CN106987575A - A kind of preparation method of biology enzyme - Google Patents
A kind of preparation method of biology enzyme Download PDFInfo
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- CN106987575A CN106987575A CN201710232255.4A CN201710232255A CN106987575A CN 106987575 A CN106987575 A CN 106987575A CN 201710232255 A CN201710232255 A CN 201710232255A CN 106987575 A CN106987575 A CN 106987575A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01011—Pectinesterase (3.1.1.11)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
Abstract
The present invention relates to a kind of preparation method of biology enzyme, methods described comprises the following steps：(1) pre-process：It will be beaten after fruit cleaning；(2) digest：Enzyme is added in fruit pulp into step (1), pH is adjusted and stirs, separation of solid and liquid, collect supernatant；(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing using milipore filter, and first time hyperfiltration treatment collects permeate, will transmit through liquid and carries out second of hyperfiltration treatment, collects trapped fluid；(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology enzyme.Preparation method of the present invention can be extracted to the biology enzyme of various fruits, and the rate of the biology enzyme of extraction is high, and bioactivity is high, the inventive method specific can be directed to the extraction that peach carries out biology enzyme, the effect of extraction is best, the yield highest of biology enzyme, bioactivity highest.
Description
Technical field
The invention belongs to food processing field, be related to a kind of preparation method of biology enzyme, in particular it relates to it is a kind of using peach as
Raw material is by step by step arithmetic and prepares the method that cellulase and pectin such as digest at the biology enzyme.
Background technology
Peach (Prunus persica) is the fruit of rose family peach family plant seed, and shape includes avette, wide oval or oblate
Shape, color and luster change by pale green white to orange-yellow, pulp is white, light green white, yellow, orange-yellow or red, the pulp of peach
Succulence is savory, sweet tea or sour-sweet.Glucose, fructose, protein, vitamin, carrotene, calcium, phosphorus, iron are rich in the pulp of peach
Deng composition；Peach has long cultivation history in China, deep to be welcome by domestic and international market.
Due to the thin skin of peach, its collecting period is general in high temperature season and picking time Relatively centralized, and under normal temperature condition by
In it is internal or external it is perishable cause it is not shelf-stable；In addition, peach is more sensitive to temperature, easily occur under the conditions of common refrigeration cold
Evil, wadding such as lose at the phenomenon, and brown stain or gelatinization phenomenon easily occur for its pulp, and quality is thick and dries, and color and luster is dark and gloomy, and fruit juice is sticky, it is difficult to
Crowded juice has a strong impact on its commodity value and economic benefit.Therefore, peach be often formed to can, peach juice, dried peach, peach preserved fruit or
The product forms such as peach jam, can evade the problems such as being difficult to storage, these value-added contents of product are not high, nutrients to a certain extent
Matter reserving degree etc. still needs to improve.
There is a certain amount of pectin and cellulose in peach, cellulose is the cell membrane important composition material of peach, and pectin is thin
There is substantial amounts of pectase and cellulase in the important component peach of utricle colloid.Peach normal mature, which softens and damages to plants caused by sudden drop in temperature wadding, loses process
In, catalytic action can occur for pectase and cellulase, cause the component contents such as peach inner cellulose and pectin degrading and cellulose
Increase, therefore peach is difficult to long term storage under normal condition.
At present, domestic and international bioactive enzyme majority derives from animal blood, liver, there is abundant resource in China, existing
Have technology for from blood or blood related raw material extract bioactive enzyme method more have been reported that.The A of CN 102690794 are public
A kind of method for preparing catalase is opened, this method is homogenized, using ultrasonic wave using fresh beef liver as raw material after being shredded
The techniques such as extraction, ion-exchange chromatography, gel permeation chromatography, freeze-drying produce high enzyme activity hydrogen peroxide enzyme product.CN
104593339 A disclose a kind of bioactive enzyme and its extraction process, and the invention bioactive enzyme is extracted from erythrocyte
Obtain, Sephadex G-25 sephadex columns and the affine post of metal-chelating are purified to bioactive enzyme.But prior art
In, the biology enzyme extracted by liver, extraction efficiency is low, is not suitable for the extraction of the biology enzyme of the raw materials such as fruit.
Therefore it provides one kind is using fruit as raw material, the method for particularly extracting biology enzyme using peach as originally is one
Individual urgent problem to be solved.
The content of the invention
It is difficult to store for fruit and traditional processing mode prepares that value-added content of product is low and nutriment reservation degree
Low problem, the present invention is intended to provide a kind of method that biology enzyme is prepared by raw material of fruit, methods described step is simple, it is biological
The purity of enzyme is high.
For up to this purpose, present invention employs following technical scheme：
In a first aspect, the invention provides a kind of preparation method of biology enzyme, comprising the following steps：
(1) pre-process：It will be beaten after fruit cleaning；
(2) digest：Enzyme is added in fruit pulp into step (1), pH is adjusted and stirs, separation of solid and liquid, collect supernatant；
(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing, first time ultrafiltration using milipore filter
Permeate is collected in processing, be will transmit through liquid and is carried out second of hyperfiltration treatment, collects trapped fluid；
(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology
Enzyme.
In the present invention, by adding biology enzyme so that the extraction yield of biology enzyme is higher, by two times of ultrafiltration membrane filtration, go
The removal of impurity so that extract biology enzyme more homogeneous.
Preferably, step (1) described fruit is any one in peach, apple, banana or pineapple or at least two group
Close, preferably peach.
In the present invention, the peach can be selected from, but not limited to, honey peach, peento or nectarine.
Preparation method of the present invention can be extracted to the biology enzyme of various fruits, specific to carry out biology enzyme for peach
Extraction effect preferably, the yield highest of biology enzyme, the active highest of biology enzyme.
Preferably, the mashing described in step (1) is specifically included：Fruit meat and fruit core are separated, fruit weight 1- is added
50 times of water, preferably 1-30 times of water, mashing processing is carried out using Mechanical Method by fruit meat.
In the present invention, fruit meat is carried out mashing and is processed as the conventional methods of beating in this area, ability by described Mechanical Method
Field technique personnel can be selected as needed, and particular determination is not done herein, and the present invention is carried out fruit meat using colloid mill
Mashing is handled.
Preferably, the temperature of the mashing processing is 0-20 DEG C, for example can be 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C, 6
DEG C, 7 DEG C, 8 DEG C, 9 DEG C, 10 DEG C, 11 DEG C, 12 DEG C, 13 DEG C, 14 DEG C, 15 DEG C, 16 DEG C, 17 DEG C, 18 DEG C, 19 DEG C or 20 DEG C, be preferably
2-15 DEG C, preferably 4-10 DEG C.
The amount of the water is 1 times of fruit weight, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times,
12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27
Again, 28 times, 29 times, 30 times, 32 times, 33 times, 35 times, 36 times, 38 times, 40 times, 42 times, 43 times, 45 times, 46 times, 48 times or 50
Times.
Preferably, the mass ratio of step (2) enzyme and fruit is 1:(50-10000), for example, can be 1:50、1:60、
1:70、1:80、1:90、1:100、1:120、1:150、1:200、1:250、1:300、1:350、1:400、1:500、1:600、1:
700、1:800、1:900、1:1000、1:1200、1:1500、1:1600、1:1800、1:1900、1:2000、1:2500、1:
3000、1:3500、1:4000、1:4500、1:5000、1:5500、1:6000、1:6500、1:7000、1:7500、1:8000、1:
8500、1:9000、1:9500 or 1:10000, preferably 1:(100-8000), more preferably 1:(500-6000).
Preferably, the enzyme is any one in lignin-degrading enzymes, manganese peroxidase or laccase or at least two
Combination.
Preferably, the pH described in step (2) is 2-11, for example, can be 2,3,4,5,6,7,8,9,10 or 11 preferably 3-
8, more preferably 3.5-6.
Preferably, the temperature of the stirring described in step (2) is 10-70 DEG C, for example can be 10 DEG C, 11 DEG C, 12 DEG C, 13
℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、25℃、26℃、28℃、30℃、32
DEG C, 35 DEG C, 38 DEG C, 40 DEG C, 42 DEG C, 45 DEG C, 48 DEG C, 50 DEG C, 52 DEG C, 55 DEG C, 58 DEG C, 60 DEG C, 62 DEG C, 65 DEG C, 68 DEG C or 70
DEG C, preferably 20-60 DEG C.
Preferably, the mixing time described in step (2) be 1-20h, for example can be 1h, 2h, 3h, 4h, 5h, 6h, 7h,
8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h, preferably 3-16h.
Preferably, the molecular cut off of the milipore filter of the first time hyperfiltration treatment is 50-300kDa, for example, can be
50kDa、51kDa、52kDa、53kDa、55kDa、60kDa、65kDa、70kDa、75kDa、80kDa、85kDa、90kDa、
95kDa, 100kDa, 110kDa, 120kDa, 150kDa, 160kDa, 180kDa, 200kDa, 220kDa, 250kDa, 280kDa or
300kDa, preferably 55-200kDa, more preferably 60-150kDa.
Preferably, the molecular cut off of the milipore filter of second of hyperfiltration treatment is 3-40kDa, for example, can be
3kDa、4kDa、5kDa、6kDa、7kDa、8kDa、9kDa、10kDa、11kDa、12kDa、13kDa、14kDa、15kDa、16kDa、
17kDa、18kDa、19kDa、20kDa、21kDa、22kDa、23kDa、24kDa、25kDa、26kDa、27kDa、28kDa、
29kDa, 30kDa, 31kDa, 32kDa, 33kDa, 34kDa, 35kDa, 36kDa, 37kDa, 38kDa, 39kDa or 40kDa, preferably
For 10-35kDa, more preferably 15-30kDa.
In the present invention, the step of enzymolysis is with two times of ultrafiltration is carried out by specific enzyme and coordinated, plays a part of Synergistic,
So that the biology enzyme extracted is more homogeneous, bioactivity is high.
Preferably, the temperature of the ultrafiltration is 20-80 DEG C, for example can be 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 25 DEG C, 26
℃、28℃、30℃、32℃、35℃、38℃、40℃、42℃、45℃、48℃、50℃、52℃、55℃、58℃、60℃、62
DEG C, 65 DEG C, 68 DEG C, 70 DEG C, 72 DEG C, 75 DEG C, 78 DEG C or 80 DEG C, preferably 30-60 DEG C.
In the present invention, described spray drying is the ordinary skill in the art, and those skilled in the art can be as needed
Alternative condition is carried out, particular determination is not done herein.
Pectinesterase, polygalacturonase, inscribe Portugal are included in the present invention, the biology enzyme extracted by the above method
Dextranase, exoglucanase and beta-glucosidase.
In the present invention, in order to which biology enzyme is further purified, ion-exchangeable chromatography, hydrophobic chromatography or gel filtration
The methods such as chromatography carry out subsequent treatment, and those skilled in the art can carry out selecting further purification process as needed,
This does not do particular determination, and biology enzyme after treatment can be further purified, and obtains the higher biology enzyme of purity.
According to the present invention, the preparation method of the biology enzyme comprises the following steps：
(1) pre-process：After fruit cleaning, separation fruit meat and fruit core add the water of 1-50 times of fruit weight, use
Fruit meat is carried out mashing processing by Mechanical Method；
(2) digest：The mass ratio added in fruit pulp into step (1) with fruit is 1:The enzyme of (50-10000), is adjusted
PH to 2-11 is saved, 10-70 DEG C of stirring 1-20h, separation of solid and liquid collects supernatant；
(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing, first time ultrafiltration using milipore filter
Processing uses molecular cut off for 50-300kDa milipore filter, collects permeate, will transmit through second of hyperfiltration treatment of liquid progress and adopts
The milipore filter for being 3-40kDa with molecular cut off, collects trapped fluid；
(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology
Enzyme；
Wherein, step (1) described fruit be peach, apple, banana or pineapple in any one or at least two combination,
Step (2) described enzyme is any one in lignin-degrading enzymes, manganese peroxidase or laccase or at least two combination.
As optimal technical scheme, the preparation method of the biology enzyme comprises the following steps：
(1) pre-process：After peach is cleaned, separation peach meat and peach-pit add the water of 1-30 times of fruit weight, using Mechanical Method
Fruit meat is subjected to mashing processing；
(2) digest：The mass ratio added in fruit pulp into step (1) with fruit is 1:The lignin of (500-6000)
Digestive enzyme and manganese peroxidase, adjust pH to 3.5-6, and 20-60 DEG C of stirring 3-16h, separation of solid and liquid collects supernatant；
(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing, first time ultrafiltration using milipore filter
Processing uses molecular cut off for 60-150kDa milipore filter, collects permeate, will transmit through second of hyperfiltration treatment of liquid progress and adopts
The milipore filter for being 15-30kDa with molecular cut off, collects trapped fluid；
(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology
Enzyme.
Compared with prior art, the present invention at least has the advantages that：
(1) preparation method of the present invention can be extracted to the biology enzyme of various fruits, and the yield of the biology enzyme of extraction is high,
Up to more than 0.65%, bioactivity is high, but the inventive method being capable of the specific extraction that biology enzyme is carried out for peach, extraction
Effect preferably, the yield highest of biology enzyme, up to more than 1.2%, bioactivity highest；
(2) the inventive method technique is simple, and step is few, easily operation, and pectinesterase, poly are included in the biology enzyme of extraction
Galacturonic acid enzyme, endoglucanase, exoglucanase and beta-glucosidase, being widely used for the enzyme, can be carried out
Industrialization large-scale production.
Embodiment
For ease of understanding the present invention, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is only to aid in understanding the present invention, is not construed as the concrete restriction to the present invention.
Embodiment 1
Raw material is Shenzhou City's honey peach newly plucked, 100kg.
(1) pre-process：Fresh Shenzhou City's honey peach 100kg of harvesting is taken and as mete-wand, using peach crushing and beating machine
Peach is handled, 1000kg clear water is added, peach pulp is broken into pulpous state using colloid mill under the conditions of 0-20 DEG C, is obtained
About 1090kg peach slurry；
(2) digest：180g lignin peroxidases and manganese peroxidase are added in the peach slurry obtained to step (1)
Mixture, pH is adjusted to 4.5-5.0, the stir process 8h under the conditions of 35 DEG C；
(3) separation of solid and liquid：Mixture after the enzymolysis for being obtained step (2) using perforated wall centrifuge carries out solid-liquid point
From collection clear liquid obtains 1050kg permeate；
(4) UF membrane 1：Carried out using molecular cut off for the clear liquid that 70kDa milipore filter obtains step (3) at ultrafiltration
Reason, collects permeate, obtains permeate 850kg；
(5) UF membrane 2：Carried out using molecular cut off for the clear liquid that 30kDa milipore filter obtains step (4) at ultrafiltration
Reason, collects trapped fluid, obtains 60kg trapped fluids；
(6) it is freeze-dried：The ultra-filter retentate that step (5) is obtained is freeze-dried, the dried frozen aquatic products 1.7kg of acquisition.
Analysis result shows gross protein 1.4kg in the dried frozen aquatic products, and high performance liquid chromatography testing result shows dried frozen aquatic products
In contain pectinesterase, polygalacturonase, endoglucanase, exoglucanase and beta-glucosidase.Pass through
Using 3,5-dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 3056U/g.
Embodiment 2
Raw material is the honey peach of placement 3 days after harvesting.
(1) pre-process：Take and the honey peach 100kg of 3 days is placed after harvesting and as mete-wand, using peach crushing and beating
Machine is handled peach, adds 1000kg clear water, peach pulp is broken into pulpous state using colloid mill under the conditions of 0-20 DEG C, obtained
Obtain about 1085kg peach slurry；
(2) digest：The mixture of 100g laccases is added in the peach slurry obtained to step (1), pH is adjusted to 3.5,60
Stir process 10h under the conditions of DEG C；
(3) separation of solid and liquid：Mixture after the enzymolysis for being obtained step (2) using plate and frame filter press carries out separation of solid and liquid,
The clear liquid of collection obtains 1045kg permeate through tubular type centrifugal treating；
(4) UF membrane 1：Carried out using molecular cut off for the clear liquid that 65kDa milipore filter obtains step (3) at ultrafiltration
Reason, collects permeate, obtains permeate 900kg；
(5) UF membrane 2：Carried out using molecular cut off for the clear liquid that 20kDa milipore filter obtains step (4) at ultrafiltration
Reason, collects trapped fluid, obtains 55kg trapped fluids；
(6) it is freeze-dried：The ultra-filter retentate that step (5) is obtained is freeze-dried, the dried frozen aquatic products 1.5kg of acquisition.
Analysis result shows gross protein 1.2kg in the dried frozen aquatic products, and high performance liquid chromatography testing result shows dried frozen aquatic products
In contain pectinesterase, polygalacturonase, endoglucanase, exoglucanase and beta-glucosidase.Pass through
Using 3,5-dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 3142U/g.
Embodiment 3
Raw material is the honey peach of placement 3 days after harvesting.
(1) pre-process：Take and the honey peach 100kg of 3 days is placed after harvesting and as mete-wand, using peach crushing and beating
Machine is handled peach, adds 100kg clear water, and peach pulp is broken into pulpous state using colloid mill under the conditions of 0 DEG C, obtained about
190kg peach slurry；
(2) digest：100g lignin-degrading enzymes are added in the peach slurry obtained to step (1), pH is adjusted to 2,10
Stir process 20h under the conditions of DEG C；
(3) separation of solid and liquid：Mixture after the enzymolysis for being obtained step (2) using plate and frame filter press carries out separation of solid and liquid,
The clear liquid of collection obtains 165kg permeate through tubular type centrifugal treating；
(4) UF membrane 1：Carried out using molecular cut off for the clear liquid that 50kDa milipore filter obtains step (3) at ultrafiltration
Reason, collects permeate, obtains permeate 105kg；
(5) UF membrane 2：Carried out using molecular cut off for the clear liquid that 3kDa milipore filter obtains step (4) at ultrafiltration
Reason, collects trapped fluid, obtains 45kg trapped fluids；
(6) it is freeze-dried：The ultra-filter retentate that step (5) is obtained is freeze-dried, the dried frozen aquatic products 1.1kg of acquisition.
Analysis result shows gross protein 0.9kg in the dried frozen aquatic products, and high performance liquid chromatography testing result shows dried frozen aquatic products
In contain pectinesterase, polygalacturonase, endoglucanase, exoglucanase and beta-glucosidase.Pass through
Using 3,5-dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 2224U/g.
Embodiment 4
Raw material is the nectarine of placement 3 days after harvesting.
(1) pre-process：Take and the nectarine 100kg of 3 days is placed after harvesting and as mete-wand, using peach crushing and beating machine
Peach is handled, 5000kg clear water is added, peach pulp is broken into pulpous state using colloid mill under the conditions of 20 DEG C, obtained about
5030kg peach slurry；
(2) digest：2000g manganese peroxidases are added in the peach slurry obtained to step (1), pH is adjusted to 11, at 70 DEG C
Under the conditions of stir process 1h；
(3) separation of solid and liquid：Mixture after the enzymolysis for being obtained step (2) using plate and frame filter press carries out separation of solid and liquid,
The clear liquid of collection obtains 4860kg permeate through tubular type centrifugal treating；
(4) UF membrane 1：Using molecular cut off ultrafiltration is carried out for the clear liquid that 300kDa milipore filter obtains step (3)
Processing, collects permeate, obtains permeate 4320kg；
(5) UF membrane 2：Carried out using molecular cut off for the clear liquid that 40kDa milipore filter obtains step (4) at ultrafiltration
Reason, collects trapped fluid, obtains 40kg trapped fluids；
(6) it is freeze-dried：The ultra-filter retentate that step (5) is obtained is freeze-dried, the dried frozen aquatic products 1.2kg of acquisition.
Analysis result shows gross protein 0.8kg in the dried frozen aquatic products, and high performance liquid chromatography testing result shows dried frozen aquatic products
In contain pectinesterase, polygalacturonase, endoglucanase, exoglucanase and beta-glucosidase.Pass through
Using 3,5-dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 2573U/g.
Embodiment 5
Raw material is the pineapple of placement 3 days after harvesting.
(1) pre-process：Take and the pineapple 100kg of 3 days is placed after harvesting and as mete-wand, using pineapple crushing and beating machine
Pineapple is handled, 100kg clear water is added, pineapple pulp is broken into pulpous state using colloid mill under the conditions of 0 DEG C, obtained about
190kg pineapple slurry；
(2) digest：100g lignin-degrading enzymes are added in the pineapple slurry obtained to step (1), pH is adjusted to 2,
Stir process 20h under the conditions of 10 DEG C；
(3) separation of solid and liquid：Mixture after the enzymolysis for being obtained step (2) using plate and frame filter press carries out separation of solid and liquid,
The clear liquid of collection obtains 150kg permeate through tubular type centrifugal treating；
(4) UF membrane 1：Carried out using molecular cut off for the clear liquid that 50kDa milipore filter obtains step (3) at ultrafiltration
Reason, collects permeate, obtains permeate 102kg；
(5) UF membrane 2：Carried out using molecular cut off for the clear liquid that 3kDa milipore filter obtains step (4) at ultrafiltration
Reason, collects trapped fluid, obtains 35kg trapped fluids；
(6) it is freeze-dried：The ultra-filter retentate that step (5) is obtained is freeze-dried, the dried frozen aquatic products 1kg of acquisition.
Analysis result shows gross protein 0.65kg in the dried frozen aquatic products, and high performance liquid chromatography testing result shows dried frozen aquatic products
In contain pectinesterase, polygalacturonase, endoglucanase, exoglucanase and beta-glucosidase.Pass through
Using 3,5-dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 1567U/g.
Embodiment 6
Raw material is places 3-5 days after ripe apple-picking, 100kg.
(1) pre-process：Take and the apple 100kg of 3 days is placed after harvesting and as mete-wand, using apple crushing and beating machine
Apple is handled, 100kg clear water is added, apple grunt is broken into pulpous state using colloid mill under the conditions of 0 DEG C, obtained about
190kg apple pulp；
(2) digest：100g lignin-degrading enzymes are added in the apple pulp obtained to step (1), pH is adjusted to 2,
Stir process 20h under the conditions of 10 DEG C；
(3) separation of solid and liquid：Mixture after the enzymolysis for being obtained step (2) using plate and frame filter press carries out separation of solid and liquid,
The clear liquid of collection obtains 172kg permeate through tubular type centrifugal treating；
(4) UF membrane 1：Carried out using molecular cut off for the clear liquid that 50kDa milipore filter obtains step (3) at ultrafiltration
Reason, collects permeate, obtains permeate 123kg；
(5) UF membrane 2：Carried out using molecular cut off for the clear liquid that 3kDa milipore filter obtains step (4) at ultrafiltration
Reason, collects trapped fluid, obtains 40kg trapped fluids；
(6) it is freeze-dried：The ultra-filter retentate that step (5) is obtained is freeze-dried, the dried frozen aquatic products 1.2kg of acquisition.
Analysis result shows gross protein 0.68kg in the dried frozen aquatic products, and high performance liquid chromatography testing result shows dried frozen aquatic products
In contain pectinesterase, polygalacturonase, endoglucanase, exoglucanase and beta-glucosidase.Pass through
Using 3,5-dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 1747U/g.
Comparative example 1
Compared with Example 1, it is other same as Example 1 in addition to no second of UF membrane of process.
The dried frozen aquatic products 6.5kg of acquisition, analysis result shows gross protein 0.32kg in the dried frozen aquatic products.By using 3,5-
Dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 352U/g.
Comparative example 2
Compared with Example 1, it is other same as Example 1 in addition to no process first time UF membrane.
The dried frozen aquatic products 1.1kg of acquisition, analysis result shows gross protein 0.41kg in the dried frozen aquatic products.By using 3,5-
Dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 635U/g.
Comparative example 3
Compared with Example 1, carried out except no by second of UF membrane before first time UF membrane using chromatographic column
It is other same as Example 1 outside adsorbing separation.
The dried frozen aquatic products 3.6kg of acquisition, analysis result shows gross protein 0.46kg in the dried frozen aquatic products.By using 3,5-
Dinitrosalicylic Acid Colorimetry determines the enzyme activity of dried frozen aquatic products, and the enzyme activity of the biology enzyme of extraction is up to 1034U/g.
From comparative example 1-3 and the contrast of embodiment 1 as can be seen that the inventive method extract after biology enzyme in yield and enzyme
Bioactivity is apparently higher than comparative example 1-3.
In summary, preparation method of the present invention can be extracted to the biology enzyme of various fruits, the biology enzyme of extraction
Yield is high, and bioactivity is high, but the inventive method specific can be directed to the extraction that peach carries out biology enzyme, and the effect of extraction is most
It is good, the yield highest of biology enzyme, bioactivity highest, moreover, if from comparative example as can be seen that reducing the mistake of milipore filter
Number of times is filtered, or makes into separate otherwise, its effect is substantially not as the present invention.
Applicant states that the present invention illustrates the technological process of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned detailed process flow, that is, does not mean that the present invention has to rely on above-mentioned detailed process flow and could implemented.Affiliated skill
The technical staff in art field is it will be clearly understood that any improvement in the present invention, equivalence replacement to each raw material of product of the present invention and auxiliary
The addition of co-ingredients, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and being open.
Claims (9)
1. a kind of preparation method of biology enzyme, it is characterised in that comprise the following steps：
(1) pre-process：It will be beaten after fruit cleaning；
(2) digest：Enzyme is added in fruit pulp into step (1), pH is adjusted and stirs, separation of solid and liquid, collect supernatant；
(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing, first time hyperfiltration treatment using milipore filter
Permeate is collected, liquid is will transmit through and carries out second of hyperfiltration treatment, collect trapped fluid；
(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology enzyme.
2. preparation method according to claim 1, it is characterised in that step (1) described fruit be peach, apple, banana or
In pineapple any one or at least two combination, preferably peach；
Preferably, the mashing described in step (1) is specifically included：Fruit meat and fruit core are separated, 1-50 times of fruit weight is added
Water, preferably 1-30 times of water, using Mechanical Method by fruit meat carry out mashing processing；
Preferably, the temperature of the mashing processing is 0-20 DEG C, preferably preferably 2-15 DEG C, 4-10 DEG C.
3. preparation method according to claim 1 or 2, it is characterised in that the mass ratio of step (2) enzyme and fruit is
1:(50-10000), preferably 1:(100-8000), more preferably 1:(500-6000)；
Preferably, the enzyme is any one in lignin-degrading enzymes, manganese peroxidase or laccase or at least two group
Close.
4. the preparation method according to any one of claim 1-3, it is characterised in that the pH described in step (2) is 2-11,
Preferably 3-8, more preferably 3.5-6.
5. the preparation method according to any one of claim 1-4, it is characterised in that the temperature of the stirring described in step (2)
Spend for 10-70 DEG C, preferably 20-60 DEG C；
Preferably, the mixing time described in step (2) is 1-20h, preferably 3-16h.
6. the preparation method according to any one of claim 1-5, it is characterised in that the first time hyperfiltration treatment it is super
The molecular cut off of filter membrane is 50-300kDa, more preferably preferably 55-200kDa, 60-150kDa.
7. the preparation method according to any one of claim 1-6, it is characterised in that second of hyperfiltration treatment it is super
The molecular cut off of filter membrane is 3-40kDa, more preferably preferably 10-35kDa, 15-30kDa；
Preferably, the temperature of the two times of ultrafiltration is 20-80 DEG C, preferably 30-60 DEG C.
8. the preparation method according to any one of claim 1-7, it is characterised in that comprise the following steps：
(1) pre-process：After fruit cleaning, separation fruit meat and fruit core add the water of 1-50 times of fruit weight, using machinery
Fruit meat is carried out mashing processing by method；
(2) digest：The mass ratio added in fruit pulp into step (1) with fruit is 1:The enzyme of (50-10000), adjusts pH
To 2-11,10-70 DEG C of stirring 1-20h, separation of solid and liquid collects supernatant；
(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing, first time hyperfiltration treatment using milipore filter
Molecular cut off is used for 50-300kDa milipore filter, permeate is collected, second of hyperfiltration treatment use of liquid progress is will transmit through and cuts
The milipore filter that molecular weight is 3-40kDa is stayed, trapped fluid is collected；
(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology enzyme；
Wherein, step (1) described fruit is any one in peach, apple, banana or pineapple or at least two combination, step
(2) enzyme is any one in lignin-degrading enzymes, manganese peroxidase or laccase or at least two combination.
9. the preparation method according to any one of claim 1-8, it is characterised in that comprise the following steps：
(1) pre-process：After peach is cleaned, separation peach meat and peach-pit adds the water of 1-30 times of fruit weight, using Mechanical Method by water
Pulp carries out mashing processing；
(2) digest：The mass ratio added in fruit pulp into step (1) with fruit is 1:The lignin degradation of (500-6000)
Enzyme and manganese peroxidase, adjust pH to 3.5-6, and 20-60 DEG C of stirring 3-16h, separation of solid and liquid collects supernatant；
(3) UF membrane：The supernatant obtained to step (2) carries out two times of ultrafiltration processing, first time hyperfiltration treatment using milipore filter
Molecular cut off is used for 60-150kDa milipore filter, permeate is collected, second of hyperfiltration treatment use of liquid progress is will transmit through and cuts
The milipore filter that molecular weight is 15-30kDa is stayed, trapped fluid is collected；
(4) it is spray-dried：The trapped fluid that step (3) is obtained is spray-dried, and dried dried frozen aquatic products is biology enzyme.
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| CN1944640A (en) * | 2006-08-03 | 2007-04-11 | 谢丹青 | Physical extracting method for papain |
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| CN1944640A (en) * | 2006-08-03 | 2007-04-11 | 谢丹青 | Physical extracting method for papain |
| CN103305492A (en) * | 2013-06-09 | 2013-09-18 | 诸辉 | Method for extracting high-purity pharmaceutical grade bromelain |
| CN104293750A (en) * | 2014-10-15 | 2015-01-21 | 重庆骄王天然产物股份有限公司 | Method for efficiently extracting sterile papain |
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| HUI-LEI YU，ET AL.: "Identification, purification and characterization of β-glucosidase from apple seed as a novel catalyst for synthesis of O-glucosides", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
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