CN107699549A - A kind of pectase and preparation method thereof - Google Patents
A kind of pectase and preparation method thereof Download PDFInfo
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- CN107699549A CN107699549A CN201711163178.8A CN201711163178A CN107699549A CN 107699549 A CN107699549 A CN 107699549A CN 201711163178 A CN201711163178 A CN 201711163178A CN 107699549 A CN107699549 A CN 107699549A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- 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)
- C12N9/24—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)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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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- 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)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
Abstract
The present invention discloses a kind of pectase and preparation method thereof, belongs to bio-fermentation engineering field.Its preparation side includes preparing banana skin slag dry powder, actication of culture, prepares seed liquor, solid fermentation culture and prepare thick zyme extract, and the activation of described thalline is that aspergillus niger is put into 20 ~ 30 DEG C of Czapek's medium to cultivate 100 ~ 120 hours；Described solid fermentation culture is after mixing banana peel dry powder and wheat bran in proportion, add deionized water to be well mixed, add ammonium sulfate, dipotassium hydrogen phosphate, calcium chloride stirring, charging, after 110 ~ 125 DEG C of moist heat sterilizations 20 ~ 40 minutes, inoculating spores suspension culture 60 ~ 72 hours.The present invention solves the Utilizing question of principal by product in banana deep-processing process, and preparation technology is simple, and cost is cheap, and pectinase activity is high, can effectively improve the crushing juice rate and clarity of common fruit juice.
Description
Technical field
The invention belongs to bio-fermentation engineering field, and in particular to a kind of pectase and preparation method thereof.
Background technology
Banana belongs to Musaceae (Musaceae) Musa (Musa), is one of world's " four big fruit ", banana is China
One of southern important tropical fruit (tree), banana skin accounts for the 30 ~ 50% of its fruit weight.Research shows, containing a large amount of in banana skin
The material such as pectin, starch, carbohydrate, cellulose, hemicellulose and lignin, also containing protein, water-soluble sugar, unsaturated lipid
The nutritional ingredient such as fat acid, vitamin, inorganic salts, Ca, Mg, P and K.
According to statistics, nearly ten years, Chinese banana production rises to 2015 years from 651.81 ten thousand tons of 2005
1246.63 ten thousand tons, account for the total fruit of banana by banana skin 35 ~ 40% calculate, and can produce 436.32 ~ 498.65 ten thousand tons of banana skin.It is fragrant
For any of several broadleaf plants in addition to directly eating, pulp is processed into bananas juice, banana breast, banaina, banana figs, banana beverage, Banana Wine, banana
The products such as vinegar, banana skin be eat raw and deep-processing process in caused discarded object, banana skin is nutritious, but easily brown stain and corruption
It is rotten, it is not easy to store, easy breed bacteria, directly abandons and not only pollute environment, and cause the serious wasting of resources.Extensively
West is the high yield area of banana, and development banana industry has advantageous condition, if can be reasonably utilized banana resource,
It can then turn waste into wealth, therefore the sustainable development for effectively expanding effective protection and industrial chain of its utilization ways to environment has
There is positive meaning.
For pectase as one of four big enzyme preparation of the world, energy depolymerized pectin matter, is most important enzyme in fruit process.In agriculture
In Product processing, played an important role to improving crushing juice rate, clarity, reducing viscosity etc., especially in fruit juice manufacture, fruit wine
Brewage etc. has a very wide range of applications.At present in prepared by the fermentation of pectase, often using melanomyces as fermented bacterium,
Because fermentation of Aspergillus niger production pectase has the characteristics that technical maturity, the pectinase activity height of unit raw material production, security, its
The primary raw material of raw material is the rich pectous processing of farm products accessory substance such as pomace, orange peel powder, and it is rarely seen have utilize banana skin
Mixing wheat bran as fermentation major ingredient development pectin enzyme fermentation, isolate and purify, the correlative study of zymologic property and application.
The content of the invention
The invention aims to low, the pectase that solves the utilization rate of principal by product in existing banana deep-processing process
Preparing raw material is single, high expensive, the problems such as enzymatic activity is low, stability is poor, there is provided a kind of pectase and preparation method thereof.For reality
Now technical scheme used in the object of the invention is：
The present invention carries out the culture of banana skin slag solid fermentation using deposit number CICC40493 aspergillus nigers, extracting and developing purifies
To the pectase available for fruit juice production.A kind of preparation method of pectase, including prepare banana skin slag dry powder, actication of culture,
Prepare seed liquor, solid fermentation culture and prepare thick zyme extract, the activation of described thalline is that aspergillus niger is put into Cha Shi cultures
20 ~ 30 DEG C of base is cultivated 100 ~ 120 hours；Described solid fermentation culture is after mixing banana peel dry powder and wheat bran in proportion,
Add deionized water to be well mixed, add ammonium sulfate, dipotassium hydrogen phosphate, calcium chloride stirring, charging is damp and hot in 110 ~ 125 DEG C
After sterilizing 20 ~ 40 minutes, inoculating spores suspension culture 60 ~ 72 hours.
Preferably, specific preparation process is as follows：
（1）Prepare banana skin slag dry powder：Banana skin is sorted, smashed after being dried 10 ~ 12 hours in 80 ~ 90 DEG C, excessively 200 ~
300 mesh sieves, banana skin slag dry powder is obtained, it is standby；
（2）Actication of culture：By aspergillus niger be put into Czapek's medium in 28 DEG C incubated 100 ~ 120 hours, slant strains and three
After angle bottle expands culture strain, it is placed in 0 ~ 5 DEG C of refrigerator and preserves；
（3）Prepare seed liquor：One bottle of cultured strain is taken, adds the sterile saline that mass fraction is 0.85%, gently will
The spore of media surface is scraped, and spore suspension is placed in sterilizing triangular flask, sterile glass ball is placed in advance in bottle, fully
Spore suspension is prepared into the filtering of sterile absorbent cotton after shaking, it is 5 × 10 to control suspension spore count5~6×105 Individual/mL bacterium
Liquid, as seed liquor；
（4）Solid fermentation culture：By banana skin slag dry powder and wheat bran with mass ratio 1 ~ 3:8 ~ 10 mixing, add 1.5 ~ 2 times go from
Sub- water, it is sufficiently mixed uniformly, adds 0.15 ~ 0.25% ammonium sulfate, 0.01 ~ 0.03% dipotassium hydrogen phosphate, 0.1 ~ 0.2% calcium chloride
Stirring, then fed by 5 ~ 10% charges, 110 ~ 125 DEG C of moist heat sterilizations are after 20 ~ 40 minutes, inoculation seed liquor, inoculum concentration 1 ~
2mL, incubation time 60 ~ 72 hours, Qu Yici was respectively turned in 12,24,36 hours in incubation；
（5）Prepare thick zyme extract：After fermentation, the pure water of 6 ~ 8 times of culture medium quality is added, opens ultrasonic extractor progress
Extraction, 32 DEG C, ultrasonic power 20KHZ of Extracting temperature, extract 1 hour, after extraction, 4000r/ minutes refrigerated centrifuge 10 divides
Clock, it is the thick zyme extract of pectin to take supernatant.
Preferably, preparation method also include isolate and purify step by step, using ultrafiltration concentration, ammonium sulfate precipitation, dialysis and
DEAE-52 ion-exchange chromatographies are isolated and purified.
Preferably, the physical and chemical index of the banana skin slag dry powder, which controls, is：Total reducing sugar 40 ~ 45%, pectin 14 ~ 16%, protein 2
~ 3%, total acid 2 ~ 3%, moisture 4 ~ 4.5%, crude fat 3.8 ~ 4.5%, ash content 7 ~ 9% and other compositions 15 ~ 27.2%.
Preferably, described step（4）The amount that adding deionized water is is entered with the quality of banana skin slag dry powder and wheat bran
Row calculates, and ammonium sulfate, dipotassium hydrogen phosphate and the calcium chloride of addition are with the total amount after banana skin slag dry powder, wheat bran and deionized water
Calculate.
Preferably, the thick zyme extract enzyme of described pectin is 190 ~ 195U/mg albumen than control living.
Preferably, the initial concentration of described ammonium sulfate precipitation is 30%, final concentration of 80%.
Preferably, described ultrafiltration concentration is to pour into pectin thick zyme extract in ultrafiltration cup to be concentrated by ultrafiltration, concentration
Liquid product be stoste 1/10th, and ultrafiltration retaining molecular weight is 1 ten thousand to one ten thousand two；
Described ammonium sulfate precipitation is that the thick zyme extract of pectin after concentration is put into frozen water to mix in bath, into enzyme liquid slowly
Ammonium sulfate powder is added to saturation；Stirring while adding during operation, stirring is as far as possible slow and uniform, in order to avoid the generation of foam；
Standing 12-14 hours in refrigerator are put into, 4 DEG C of 4000r refrigerated centrifuges obtain thick enzyme precipitation for 10 minutes；
Described dialysis is that thick enzyme precipitation is dissolved in the HAc-NaAc buffer solutions of 0.02M pH=4.8, loads molecular cut off
In 14000 bag filter, to be placed in 5 DEG C of refrigerators, dialysed 24 hours in pure water, after thick zyme extract be freeze-dried
Thick enzyme powder；
Described DEAE-52 ion-exchange chromatographies are by the thick enzyme powder after freeze-drying, are dissolved in 0.02M pH=4.2
In HAc-NaAc buffer solutions, it is slowly added into dropper on the DEAE-52 chromatographic columns balanced, first elutes 4 cylinders with buffer solution
Product, then linear elution is carried out with the identical buffer solution of the NaCL gradients Han 0 ~ 1.0M, flow velocity is 1mL/ minutes, and often pipe 4mL, is adopted
Collected with part automatic collector, detect protein content and pectase enzyme activity by pipe, collect enzyme activity composition.
Preferably, described freeze-drying is at -40 DEG C by thick zyme extract, is freeze-dried 24 hours, and it is dry to obtain freezing
Dry thick enzyme powder.
The present invention also provides more than one pectases prepared by the preparation method.
The prominent substantive distinguishing features of the present invention and significant progress are：
（1）One of raw material of the present invention is banana skin, accounts for the 30 ~ 50% of fruit total amount, and resource very abundant, nutritive value is high,
Not only waste of resource is abandoned, environment can also be polluted.Banana skin is brought as raw material, can be turned waste into wealth, is solved
By-product utilization problem in banana deep-processing process.Processing method banana skin processing method of the present invention is without using acid, alkali, third
The organic solvents such as ketone, reduce the secondary pollution in banana skin deep processing, and utilize the mistake of banana skin slag pectinase by fermentation
Poisonous and harmful reagent need not be used in journey, secondary pollution will not be produced to environment.In addition, banana skin is processed into product makes cost
Obvious to reduce, technical process is simple, and flow is short, and reaction speed is fast, and caused waste residue is few in production process, banana skin utilization rate
It is high.
（2）The inventive method whole flow process is extracted by special equipment, prepares and extraction process is not added and any had
Separated, purified on the premise of evil organic solvent, thus make activity produced by the invention high, reach improve pectase for
Food, medicine, the application of daily use chemicals and textile industry, improve the security used, reliability；Most important is significantly to provide
The crushing juice rate and clarity of common fruit juice.Also include freeze-drying step in method, solve that liquid enzyme stability is poor, guarantees the quality
The shortcomings of phase is short, enzyme activity is easily lost so that pectase is easy to preserve, subsequently to need to carry out again according to what is tested or produce
It can guarantee that the vigor of enzyme keeps preferably horizontal when further refining.
（3）The microorganism of existing pectase can not grow producing enzyme on simple banana skin slag, and preparation method of the present invention,
Using banana skin slag as the sample of culture medium, mixed by banana peel dry powder and wheat bran, solve banana skin and suppress because acidity is high
The growth of microbes producing cellulase, the problem of producing pectase micro-organisms pectase can not be utilized by overcoming pure banana skin slag, be led to
The optimization for crossing culture medium and condition of culture forms the cheap preparation technology that can obtain active pectin enzyme of a set of cost.
（4）The thalline of Cord blood can recover thalline in a dormant state, using the thalline activation medium of the present invention and live
Power, by expanding culture step by step, in order to obtain pure and strong culture, that is, obtain training flush, that inoculation quantity is enough
Thing is supported, allows strain gradually to adapt to fermentation medium culture environment.
（5）The present invention is mixed using banana peel dry powder and wheat bran, is added ammonium sulfate, dipotassium hydrogen phosphate, calcium chloride and is carried out
Solid culture, because pectase as a kind of induced enzyme needs inducer pectin to generate, pectic substance enriches in banana skin powder,
Wheat bran can allow fermentation medium to become the quantity delivered of fluffy increase oxygen, and banana skin and wheat bran are with 1 ~ 3:8 ~ 10 mixing can be good
Formed complementary；Ammonium sulfate is conventional nitrogen source, and dipotassium hydrogen phosphate and calcium chloride are the growth factors of microorganism, to micro-organisms
Nitrogen source and inorganic salts are provided, promote the effect of pectin enzymatic synthesis, while potassium dihydrogen phosphate and calcium chloride have regulation culture medium
The effect of basicity.
Embodiment
The present invention program is described in further detail with reference to embodiment, the description below merely to explain the present invention, and
Its content is not defined.
Embodiment 1：Aspergillus niger（CICC40493）Utilize banana skin slag solid fermentation culture pectase
Step：
1. banana skin slag pre-processes：Banana skin, sort, drying（80 ~ 90 DEG C, dry 10 ~ 12 hours）, smash, cross 200 ~ 300 mesh
Sieve, standby, banana skin slag dry powder physical and chemical index control range is shown in Table 1.
The banana skin slag dry powder physical and chemical index of table 1
| Total reducing sugar (%) | 40~45 |
| Pectin (%) | 14~16 |
| Protein (%) | 2~3 |
| Total acid (%) | 2~3 |
| Moisture (%) | 4~4.5 |
| Crude fat (%) | 3.8~4.5 |
| Ash content (%) | 7~9 |
| Other compositions（%） | 15~27.2 |
2. fungi preservation and seed expand culture
Fungi preservation uses CM0015 czapek's mediums with expanding culture.Condition of culture：28 DEG C are incubated 100 ~ 120 hours,
Slant strains and triangular flask expand culture strain and are placed in 0 ~ 5 DEG C of refrigerator preservation.
3. the preparation of aspergillus niger CICC40493 spore suspensions
One bottle of cultured strain is taken, the sterile saline that mass fraction is 0.85% is added, gently by media surface
Spore is scraped, and spore suspension is placed in sterilizing triangular flask, several sterile glass balls are placed in advance in bottle, are used after shake well
The filtering of sterile absorbent cotton is prepared into spore suspension, and it is 5 × 10 to control bacteria suspension spore count5~6×105 Individual/mL bacterium solutions.
4. solid state fermentation pectase
Solid state fermentation basal medium：Banana skin slag dry powder is with wheat bran with mass ratio 1 ~ 3:8 ~ 10 mixing, add 1.5 ~ 2 times of quality
Deionized water（In terms of butt quality）, it is sufficiently mixed uniformly, adds 0.15 ~ 0.25% ammonium sulfate, 0.01 ~ 0.03% phosphoric acid hydrogen
Dipotassium, 0.1 ~ 0.2% calcium chloride（Ammonium sulfate, dipotassium hydrogen phosphate, calcium chloride addition are based on wet basis）Stir, by 5 ~ 10% dresses
Doses feeds, 121 DEG C of moist heat sterilizations 30 minutes, and under natural pH, inoculating spores suspension, concentration is 5 × 105 ~6×105Individual/mL
Bacterium solution, 1 ~ 2mL of inoculum concentration, incubation time 60 ~ 72 hours, Qu Yici was respectively turned in 12,24,36 hours in incubation.
5. the preparation of thick zyme extract
After fermentation, the pure water of 6 ~ 8 times of culture medium quality is added, ultrasonic extractor is opened and is extracted（32 DEG C of Extracting temperature,
Ultrasonic power 20KHZ, extract 1 hour）, 4000r/ minutes refrigerated centrifuge 10 minutes after extraction, it is fruit to take supernatant
The thick zyme extract of glue, enzyme is than 190 ~ 195U/mg albumen living.
Embodiment 2：The extraction of pectase and isolate and purify
1. the concentration of thick zyme extract
Thick zyme extract is poured into ultrafiltration cup and is concentrated by ultrafiltration, the volume of concentrate is stoste 1/10th or so, milipore filter
Molecular cut off is 10,000 or so.
2. ammonium sulfate precipitation
The thick zyme extract of pectin after concentration is put into frozen water to mix in bath, ammonium sulfate powder is slowly added into enzyme liquid to certain
Saturation degree.Stirring while adding during operation, stirring is as far as possible slow and uniform, in order to avoid the generation of foam.It is put into refrigerator and stands
Overnight, 4 DEG C of 4000r refrigerated centrifuges obtain enzyme precipitation for 10 minutes.
The initial concentration of ammonium sulfate precipitation is 30%, final concentration of 80%.
3. dialysis desalting
Thick enzyme precipitation is dissolved in the HAc-NaAc buffer solutions of a small amount of 0.02M pH=4.8, loading molecular cut off is
In 14000 bag filter, it is placed in 5 DEG C of refrigerators, is dialysed 24 hours in pure water.By the enzyme liquid constant volume dialysed to certain volume.
For enzyme than 267.16U/mg albumen living, purification is 1.39 times after measuring desalination.
4. it is freeze-dried
- 40 DEG C, it is freeze-dried 24 hours, obtains and be freeze-dried thick enzyme powder.
5. DEAE-52 ion-exchange chromatographies separate
The thick enzyme powder after desalination freeze-drying is weighed, is dissolved in the HAc-NaAc buffer solutions of 0.02M pH=4.2, it is slow with dropper
Slowly the DEAE-52 balanced with the HAc-NaAc buffer solutions of 0.02M pH=4.8 is added（1.6×30cm）On chromatographic column, first with slow
Fliud flushing elutes 4 column volumes, then carries out linear elution, flow velocity 1mL/ with the identical buffer solution of the NaCl gradients Han 0 ~ 1.0M
Minute, every pipe 4mL, collected using part automatic collector, protein content and pectase enzyme activity detected by pipe, collect enzyme activity into
Point.
Pectase can obtain 4 obvious albumen cutting edges of a knife or a sword after DEAE-52 column chromatographies.First cutting edge of a knife or a sword is direct buffering
What liquid afforded, second to the 4th cutting edge of a knife or a sword is the cutting edge of a knife or a sword eluted by 0 ~ 1.0M NaCl, is named as E1, E2, E3, E4 successively, is received
Collect each protein peak, determine the protein of each component after PEG 20000 concentration and enzyme activity is shown in Table 2.
The each component protein content of table 2 and enzyme activity
| Component | E1 | E2 | E3 | E4 |
| Protein content mg | 0.06 | 2.01 | 5.45 | 6.25 |
| Enzyme activity U | Do not measure | 7413.14 | 9290.92 | 5994.05 |
| Enzyme is than U/mg albumen living | Do not measure | 3688.13 | 1704.76 | 959.05 |
Note：List data is by taking 100mg applied sample amounts as an example
The purification that E2, E3, E4 contrast thick enzyme is respectively 19.21,8.88,4.99, and the rate of recovery is followed successively by 6.20%, 16.82%
With 19.51%.
Embodiment 3：Zymologic property
1. optimal pH
Different pH buffer solution is prepared respectively, under the conditions of 25 DEG C, the enzyme activity of thick zyme extract is determined respectively, it is determined that most suitable
Enzyme reaction pH.
The pH of the enzyme optimal reaction is 4.In pH ＜ 4, enzyme activity is very low, and in the range of 4.5 ~ 6.5, enzyme activity reduces pH, but becomes
Change amplitude is smaller.The suitable pH scopes of the enzyme are 4.5-6.5, and experiment proves that the enzyme suitably reacts in the environment of weak acid.
2. pH stability
Enzyme is respectively placed in different pH buffer solution, 37 DEG C are incubated 1 hour, and measure enzyme activity loses situation, with most stable place
Enzyme activity is 100%, and the enzyme activity under other pH is represented with remaining enzyme activity.
Enzyme is most stable when pH is 4.5.Remaining enzyme activity is more than 90% in the range of pH4.5-6.0, pH stability
Preferably, it is larger in the loss of the enzyme activities of pH ﹤ 4.5, when illustrating to work as pH ﹤ 4.5, less stable.
3. optimum temperature
Different temperatures is taken, determines the enzyme activity of thick zyme extract respectively, it is determined that most suitable enzyme reaction temperature.
E3 optimum temperature is 50 DEG C, and enzyme activity is higher in the range of 40-55 DEG C of temperature, and change it is smaller, and in temperature ﹤
Enzyme activity declines larger at 35 DEG C or ﹥ 55 DEG C, therefore the suitable temperature range of the enzyme is 45-55 DEG C.
4. temperature stability
By enzyme respectively in certain temperature for a period of time（0 ~ 120 minute）, using uninsulated enzyme activity as 100%, other enzyme activity
Calculated by residual enzyme work
30 minutes are incubated at 40 DEG C, remaining enzyme activity decline is smaller, is 97.21%, with the extension of soaking time, under remaining enzyme activity
Drop is rapid, is only 60.83% at 60 minutes.It is incubated at a temperature of 50 and 60 DEG C, in 30 minutes, remaining enzyme activity declines just non-
It is often rapid, respectively 66.12% and 55.10%.30 minutes are incubated at 50 DEG C of optimum temperature, remaining enzyme activity all declines rapidly,
Therefore the new enzyme of in good time addition is wanted in industrialized production, to ensure the speed of enzyme reaction.
5. the influence of metal ion
Different metal salt is received into buffer solution with citric acid-citric acid that pH is 4.8, being configured to amount of substance concentration is
The buffer solution of 2mmol/L metalline, determine the vigor of thick zyme extract respectively at 25 DEG C.
Cu2+、Fe2+、Zn2+There are activation, wherein Cu to pectase enzyme activity2+Effect is obvious；Ca2+Have substantially to the enzyme
Inhibitory action, Ba2+ 、Mg2+To the enzyme without obvious effect.
Conclusion：The optimal pH of the thick enzyme of pectin is 4.5, and suitable pH scopes are 4.5-6.5.Optimum temperature is 50 DEG C, and it is suitable
Suitable temperature range is narrower；Half an hour is incubated under optimum temperature, enzyme activity decline it is rapid, therefore should be noted that in reaction it is in good time
Enzyme liquid is added, to ensure the speed of reaction；Cu2+、Fe2+、Zn2+There are activation, wherein Cu to pectase enzyme activity2+Effect is obvious；
Ca2+ There are obvious inhibitory action, Ba to the enzyme2+ 、Mg2+To the enzyme without obvious effect；Km is 0.27%.
Embodiment 4：Pectase specific activity of enzyme compares
Banana skin slag dry powder is with wheat bran with mass ratio 3:7 mixing, add the deionized water of 1.5 times of quality（In terms of butt quality）,
It is sufficiently mixed uniformly, adds 0.18% ammonium sulfate, stir, is fed by 4% charge, 121 DEG C of moist heat sterilizations 30 minutes, from
Under right pH, inoculum density is 5 × 105 Individual/mL spore suspension bacterium solution 1mL, incubation time 72 hours, the pectase enzyme ratio of acquisition
Vigor 145.46U/mg albumen.
Banana skin slag dry powder is with wheat bran with mass ratio 3:7 mixing, add the deionized water of 1.5 times of quality（With butt quality
Meter）, it is sufficiently mixed uniformly, adds 0.18% ammonium sulfate, 0.01% dipotassium hydrogen phosphate, 0.1% calcium chloride, stir, by 4% dress
Doses feeds, 121 DEG C of moist heat sterilizations 30 minutes, natural pH, and inoculum density is 5 × 105 Individual/mL spore suspension bacterium solution 1mL, training
To support 72 hours time, the thick enzyme of pectase of acquisition is 192.14U/mg albumen than work,
As a result show, the specific activity of enzyme that with the addition of the pectase crude extract of ammonium sulfate, hydrogen sulfate dipotassium and calcium chloride improves
32.09%。
Embodiment 5：Application of the pectase in fruit juice is prepared
Banana, apple, dragon fruit are removed the peel respectively after shredding by 2:After the ratio of 1 solid-to-liquid ratio is beaten, Juice pH is adjusted
Scope adds the thick zyme extract of pectin prepared by 1% ~ 2.5% embodiment 1,90 points is digested at 50 DEG C respectively between 4.5 ~ 6.5
Clock, added the thick zyme extract of pectin prepared by 0.5,1.0 and 1.25% embodiment 1 again respectively when 45 minutes, while with commercially available
Dong Henghua roads pectase be contrast experiment under similarity condition.Banana, apple, dragon fruit add the juice of pectase of the present invention
Rate and light transmittance are shown in Table 3；Banana, apple, the crushing juice rate of dragon fruit addition commercially available Dong Henghua roads pectase and light transmittance are shown in Table 4.
Crushing juice rate and light transmittance after 3 each raw material of table addition pectase of embodiment 1
4 each raw material of table adds the crushing juice rate and light transmittance after commercially available pectase
As a result show：The crushing juice rate that with the addition of the banana of pectase of the present invention reaches 50 ~ 65%, and does not add the contrast of pectase, goes out
Juice rate improves 1.5 ~ 2 times, and 8% ~ 11% is improved than commercially available pectase crushing juice rate；It with the addition of the saturating of pectase bananas juice of the present invention
Light rate reaches 55 ~ 75%, and than being not added with improving 3.5 ~ 5 times, 7% ~ 12% is improved than commercially available pectase light transmittance.It with the addition of this
The crushing juice rate of the cider of invention pectase reaches 52 ~ 73%, and does not add the contrast of pectase, and crushing juice rate improves 1 ~ 1.5 times,
8% ~ 10% is improved than commercially available pectase crushing juice rate；The light transmittance that with the addition of the cider of pectase of the present invention reaches 55 ~ 85%,
Than being not added with improving 2 ~ 4 times, 7% ~ 9% is improved than commercially available pectase light transmittance.It with the addition of the dragon fruit of pectase of the present invention
The crushing juice rate of juice reaches 55 ~ 72%, and does not add the contrast of pectase, and crushing juice rate improves 1.1 ~ 1.8 times, gone out than commercially available pectase
Juice rate improves 7% ~ 12%, and the dragon fruit light transmittance that with the addition of pectase of the present invention reaches 53 ~ 80%, than be not added with improving 3.5 ~
4.5 times, 9% ~ 10% is improved than commercially available pectase light transmittance.
Embodiment 6 determines the stability of pectase
By step in embodiment 2 ~ pectase and commercially available Dong Henghua roads pectase be placed in 50 DEG C, under conditions of pH4.5, insulation
After 1 hour, remaining enzyme activity is determined, as a result as shown in table 5.
The remaining enzyme activity of the pectase of table 5
| Pectase | Embodiment 2 | Embodiment 2 is 2. | Embodiment 2 is 3. | Dong Henghua roads pectase |
| Remaining enzyme activity | 58% | 60% | 62% | 53% |
As a result show：The enzyme liquid of the present invention is higher than the remaining enzyme activity of commercially available pectase, and pectin enzyme stability of the invention is notable
Improve.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.That is made within the spirit and principles of the invention any repaiies
Change, equivalent substitution, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of pectase, including prepare banana skin slag dry powder, actication of culture, prepare seed liquor, solid fermentation
Cultivate and prepare thick zyme extract, it is characterised in that described thalline activation is that aspergillus niger is put into 20 ~ 30 DEG C of Czapek's medium
Culture 100 ~ 120 hours；Described solid fermentation culture is after mixing banana peel dry powder and wheat bran in proportion, adds deionization
Water is well mixed, and adds ammonium sulfate, dipotassium hydrogen phosphate, calcium chloride stirring, charging, in 110 ~ 125 DEG C of 20 ~ 40 points of moist heat sterilizations
Zhong Hou, inoculating spores suspension culture 60 ~ 72 hours.
2. the preparation method of pectase according to claim 1, it is characterised in that specific preparation process is as follows：
（1）Prepare banana skin slag dry powder：Banana skin is sorted, smashed after being dried 10 ~ 12 hours in 80 ~ 90 DEG C, excessively 200 ~
300 mesh sieves, banana skin slag dry powder is obtained, it is standby；
（2）Actication of culture：By aspergillus niger be put into Czapek's medium in 28 DEG C incubated 100 ~ 120 hours, slant strains and three
After angle bottle expands culture strain, it is placed in 0 ~ 5 DEG C of refrigerator and preserves；
（3）Prepare seed liquor：One bottle of cultured strain is taken, adds the sterile saline that mass fraction is 0.85%, gently will
The spore of media surface is scraped, and spore suspension is placed in sterilizing triangular flask, sterile glass ball is placed in advance in bottle, fully
Spore suspension is prepared into the filtering of sterile absorbent cotton after shaking, it is 5 × 10 to control suspension spore count5~6×105 Individual/mL bacterium
Liquid, as seed liquor；
（4）Solid fermentation culture：By banana skin slag dry powder and wheat bran with mass ratio 1 ~ 3:8 ~ 10 mixing, add 1.5 ~ 2 times go from
Sub- water, it is sufficiently mixed uniformly, adds 0.15 ~ 0.25% ammonium sulfate, 0.01 ~ 0.03% dipotassium hydrogen phosphate, 0.1 ~ 0.2% calcium chloride
Stirring, then fed by 5 ~ 10% charges, 110 ~ 125 DEG C of moist heat sterilizations are after 20 ~ 40 minutes, inoculation seed liquor, inoculum concentration 1 ~
2mL, incubation time 60 ~ 72 hours, Qu Yici was respectively turned in 12,24,36 hours in incubation；
（5）Prepare thick zyme extract：After fermentation, the pure water of 6 ~ 8 times of culture medium quality is added, opens ultrasonic extractor progress
Extraction, 32 DEG C, ultrasonic power 20KHZ of Extracting temperature, extract 1 hour, after extraction, 4000r/ minutes refrigerated centrifuge 10 divides
Clock, it is the thick zyme extract of pectin to take supernatant.
3. the preparation method of pectase according to claim 2, it is characterised in that preparation method is also pure including separating step by step
Change, isolated and purified using ultrafiltration concentration, ammonium sulfate precipitation, dialysis and DEAE-52 ion-exchange chromatographies.
4. the preparation method of pectase according to claim 2, it is characterised in that the physical and chemical index of the banana skin slag dry powder
Control and be：Total reducing sugar 40 ~ 45%, pectin 14 ~ 16%, protein 2 ~ 3%, total acid 2 ~ 3%, moisture 4 ~ 4.5%, crude fat 3.8 ~ 4.5%, ash
Divide 7 ~ 9% and other compositions 15 ~ 27.2%.
5. the preparation method of pectase according to claim 2, it is characterised in that described step（4）Add deionized water
The amount for being is calculated with the quality of banana skin slag dry powder and wheat bran, and ammonium sulfate, dipotassium hydrogen phosphate and the calcium chloride of addition are
Calculated with the total amount after banana skin slag dry powder, wheat bran and deionized water.
6. the preparation method of pectase according to claim 2, it is characterised in that the thick zyme extract enzyme of described pectin is than living
Control as 190 ~ 195U/mg albumen.
7. the preparation method of pectase according to claim 3, it is characterised in that described ammonium sulfate precipitation it is first dense
Spend for 30%, final concentration of 80%.
8. the preparation method of pectase according to claim 3, it is characterised in that described ultrafiltration concentration is by the thick enzyme of pectin
Extract solution is poured into ultrafiltration cup and is concentrated by ultrafiltration, and the volume of concentrate is stoste 1/10th, and ultrafiltration retaining molecular weight is one
Ten thousand to one ten thousand two；
Described ammonium sulfate precipitation is that the thick zyme extract of pectin after concentration is put into frozen water to mix in bath, into enzyme liquid slowly
Ammonium sulfate powder is added to saturation；Standing 12-14 hours in refrigerator are put into, 4 DEG C of 4000r refrigerated centrifuges obtain thick enzyme for 10 minutes and sunk
Form sediment；
Described dialysis is that thick enzyme precipitation is dissolved in the HAc-NaAc buffer solutions of 0.02M pH=4.8, loads molecular cut off
In 14000 bag filter, to be placed in 5 DEG C of refrigerators, dialysed 24 hours in pure water, after thick zyme extract be freeze-dried
Thick enzyme powder；
Described DEAE-52 ion-exchange chromatographies are by the thick enzyme powder after freeze-drying, are dissolved in 0.02M pH=4.2
In HAc-NaAc buffer solutions, it is slowly added into dropper on the DEAE-52 chromatographic columns balanced, first elutes 4 cylinders with buffer solution
Product, then linear elution is carried out with the identical buffer solution of the NaCl gradients Han 0 ~ 1.0M, flow velocity is 1mL/ minutes, and often pipe 4mL, is adopted
Collected with part automatic collector, detect protein content and pectase enzyme activity by pipe, collect enzyme activity composition.
9. the preparation method of pectase according to claim 8, it is characterised in that described freeze-drying is to extract thick enzyme
Liquid is freeze-dried 24 hours at -40 DEG C, is obtained and is freeze-dried thick enzyme powder.
A kind of 10. pectase prepared such as claim 1 ~ 9 any one preparation method.
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| CN111500656A (en) * | 2020-04-14 | 2020-08-07 | 广西农业职业技术学院 | Method for extracting low-molecular-weight pectin from banana peel |
| CN112831086A (en) * | 2021-01-18 | 2021-05-25 | 刘永昶 | Preparation method and application of special solid strain for liquid fermentation culture |
| CN113249359A (en) * | 2021-05-24 | 2021-08-13 | 山西农业大学 | Method for extracting mulberry leaf functional components by using enzyme |
| CN113897343A (en) * | 2021-09-10 | 2022-01-07 | 常州大学 | Method for producing pectinase by aspergillus niger solid-state fermentation |
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| CN112831086A (en) * | 2021-01-18 | 2021-05-25 | 刘永昶 | Preparation method and application of special solid strain for liquid fermentation culture |
| CN113249359A (en) * | 2021-05-24 | 2021-08-13 | 山西农业大学 | Method for extracting mulberry leaf functional components by using enzyme |
| CN113249359B (en) * | 2021-05-24 | 2023-05-30 | 山西农业大学 | Method for extracting mulberry leaf functional ingredient by utilizing enzyme |
| CN113897343A (en) * | 2021-09-10 | 2022-01-07 | 常州大学 | Method for producing pectinase by aspergillus niger solid-state fermentation |
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