alginate micro ball particle diameter of calcification is big, stability is poor, easily polyion effect or
Calcium ion diffusion and potassium, sodium ion largely assemble when soften occur " leakage enzyme " phenomenon；Also, immobilization process separates work in enzyme
Carried out after the completion of sequence, it is necessary to add extra technique in original enzyme production procedure, cause immobilization technology cost higher；And
And the enzyme preparation prepared is embedded when in use by immobilization technology, it can not realize enzyme because enzyme is embedded in matrix
Sustained release and controlled release, therefore can be only applied to have in the liquid food of good diffusion, in the larger liquid of viscosity and it can not consolidate
Played a role in state food.
Patent of invention CN102108352A discloses a kind of process for fixation of new bromelain, and the invention is gathered with shell
Sugar is carrier, and with glutaraldehyde as cross linker, chitosan is prepared into microballoon, then fixes bromelain using chitosan microball
Enzyme, the mechanical strength of carrier was both improved, and be also beneficial to improve load enzyme amount, enzyme activity and the enzyme rate of recovery of immobilised enzymes, simultaneously
Also enzyme is made to be easily isolated with hydrolysate.But the invention is commonly used as crosslinking agent, glutaraldehyde using glutaraldehyde and makees bactericide,
There is strong impulse to glasses, skin and mucous membrane and be not easy to remove, thus have impact on the application of the invention in the food industry；
The invention needs to use extra technique on the separation purifying technique basis of bromelain；Also, due to the invention
Enzyme preparation can not realize the sustained release and controlled release of enzyme, and this similarly limits the enzyme preparation of invention preparation in the larger liquid of viscosity
And the application in solid-state food.
Therefore, explore that a kind of operating procedure is simple, cost is cheap, pollution-free, stability is high and with sustained release and controlled release work(
Bromenzyme of energy and preparation method thereof, it is significant for the application of expansion bromelain.
The content of the invention
For above mentioned problem existing for Bromenzyme in the prior art, the invention provides a kind of new pineapple egg
White enzyme preparation and preparation method thereof.
For achieving the above object, the present invention is achieved using following technical proposals：
A kind of preparation method of new Bromenzyme, comprises the following steps：
(1) pineapple peel is cleaned, squeezed the juice, filtered, after adjusting filtrate pH value, added chitosan solution, be uniformly mixed,
4 DEG C of refrigerators place 24h, take supernatant to obtain pineapple juice after centrifugation；
(2) preparing natural polyanionic polysaccharide solution, in 4 DEG C of fully hydrations overnight；
(3) pineapple juice, polyanionic polysaccharide solution to same pH are adjusted respectively, pineapple juice is molten with polyanionic polysaccharide
Liquid is sufficiently mixed, and stirring, makes the bromelain in pineapple juice interact to be formed by electrostatic attraction with polyanionic polysaccharide
Bromelain-polyanionic polysaccharide complex precipitate；
(4) bromelain-polyanionic polysaccharide complex precipitate is collected by centrifugation, through being dried to obtain bromelain system
Agent.
Further, the middle citric acid of the step (1) or malic acid solution adjust filtrate pH value to 2.5-4.0, addition etc.
1% chitosan solution of volume；20min is centrifuged under the conditions of 10000r/min in the step (1), takes supernatant to be clarified
Pineapple juice.
Further, the natural polyanions polysaccharide in the step (2) includes sodium alginate, pectin, carboxymethyl cellulose
One or more of mixtures in sodium, Arabic gum, xanthans.
Further, citric acid or malic acid solution are used in the step (3) respectively by pineapple juice and polyanionic polysaccharide
Solution ph is adjusted to 2.5-4.0；To be diluted in the step (3) 10% pineapple juice and 0.5%-2% (w/v) it is poly- it is cloudy from
Sub- polysaccharide solution is sufficiently mixed in equal volume, the stirring reaction 10-30min at 4-45 DEG C.
Further, it is more to be collected using 4000r/min centrifugation 20min for bromelain-polyanion in the step (4)
Saccharide complex precipitates.
The drying means of the step (4) is freeze-drying, is dried in vacuo, one kind in spray drying.
Further, the enzyme activity rate of recovery in the step (4) in bromelain-polyanionic polysaccharide complex precipitate is
76.7%-83.4%, the enzyme activity rate of recovery is 14.5%-20.6% in the supernatant of centrifugation, and enzyme activity loss late is 2.1%-
2.7%.
Further, the bromelain system being prepared according to the preparation method of the new Bromenzyme of the present invention
Agent.
Further, the optimal reactive temperature of the Bromenzyme is 55-65 DEG C；The Bromenzyme
Optimal reaction pH is 6.6-7.2；After the Bromenzyme heats 2h at 70 DEG C, remaining enzyme activity is 62%-73%.
Further, the Bromenzyme can discharge all bromelains in the basic conditions in 30min；
The Bromenzyme can discharge all bromelains under pH 5.0 and the acid condition of the above in 2h；The spinach
Trailing plants protease preparation is in pH 2.5-4.0 without enzyme r e lease；The presence of salt can accelerate the release of enzyme.
Compared with prior art, advantages of the present invention and advantageous effects are：
(1) waste material being largely dropped in pineapple industry can be prepared into Bromenzyme by the present invention, make pineapple
Product higher value application, solves the problems, such as environmental pollution and the wasting of resources.
(2) relative in the prior art using immobilization technology come Encapsulated Enzyme, the present invention is waiting electricity using bromelain
The following positively charged of point, electrostatic interaction can occur with negatively charged polyanionic polysaccharide and form compound polyelectrolyte, make
Both are uniformly distributed in the composite, with protect sulfydryl in bromelain, amino, trp residue and histidine residues from
Such environmental effects, the storage-stable of enzyme is set to have obtained significant raising.At present, it has not been found that using polyanionic polysaccharide
The research that compound polyelectrolyte preparation is formed with bromelain is reported.
(3) bromelain prepared relative to Immobilization in Sodium Alginate, the bromelain grain prepared by the present invention
Footpath is small, high safety, and enzyme stability is high, cannot be only used for liquid food, it may also be used for viscosity larger liquid and solid-state food,
Also, the preparation condition of the present invention is simple to operation, and equipment investment is few, is easy to industrialized production.
(4) present invention prepares Bromenzyme by electrostatic interaction between polyelectrolyte, and the method need not appoint
What organic solvent, catalyst or chemical cross-linking agent, there is safe, good biocompatibility, that technique is simple, cost is cheap etc. is aobvious
Advantage is write, therefore, the heat endurance of bromelain can be improved to prepare Bromenzyme by compound polyelectrolyte,
Extend its storage period and validity period, the production efficiency of processing food can be improved, reduce cost.
(5) present invention adds appropriate polyanionic polysaccharide in pineapple juice, by adjusting pineapple juice and polysaccharide ratio, mixing
PH value of solution etc. is closed to form insoluble compound polyelectrolyte, by being drying to obtain Bromenzyme, can not only be reached
The purpose of concentrating and separating bromelain, but also the stability of enzyme can be improved simultaneously and obtain required controlled release properties, can
The deficiency of existing Bromenzyme technology is made up, therefore, there is good application in the exploitation of novel foodstuff enzyme preparation
Potentiality.
(6) of the invention compared with traditional kaolin absorption method, the tannin precipitation method and ultrafiltration concentration method etc., technique simply may be used
OK, security is good, and the enzyme activity rate of recovery is high, stability is high, also, the present invention has stronger selectivity, can be by controlling solution
PH value being separated bromelain enzyme selectivity from solution, therefore be remarkably improved the vigor of enzyme preparation.
(7) pH value is adjusted to less than 4.0 in the preparation of pineapple juice, makes filter by the present invention using chitosan as fining agent
Bromelain in liquid becomes positively charged, and therefore, bromelain will not react to form precipitation and produce damage with chitosan
Lose, to ensure the purity of bromelain and organic efficiency.
(8) present invention uses the regulation that pH is carried out than milder citric acid or malic acid, safe, will not be to food
Flavor adversely affect.
Summary, the present invention use polyanionic polysaccharide and bromelain, produced by electrostatic adelphotaxy
The principle of precipitation reaches the purpose of bromelain separation and preparation, and the enzyme preparation formulation can both keep bromelain to exist
Stability during saving and processing, moreover it is possible to overcome bromelain to the shortcomings that thermo-responsive, oxidizable, and delay with good
Release and controlled release properties, moreover, the preparation method of the present invention is simple to operate, security is good, and production cost is low, and selectivity is stronger, enzyme
The rate of recovery living is high, and equipment investment is few, is easy to industrialized production.
Brief description of the drawings
After the embodiment of the present invention is read in conjunction with the figure, the other features and advantages of the invention will become more clear
Chu.
Fig. 1 is the schematic flow sheet of the present invention；
Fig. 2 is the form schematic diagram of the Bromenzyme in the present invention.
Embodiment
Technical scheme is described in further detail with reference to the accompanying drawings and detailed description.
2% (w/v) polyanionic polysaccharide solution：2g polyanionic polysaccharides accurately are weighed, add appropriate deionized water, are used
Constant temperature blender with magnetic force is stirred to abundant dissolving, is settled to 100mL with deionized water, is obtained 2% polyanionic polysaccharide storing solution,
4 DEG C of refrigerator overnights are positioned over fully to be hydrated.Using preceding required concentration is diluted to deionized water.
For prepare 2% (w/v) polyanionic polysaccharide solution natural polyanions polysaccharide include sodium alginate, pectin,
One or more of mixtures in sodium carboxymethylcellulose, Arabic gum, xanthans.
Embodiment 1
100g pineapple peels are taken, are cleaned, add 100mL deionized waters, squeezes the juice, after nylon cloth filtering, is adjusted with citric acid clear
Liquid pH value adds 1% isometric chitosan solution, is uniformly mixed to 3.0.4 DEG C of refrigerators place 24h, 10000r/min
Supernatant is taken after centrifugation 20min, with deionized water constant volume to 100mL, that is, obtains 10% pineapple juice.4 DEG C of refrigerators save backup.
2% (w/v) sodium alginate soln is diluted to 0.5% (w/v) with deionized water, respectively by 10% pineapple juice and
0.5% (w/v) sodium alginate soln adjusts pH value to 3.0 with citric acid solution, then that identical pH two kinds of solution are isometric
Mix, 30min is stirred at 4 DEG C, 4000r/min centrifugation 20min, collects bromelain-sodium alginate compound polyelectrolyte
Precipitation, freeze-drying obtain Bromenzyme.The enzyme activity rate of recovery under the conditions of this in complex precipitate is 76.7%, centrifugation
Supernatant in the enzyme activity rate of recovery be 20.6%, enzyme activity loss late be 2.7%.
The enzyme preparation optimal reactive temperature is 55 DEG C, and 5 DEG C are improved relative to commodity Bromenzyme；Optimal reaction pH
For 6.6,0.6 is improved relative to commodity Bromenzyme, is resistant to broader pH scopes；2h, commodity spinach are heated at 70 DEG C
Trailing plants protease preparation residue enzyme activity 29%, the present invention in Bromenzyme residue enzyme activity 65%.The enzyme preparation is in alkali
All bromelains can be discharged in 30min under the conditions of property, discharge institute in 2h under pH more than 5.0 acid condition
There is a bromelain, without enzyme r e lease during pH 3.0 or so；The presence of salt can accelerate the release of enzyme.Bromelain only works as medium
In pH value or ionic strength can be triggered and discharge when can result in complex dissociation, and the trigger condition can
To be adjusted by changing the preparation condition of the enzyme preparation.
Embodiment 2
100g pineapple peels are taken, are cleaned, add 100mL deionized waters, squeezes the juice, after nylon cloth filtering, is adjusted with malic acid clear
Liquid pH value adds 1% isometric chitosan solution, is uniformly mixed to 2.5.4 DEG C of refrigerators place 24h, 10000r/min
Supernatant is taken after centrifugation 20min, with deionized water constant volume to 100mL, that is, obtains 10% pineapple juice.4 DEG C of refrigerators save backup.
2% (w/v) pectin solution is diluted to 1% (w/v) with deionized water, respectively by 10% pineapple juice and 1% (w/
V) pectin solution adjusts pH value to 2.5 with malic acid solution, then mixes identical pH two kinds of solution in equal volume, is stirred at 35 DEG C
10min is mixed, 4000r/min centrifugation 20min, collects bromelain-Pectin Polyelectrolyte complex precipitate, vacuum drying obtains
Bromenzyme.The enzyme activity rate of recovery under the conditions of this in complex precipitate is 78.5%, the enzyme activity in the supernatant of centrifugation
The rate of recovery is 19.2%, and enzyme activity loss late is 2.3%.
The enzyme preparation optimal reactive temperature is 60 DEG C, and 10 DEG C are improved relative to commodity Bromenzyme；Optimal reaction
PH is 6.8, improves 0.8 relative to commodity Bromenzyme, is resistant to broader pH scopes；2h, commodity are heated at 70 DEG C
Bromenzyme residue enzyme activity 29%, the present invention in Bromenzyme residue enzyme activity 66%.The enzyme preparation exists
All bromelains can be discharged under alkalescence condition in 30min, are discharged under pH more than 5.0 acid condition in 2h
All bromelains, without enzyme r e lease during pH 2.5 or so；The presence of salt can accelerate the release of enzyme.
Embodiment 3
100g pineapple peels are taken, are cleaned, add 100mL deionized waters, squeezes the juice, after nylon cloth filtering, is adjusted with citric acid clear
Liquid pH value adds 1% isometric chitosan solution, is uniformly mixed to 4.0.4 DEG C of refrigerators place 24h, 10000r/min
Supernatant is taken after centrifugation 20min, with deionized water constant volume to 100mL, that is, obtains 10% pineapple juice.4 DEG C of refrigerators save backup.
2% (w/v) carboxymethylcellulose sodium solution is diluted to 1% (w/v) with deionized water, respectively by 10% pineapple
Juice and 1% (w/v) carboxymethylcellulose sodium solution adjust pH value to 4.0 with citric acid solution, then molten by two kinds of identical pH
Liquid is mixed in equal volume, and 20min is stirred at 40 DEG C, 4000r/min centrifugation 20min, collects bromelain-sodium carboxymethylcellulose
Compound polyelectrolyte precipitates, and vacuum drying obtains Bromenzyme.Enzyme activity recovery under the conditions of this in complex precipitate
Rate is 83.4%, and the enzyme activity rate of recovery in the supernatant of centrifugation is 14.5%, and enzyme activity loss late is 2.1%.
The enzyme preparation optimal reactive temperature is 65 DEG C, and 15 DEG C are improved relative to commodity Bromenzyme；Optimal reaction
PH is 7.2, improves 1.2 relative to commodity Bromenzyme, is resistant to broader pH scopes；2h, commodity are heated at 70 DEG C
Bromenzyme residue enzyme activity 29%, the present invention in Bromenzyme residue enzyme activity 73%.The enzyme preparation exists
All bromelains can be discharged under alkalescence condition in 30min, are discharged under pH 5.0 and the acid condition of the above in 2h
Go out all bromelains, without enzyme r e lease during pH 4.0 or so；The presence of salt can accelerate the release of enzyme.
Embodiment 4
100g pineapple peels are taken, are cleaned, add 100mL deionized waters, squeezes the juice, after nylon cloth filtering, is adjusted with citric acid clear
Liquid pH value adds 1% isometric chitosan solution, is uniformly mixed to 3.0.4 DEG C of refrigerators place 24h, 10000r/min
Supernatant is taken after centrifugation 20min, with deionized water constant volume to 100mL, that is, obtains 10% pineapple juice.4 DEG C of refrigerators save backup.
10% pineapple juice and 2% (w/v) gumwater are adjusted into pH value to 3.0 with citric acid solution respectively, then
Identical pH two kinds of solution are mixed in equal volume, 15min is stirred at 45 DEG C, 4000r/min centrifugation 20min, collects bromelain
Enzyme-Arabic gum compound polyelectrolyte precipitation, freeze-drying obtain Bromenzyme.Under the conditions of being somebody's turn to do in complex precipitate
The enzyme activity rate of recovery be 80.5%, the enzyme activity rate of recovery in the supernatant of centrifugation is 17.1%, and enzyme activity loss late is 2.4%.
The enzyme preparation optimal reactive temperature is 60 DEG C, and 10 DEG C are improved relative to commodity Bromenzyme；Optimal reaction
PH is 7.0, improves 1.0 relative to commodity Bromenzyme, is resistant to broader pH scopes；2h, commodity are heated at 70 DEG C
Bromenzyme residue enzyme activity 29%, the present invention in Bromenzyme residue enzyme activity 68%.The enzyme preparation exists
All bromelains can be discharged under alkalescence condition in 30min, are discharged under pH 5.0 and above acid condition in 2h
All bromelains, without enzyme r e lease during pH 3.0 or so；The presence of salt can accelerate the release of enzyme.
Embodiment 5
100g pineapple peels are taken, are cleaned, add 100mL deionized waters, squeezes the juice, after nylon cloth filtering, is adjusted with malic acid clear
Liquid pH value adds 1% isometric chitosan solution, is uniformly mixed to 4.0.4 DEG C of refrigerators place 24h, 10000r/min
Supernatant is taken after centrifugation 20min, with deionized water constant volume to 100mL, that is, obtains 10% pineapple juice.4 DEG C of refrigerators save backup.
2% (w/v) xanthan gum solution is diluted to 0.5% (w/v) with deionized water, respectively by 10% pineapple juice and
0.5% (w/v) xanthan gum solution adjusts pH value to 4.0 with malic acid solution, then mixes identical pH two kinds of solution in equal volume
Close, 15min is stirred at 30 DEG C, 4000r/min centrifugation 20min, bromelain-xanthans compound polyelectrolyte is collected and sinks
Form sediment, Bromenzyme is obtained through spray drying after being diluted with deionized water.Enzyme activity under the conditions of this in complex precipitate is returned
Yield is 78.5%, and the enzyme activity rate of recovery in the supernatant of centrifugation is 19.2%, and enzyme activity loss accounts for 2.3%.
The enzyme preparation optimal reactive temperature is 55 DEG C, and 5 DEG C are improved relative to commodity Bromenzyme；Optimal reaction pH
For 6.8,0.8 is improved relative to commodity Bromenzyme, is resistant to broader pH scopes；2h, commodity spinach are heated at 70 DEG C
Trailing plants protease preparation residue enzyme activity 29%, the present invention in Bromenzyme residue enzyme activity 62%.The enzyme preparation is in alkali
All bromelains can be discharged in 30min under the conditions of property, are discharged under pH 5.0 and the acid condition of the above in 2h
All bromelains, without enzyme r e lease during pH 4.0 or so；The presence of salt can accelerate the release of enzyme.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality
Example is applied the present invention is described in detail, for the person of ordinary skill of the art, still can be to foregoing implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replaced
Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.
Claims (10)
1. a kind of preparation method of new Bromenzyme, it is characterised in that comprise the following steps：
(1) pineapple peel is cleaned, squeezed the juice, filtered, after adjusting filtrate pH value, added chitosan solution, be uniformly mixed, 4 DEG C
Refrigerator places 24h, takes supernatant to obtain pineapple juice after centrifugation；
(2) preparing natural polyanionic polysaccharide solution, in 4 DEG C of fully hydrations overnight；
(3) pineapple juice, polyanionic polysaccharide solution to same pH are adjusted respectively, and pineapple juice and polyanionic polysaccharide solution are filled
Divide mixing, stirring, make the bromelain in pineapple juice interact to form pineapple by electrostatic attraction with polyanionic polysaccharide
Protease-polyanionic polysaccharide complex precipitate；
(4) bromelain-polyanionic polysaccharide complex precipitate is collected by centrifugation, through being dried to obtain Bromenzyme.
2. the preparation method of new Bromenzyme according to claim 1, it is characterised in that：The step (1)
It is middle to adjust filtrate pH value to 2.5-4.0 with citric acid or malic acid solution, add 1% isometric chitosan solution；The step
Suddenly 20min is centrifuged under the conditions of 10000r/min in (1), the pineapple juice for taking supernatant to be clarified.
3. the preparation method of new Bromenzyme according to claim 1, it is characterised in that：The step (2)
In natural polyanions polysaccharide include sodium alginate, pectin, sodium carboxymethylcellulose, Arabic gum, one kind in xanthans
Or several mixtures.
4. the preparation method of new Bromenzyme according to claim 1, it is characterised in that：The step (3)
It is middle that pineapple juice and polyanionic polysaccharide solution ph are adjusted to by 2.5-4.0 using citric acid or malic acid solution respectively；The step
Suddenly the polyanionic polysaccharide solution of the pineapple juice and 0.5%-2% (w/v) that are diluted to 10% is sufficiently mixed in equal volume in (3),
The stirring reaction 10-30min at 4-45 DEG C.
5. the preparation method of new Bromenzyme according to claim 1, it is characterised in that：The step (4)
It is middle that 20min is centrifuged using 4000r/min, collect bromelain-polyanionic polysaccharide complex precipitate.
6. the preparation method of new Bromenzyme according to claim 1, it is characterised in that：The step (4)
Drying means for freeze-drying, vacuum drying, spray drying in one kind.
7. the preparation method of new Bromenzyme according to claim 1, it is characterised in that：The step (4)
The enzyme activity rate of recovery in middle bromelain-polyanionic polysaccharide complex precipitate is 76.7%-83.4%, in the supernatant of centrifugation
The enzyme activity rate of recovery is 14.5%-20.6% in liquid, and enzyme activity loss late is 2.1%-2.7%.
8. the pineapple egg that the preparation method of the new Bromenzyme according to claim any one of 1-7 is prepared
White enzyme preparation.
9. Bromenzyme according to claim 8, it is characterised in that：The Bromenzyme it is most suitable anti-
It is 55-65 DEG C to answer temperature；The optimal reaction pH of the Bromenzyme is 6.6-7.2；The Bromenzyme exists
After heating 2h at 70 DEG C, remaining enzyme activity is 62%-73%.
10. Bromenzyme according to claim 8, it is characterised in that：The Bromenzyme is in alkalescence
Under the conditions of can discharge all bromelains in 30min；The Bromenzyme is in pH 5.0 and the acidity of the above
Under the conditions of can discharge all bromelains in 2h；The Bromenzyme is in pH 2.5-4.0 without enzyme r e lease；Food
The presence of salt can accelerate the release of enzyme.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504646A (en) * | 2018-06-07 | 2018-09-07 | 贵州沣稷生物科技有限公司 | A kind of highly effective extraction method of bromelain |
| CN109880814A (en) * | 2019-04-28 | 2019-06-14 | 中国热带农业科学院农产品加工研究所 | A method of it is extracted from pineapple bran and obtains bromelain |
| WO2021068025A1 (en) * | 2019-10-09 | 2021-04-15 | Phoenix Eagle Company Pty Ltd | Enhancement and stabilisation of proteolytic activity of proteases |
| CN114699517A (en) * | 2022-04-25 | 2022-07-05 | 南京鲸力医药科技有限公司 | Bromelain preparation and application thereof in detumescence composition |
| EP4041881A4 (en) * | 2019-10-09 | 2023-11-08 | Phoenix Eagle Company Pty Ltd | Enhancement and stabilisation of proteolytic activity of proteases |
| RU2819793C1 (en) * | 2023-10-31 | 2024-05-24 | федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет" (ФГБОУ ВО "ВГУ") | Method of producing a hybrid preparation of bromelain and chitosan ascorbate in form of a thick solution |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108352A (en) * | 2009-12-25 | 2011-06-29 | 南通远大生物科技发展有限公司 | Immobilization method of bromelain |
| CN102742810A (en) * | 2012-07-19 | 2012-10-24 | 青岛农业大学 | Garlic oil microcapsule with shell made of soybean protein isolate and chitosan and preparation method and application thereof |
| CN103421759A (en) * | 2013-07-25 | 2013-12-04 | 广西大学 | Method for using soy protein to extract bromelain |
| CN104611316A (en) * | 2015-02-13 | 2015-05-13 | 广西壮族自治区分析测试研究中心 | Method for extracting bromelin |
| CN106282144A (en) * | 2016-09-23 | 2017-01-04 | 合肥信达膜科技有限公司 | A kind of bromelain production method |
| CN106636039A (en) * | 2017-01-22 | 2017-05-10 | 嵊州市派特普科技开发有限公司 | Extraction method for bromelain |
-
2017
- 2017-09-19 CN CN201710851725.5A patent/CN107475226A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108352A (en) * | 2009-12-25 | 2011-06-29 | 南通远大生物科技发展有限公司 | Immobilization method of bromelain |
| CN102742810A (en) * | 2012-07-19 | 2012-10-24 | 青岛农业大学 | Garlic oil microcapsule with shell made of soybean protein isolate and chitosan and preparation method and application thereof |
| CN103421759A (en) * | 2013-07-25 | 2013-12-04 | 广西大学 | Method for using soy protein to extract bromelain |
| CN104611316A (en) * | 2015-02-13 | 2015-05-13 | 广西壮族自治区分析测试研究中心 | Method for extracting bromelin |
| CN106282144A (en) * | 2016-09-23 | 2017-01-04 | 合肥信达膜科技有限公司 | A kind of bromelain production method |
| CN106636039A (en) * | 2017-01-22 | 2017-05-10 | 嵊州市派特普科技开发有限公司 | Extraction method for bromelain |
Non-Patent Citations (5)
| Title |
|---|
| DÉBORA A. CAMPOS等: ""Platform design for extraction and isolation of Bromelain: Complex formation and precipitation with carrageenan"", 《PROCESS BIOCHEMISTRY》 * |
| NADIA WOITOVICH VALETTI ET AL.: ""Polyelectrolytes–protein complexes: a viable platform in the downstream processes of industrial enzymes at scaling up level"", 《J CHEM TECHNOL BIOTECHNOL》 * |
| YISHENG XU ET AL.: ""Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity"", 《BIOMACROMOLECULES》 * |
| 张琛等: ""海藻酸钠包埋法制备固定化菠萝蛋白酶"", 《生物加工过程》 * |
| 金昌海: "《果蔬贮藏与加工》", 30 September 2016, 中国轻工业出版社 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504646A (en) * | 2018-06-07 | 2018-09-07 | 贵州沣稷生物科技有限公司 | A kind of highly effective extraction method of bromelain |
| CN109880814A (en) * | 2019-04-28 | 2019-06-14 | 中国热带农业科学院农产品加工研究所 | A method of it is extracted from pineapple bran and obtains bromelain |
| WO2021068025A1 (en) * | 2019-10-09 | 2021-04-15 | Phoenix Eagle Company Pty Ltd | Enhancement and stabilisation of proteolytic activity of proteases |
| EP4041881A4 (en) * | 2019-10-09 | 2023-11-08 | Phoenix Eagle Company Pty Ltd | Enhancement and stabilisation of proteolytic activity of proteases |
| CN114699517A (en) * | 2022-04-25 | 2022-07-05 | 南京鲸力医药科技有限公司 | Bromelain preparation and application thereof in detumescence composition |
| CN114699517B (en) * | 2022-04-25 | 2023-04-14 | 南京鲸力医药科技有限公司 | Bromelain preparation and application thereof in detumescence composition |
| RU2819793C1 (en) * | 2023-10-31 | 2024-05-24 | федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет" (ФГБОУ ВО "ВГУ") | Method of producing a hybrid preparation of bromelain and chitosan ascorbate in form of a thick solution |
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