CN106434622A - Preparation method of co-immobilized enzyme - Google Patents
Preparation method of co-immobilized enzyme Download PDFInfo
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- CN106434622A CN106434622A CN201610736261.9A CN201610736261A CN106434622A CN 106434622 A CN106434622 A CN 106434622A CN 201610736261 A CN201610736261 A CN 201610736261A CN 106434622 A CN106434622 A CN 106434622A
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Abstract
The invention provides a preparation method of a co-immobilized enzyme. The preparation method comprises the following steps: (1) adding lipase A6, lipase MER and proteinase MSD into a sodium alginate solution; uniformly stirring to obtain a commixed solution; (2) dripping the commixed solution obtained by the step (1) into a CaCl2 water solution; standing and curing to obtain gel particles, wherein the prepared gel particles are of the co-immobilized enzyme. The co-immobilized enzyme prepared by the method provided by the invention has a relatively high immobilization rate, and the enzyme activity operation stability and the storage stability of the co-immobilized enzyme are relatively good.
Description
Technical field
The present invention relates to a kind of preparation method of enzyme, and in particular to a kind of preparation method of co-immobilization enzyme.
Background technology
German physiologist William Kühne proposed this concept of enzyme first in 1878.Enzyme, is commonly called as ferment, is to plant
The synthetically produced class specific proteins of thing, animal, microorganism and human body cell.Biochemical reaction speed is improved as enzyme has
Degree and the catalytic capability of reaction mass, commonly known as biocatalyzer.Compare with traditional catalyst, enzyme has high efficiency, specially
The advantages of one property, multiformity, reaction condition are gentle, is widely used in every field.But which is in acid, alkali, heat and organic solution
Deng under the conditions of easily there is the degeneration of pheron, so that enzyme activity is reduced or lose, and carried out more than enzyme reaction in the solution, be difficult after reaction
Reclaim, product separating-purifying difficulty, it is difficult to realize industrial serialization, automated production, therefore the application of enzyme engineering
Development is limited by very large.
Immobilized enzyme is with chemically or physically method, carries out respective handling to enzyme so that the stronger enzyme of script water solublity
It is combined with each other with the water-fast carrier of solid-state, or loaded body embedding, make both form a unified entirety.
After immobilization, immobilized enzyme has the more advantages of specific ionization enzyme:
(1) immobilization enzyme-to-substrate, product are more easily separated, and reaction can through the shirtsleeve operation such as centrifugation or filtration after terminating
Immobilized enzyme is reclaimed, and enzyme activity reduces less, repeatable multiple batches of use, reduces production cost；
(2) after immobilization process, general stability can improve a lot, and pH stability, heat stability etc. are improved, right
The sensitivity of inhibitor declines.
(3) immobilized enzyme is applied to automatization, continuous prodution, catalytic process easy to control, and will not be by pheron band
Enter to cause the residual of enzyme in product, simplify later purification technique, the utilization ratio of enzyme is improve, reduces production cost.
Certainly, there is shortcoming in immobilized enzyme:
(1) part enzyme activity can be lost during immobilization；
(2) enzyme immobilizatio needs equipment and carrier, increased production cost；
(3) when just thinking that substrate is solvable, immobilized enzyme is just suitable for, and is poorly suitable for macromolecule substrate；
(4) endocellular enzyme of microorganism secretion after isolation could immobilization.
With deepening continuously for immobilized enzyme research, process for fixation has slowly been transitioned into the combination of multiple methods.But
It is developed so far, does not almost have a kind of immobilization technology to be applied to any enzyme, therefore will be according to concrete enzyme viability and application
Purpose, selects suitable process for fixation.
Enzyme immobilizatio method can be divided into 4 classes:Absorption method, covalent coupling method, cross-linking method and investment.
Absorption method is one of enzyme immobilizatio method for using earliest, absorption method be using Van der Waals force, ionic bond, hydrogen
The active forces such as key, physical absorption, fixed form enzyme being fixed on carrier.Generally can be divided into physisorphtion and ionic adsorption
Method.
Lu Yuxia is fixed on lipase on large pore anion resin D201 using absorption method, and the immobilized enzyme for obtaining has relatively
Good operational stability.Be hydrolyzed reaction with the immobilized enzyme catalysis real butter for preparing, hydrolyzate soft aroma with, pure
Thick.
Covalent coupling method refers to the work(by the method for Covalent bonding together the nonessential function base on enzyme and carrier surface
The process for fixation that energy base junction is closed.
Chemical crosslink technique is by difunctional or poly functional reagent, pheron molecule to be crosslinked each other, make enzyme molecule and
Covalent bond is formed between double-functional group reagent or function groups reagent, obtains the cross-linked structure of three-dimensional.In the process, enzyme
While crosslinking between molecule, intramolecule there is also a certain degree of crosslinking.Chemical crosslink technique can be divided into cross-linking enzyme crystalline substance
Body and cross-linked enzyme aggregate.
After investment refers to that enzyme is mixed with carrier solution, there is polyreaction by initiator, by physical action enzyme
It is limited in the network structure of carrier, the method so as to realize the fixation of enzyme.Its advantage is immobilization low cost, immobilization enzyme amount
Greatly, operation is simple, fixing condition is gentle, the stability height of enzyme molecule etc., but as enzyme is fixed on the inside of carrier
In space, reaction substrate is because there is catalytic reaction in the active center that the impact of resistance to mass tranfer need to get to enzyme through diffusion.With
When, violent enzyme digestion reaction is possible to the microcapsule membrane of polymer or network structure be caused to damage, and causes enzyme to leak.In carrier
In the selection of material, polypropylene phthalein amine, gelatin, carrageenan, shitosan and sodium alginate etc. are common selections.
Noble glad grade is studied with sodium alginate as carrier, with CaCl2- chitosan solution is coagulator, using the side of embedding
Method immobilization pineapple stem protease.Test shows, the heat stability of immobilized enzyme prepared by the method and optimum temperature are improved, instead
Answer pH to alkalescence skew, zymologic property is improved.Meanwhile, the enzyme after immobilization also has good operability, repeats to make
After 4 times, enzyme activity still retains more than 80%, and immobilization effect is preferable.
The sodium alginates such as Lu Yuxia are carrier, CaCl2Solution is coagulator, using investment fixed fat enzyme.Test table
Bright, the better heat stability of immobilized enzyme prepared by the method, at 60 DEG C after heating 1.5h, the enzyme activity of immobilized enzyme only declines
30%.And, which is also with good operational stability, continuous Reusability 10 times, and the enzyme activity of immobilized enzyme still retains
More than 95%.
Co-immobilization enzyme starts from the eighties in 20th century, is two or more enzyme to be fixed in identical carrier formed
A kind of technology of co-immobilization system.Co-immobilization enzyme is based on the fixation of single enzyme, considers co-immobilization
The optimal process for fixation of enzyme, the often crosslinking method of the studied common immobilization method for using and investment.Co-immobilization enzyme with
Common immobilized enzyme is compared, and co-immobilization enzyme can give full play to the feature of different enzymes, while its catalysis characteristics is combined,
Synergism is fully demonstrated, improves catalytic efficiency.While shortening the response time and reducing reactions steps, continuous prodution is realized,
Product quality can preferably be controlled.
But the correlational study report at present, being at home and abroad related to common immobilized enzyme is simultaneously few, and focuses primarily upon saccharifying
In terms of enzyme, the relevant research with regard to co-immobilization protease and lipase is less.Lipase and protease are that industrialized production should
With in widest two kinds of enzymes, with vast potential for future development.If lipase and protease are fixed on the same vector altogether,
As enzyme reactor, it is possible to achieve industrialized production, Simplified flowsheet, reduces cost.
Easy grade is in using chitosan as carrier, using glutaraldehyde as cross-linking agent, by papain and pancreas egg
White enzyme is fixed on carrier altogether, and its activity improves 25% than single enzyme immobilization, and the method is widely used in the anti-of drinks
In the productions such as muddy, clarification.
The research and inquirement of Liu Ziqin impacts of the different protease to lipase activity, as a result find trypsin to fat
The vigor of enzyme Palatase 20000L is improved effect.While the method by studying co-immobilization, successfully by protease and fat
Fat enzyme co-immobilization can be effectively improved the vigor of protease and the vigor loss to lipase is less on macroporous resin.
Content of the invention
It is an object of the invention to overcoming the weak point of prior art presence and providing a kind of system of co-immobilization enzyme
Preparation Method, present invention also offers the co-immobilization enzyme for being prepared using the method.
For achieving the above object, the technical scheme that is taken：A kind of preparation method of co-immobilization enzyme, the preparation method
Comprise the following steps：
(1) lipase A 6, lipase MER and protease MSD are added in sodium alginate soln, is total to after stirring
Mixed liquid；
(2) blended liquid that step (1) is obtained is instilled CaCl2In aqueous solution, gel particle is formed after standing solidification, be obtained
Gel particle be the co-immobilization enzyme.
Preferably, in described step (1) gross weight of lipase A 6, lipase MER and protease MSD be
The 2.2%-3.52% of sodium weight, the weight ratio of the lipase A 6, lipase MER and protease MSD is lipase A 6：Fat
Enzyme MER：Protease MSD=2:1：0.3.
Preferably, in described step (1) gross weight of lipase A 6, lipase MER and protease MSD be
The 3.0% of sodium weight, the weight ratio of the lipase A 6, lipase MER and protease MSD is lipase A 6：Lipase MER：
Protease MSD=2:1：0.3.
Preferably, in described step (1) sodium alginate soln be pH be 6.0～8.0, mass fraction be
Sodium alginate soln, CaCl in step (2)2Aqueous solution is the CaCl that mass fraction is 3.0%～6.0%2Aqueous solution.
Preferably, in described step (1) sodium alginate soln be pH be 6.5, mass fraction be 1.5% sodium alginate molten
Liquid；CaCl in step (2)2Aqueous solution is the CaCl that mass fraction is 4.0%2Aqueous solution.The sodium alginate soln by with
Lower section method is prepared from：Sodium alginate solid is added to the phosphate buffer solution of pH 6.5, heating for dissolving, it is made into mass fraction
For 1.5% sodium alginate soln.
Preferably, it is 20～40min 20～35 DEG C, hardening time that in described step (2), solidification temperature is.
Preferably, it is 30min 30 DEG C, hardening time that in described step (2), solidification temperature is.
The invention provides the co-immobilization enzyme being prepared from using method described above.
The invention provides the purposes of co-immobilization enzyme described above in milk base material is prepared.
The beneficial effects of the present invention is：The invention provides a kind of preparation method of co-immobilization enzyme, using the method
The co-immobilization enzyme immobilizatio rate of preparation is higher, and the enzyme activity operational stability of co-immobilization enzyme and storage-stable are all relatively
Good.
Description of the drawings
Fig. 1 is bovine serum albumin canonical plotting in the embodiment of the present invention 1；
Fig. 2 is the broken line graph of impact of the pH value to co-immobilization enzyme activity and fixing rate in the embodiment of the present invention 1；
Fig. 3 is the folding of impact of the sodium alginate concentration to co-immobilization enzyme activity and fixing rate in the embodiment of the present invention 1
Line chart；
Fig. 4 is the broken line graph of impact of the enzyme amount to co-immobilization enzyme activity and fixing rate in the embodiment of the present invention 1；
Fig. 5 is the broken line of impact of the CaCl2 concentration to co-immobilization enzyme activity and fixing rate in the embodiment of the present invention 1
Figure；
Fig. 6 is the folding of impact of the co-immobilization temperature to co-immobilization enzyme activity and fixing rate in the embodiment of the present invention 1
Line chart；
Fig. 7 is the folding of impact of the co-immobilization time to co-immobilization enzyme activity and fixing rate in the embodiment of the present invention 1
Line chart；
Fig. 8 is the broken line graph of heat stability of the co-immobilization enzyme at 20 DEG C in the embodiment of the present invention 1；
Fig. 9 is the broken line graph of heat stability of the co-immobilization enzyme at 60 DEG C in the embodiment of the present invention 1；
Figure 10 is the broken line graph of the operational stability of co-immobilization enzyme in the embodiment of the present invention 1；
Figure 11 is the broken line graph of the storage-stable of co-immobilization enzyme in the embodiment of the present invention 1；
Figure 12 is the broken line graph of impact of the co-immobilization enzyme addition to acid value and sensory evaluation scores in the embodiment of the present invention 1；
Figure 13 is the broken line of impact of the enzymolysis time of co-immobilization enzyme in the embodiment of the present invention 1 to acid value and sensory evaluation scores
Figure；
Figure 14 is the broken line of impact of the hydrolysis temperature of co-immobilization enzyme in the embodiment of the present invention 1 to acid value and sensory evaluation scores
Figure.
Specific embodiment
For better illustrating the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
The present invention is entered to protease MSD, lipase A 6 and lipase MER using sodium alginate as carrier using investment
Row co-immobilization, with fix rate and co-immobilization enzyme enzyme activity as leading indicator, co-immobilization condition is optimized,
By the co-immobilization enzyme for obtaining after optimization for preparing milk base material, with acid value and sensory evaluation as leading indicator, to milk base
The preparation technology of material is optimized.
1st, materials and methods
1.1 test material
(1) butter is commercially available many U.S. fresh salt-free butter.
(2) whey powder (Lactose 60%, protein 9%, fat 15%) is purchased from Guangzhou market day cutting edge of a knife or a sword herding company limited.
(3) lipase A " day open country " 6 (Lipase A " Amano " 6), write a Chinese character in simplified form into lipase A 6, by A Manuo amano enzyme preparation
Commerce and trade (Shanghai) Co., Ltd. gives.
(4) lipase MER " day open country " (Lipase MER " Amano "), writes a Chinese character in simplified form lipase MER, by A Manuo amano enzyme system
Agent commerce and trade (Shanghai) Co., Ltd. gives.
(5) protease M " day open country " SD (Protease M " Amano " SD), writes a Chinese character in simplified form into protease MSD, wild by A Manuo days
Enzyme preparation commerce and trade (Shanghai) Co., Ltd. gives.
1.2 test reagent
It is pure that ether, dehydrated alcohol, KOH, Potassium Hydrogen Phthalate, phenolphthalein indicator etc. are commercially available analysis.
1.3 test capital equipments
(1) main experimental equipment
The instrument that table 1 is mainly used in testing
(2) Other Instruments equipment
Alkaline burette, conical flask, graduated cylinder, beaker, iron stand, medicine spoon, Glass rod, pipet, pan paper etc..
1.4 test method
1.4.1 the selection of fixation support and process for fixation
1.4.1.1 macroporous resin loading chitosan film cross-linking method
Weigh 0.1g shitosan to be dissolved in the acetic acid that 100mL mass fraction is 1%, be made into the shitosan-acetic acid of 10mg/mL
Solution.Shitosan-the acetum for weighing 10g macroporous resin and 50mL is sufficiently mixed, 50 DEG C of water-bath rotations under 1300Pa state
Turn evacuation 10min, be washed with deionized to neutrality.Add 0.125% glutaraldehyde solution of 30mL, 20 DEG C of standing crosslinkings
2h.The glutaraldehyde for removing residual is washed with deionized, till the absorption value until cleaning mixture at 280nm is less than 0.01.
The pretreated macroporous resin of 1g is accurately weighed in conical flask, 10mL deionized water wetting resin is added, accurately
Weigh 10mg resolvase to be sufficiently mixed with resin, after putting 35 DEG C of constant-temperature table 150r/min vibration absorption 1h, filter and retain filter
Liquid.Resin deionized water is repeatedly washed, and is preserved after sucking filtration at 4 DEG C.
1.4.1.2 macroreticular resin absorbing method
Weighing 10g resin to be placed in conical flask, 95% ethanol is added toward in conical flask, is soaked sucking filtration after 24h, and spend
Ionized water is rinsed.Resin sucking filtration after 5%HC1 solution soaking 4h of 25mL, is washed with deionized to neutrality.Then 25mL is used
5%NaOH solution soaking 4h after sucking filtration, be washed with deionized to neutrality.After sucking filtration, room temperature is saved backup.
The pretreated macroporous resin of 1g is accurately weighed in conical flask, 10mL deionized water wetting resin is added, accurately
Weigh 10mg resolvase to be sufficiently mixed with resin, after putting 35 DEG C of constant-temperature table 150r/min vibration absorption 1h, filter and retain filter
Liquid.Resin deionized water is repeatedly washed, and is preserved after sucking filtration at 4 DEG C.
1.4.1.3 sodium alginate investment
Accurately weigh 1g sodium alginate solid, add deionized water, heating for dissolving, it is made into 1% sodium alginate soln.Treat
After being cooled to 45 DEG C, 10mg resolvase is added, after stirring, 100mL mass fraction being added dropwise to No. 5 syringes is
3% CaCl2In solution, gel particle is formed, standing solidification 1h at 35 DEG C.Leach gel particle and retain filtrate, gel
Grain is washed with deionized, and is preserved after sucking filtration at 4 DEG C.
1.4.1.4 chitosan imbedded method
Accurately weigh 1g chitosan solid, add 1% acetum, stirring and dissolving, be made into 1% shitosan acetic acid molten
Liquid.To be cooled to after 45 DEG C, add 10mg resolvase, after stirring, be added dropwise to 100mL mass with No. 5 syringes and divide
Number is for, in 1% NaOH solution, forming gel particle, and at 35 DEG C, standing solidifies 1h.Leach gel particle and retain filtrate, gel
Granule is washed with deionized, and is preserved after sucking filtration at 4 DEG C.
1.4.2 impact of the co-immobilization condition optimizing to enzyme activity
1.4.2.1 impact of the pH value to co-immobilization enzyme activity
Weigh 0.50g sodium alginate solid respectively to add in 7 conical flasks, to add pH be 5.0 respectively, 5.5,6.0,6.5,
7.0th, 7.5,8.0 phosphate buffer solution, heating for dissolving, are made into 1.0% sodium alginate soln.After cooling, each addition
0.0080g lipase A 6,0.0040g lipase MER and 0.0012g protease MSD, after stirring, with No. 5 syringes
It is added dropwise in the CaCl2 solution that 50mL mass fraction is 3.0%, gel particle is formed, standing solidification 20min at 35 DEG C.Leach
Gel particle simultaneously retains filtrate, and gel particle is washed with deionized, standby after sucking filtration.
Weigh 10g immobilized enzyme respectively to be put in 7 conical flasks, 6g deionized water and 10g butter is then respectively adding, is put
(35 DEG C, 150r/min) reaction 1h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, takes upper strata oil
Phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
1.4.2.2 impact of the sodium alginate concentration to co-immobilization enzyme activity
Weigh sodium alginate solid 0.25g, 0.50g, 0.75g, 1.0g, 1.25g respectively, add the phosphoric acid buffer of pH 6.5
Solution, heating for dissolving, be made into mass fraction be 0.5%, 1.0%, 1.5%, 2.0%, 2.5% sodium alginate soln.Treat cold
But, after, respectively 0.0080g lipase A 6,0.0040g lipase MER and 0.0012g protease MSD are added, after stirring,
It is added dropwise to, with No. 5 syringes, the CaCl that 50mL mass fraction is 3.0%2In solution, gel particle is formed, standing solidification at 35 DEG C
20min.Leach gel particle and retain filtrate, gel particle is washed with deionized, standby after sucking filtration.
Weigh 10g immobilized enzyme respectively to be put in 5 conical flasks, 6g deionized water and 10g butter is then respectively adding, is put
(35 DEG C, 150r/min) reaction 1h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, takes upper strata oil
Phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
1.4.2.3 impact of the enzyme amount to co-immobilization enzyme activity
Weigh 0.75g sodium alginate solid in 6 conical flasks respectively, add the phosphate buffer solution of pH 6.5, heating
Dissolving, is made into the sodium alginate soln that mass fraction is 1.5%.After cooling, lipase A 6 is separately added into, and enzyme concentration is butter
The 0.080% of quality, 0.100%, 0.120%, 0.140%, 0.160%, 0.180%；Lipase MER is separately added into, enzyme-added
Measure as 0.040%, 0.050%, 0.060%, 0.070%, 0.080%, 0.090%；It is separately added into protease MSD, enzyme concentration
For 0.012%, 0.015%, 0.018%, 0.021%, 0.027%, 0.03%.After stirring, dripped with No. 5 syringes
Enter to the CaCl that 50mL mass fraction is 3.0%2In solution, gel particle is formed, standing solidification 20min at 35 DEG C.Leach solidifying
Glue granule simultaneously retains filtrate, and gel particle is washed with deionized, standby after sucking filtration.
Weigh 10g immobilized enzyme respectively to be put in 6 conical flasks, 6g deionized water and 10g butter is then respectively adding, is put
(35 DEG C, 150r/min) reaction 1h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, takes upper strata oil
Phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
1.4.2.4 CaCl2Impact of the concentration to co-immobilization enzyme activity
Weigh 0.75g sodium alginate solid in 6 conical flasks respectively, add the phosphate buffer solution of pH 6.5, heating
Dissolving, is made into the sodium alginate soln that mass fraction is 1.5%.After cooling, be separately added into 0.0140g lipase A 6,
0.0070g lipase MER and 0.0021g protease MSD, after stirring, is added dropwise to 50mL mass with No. 5 syringes and divides
Number is respectively 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0% CaCl2In solution, gel particle is formed, at 35 DEG C
Standing solidification 20min.Leach gel particle and retain filtrate, gel particle is washed with deionized, standby after sucking filtration.
Weigh 10g immobilized enzyme respectively to be put in 6 conical flasks, 6g deionized water and 10g butter is then respectively adding, is put
(35 DEG C, 150r/min) reaction 1h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, takes upper strata oil
Phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
1.4.2.5 impact of the co-immobilization temperature to co-immobilization enzyme activity
Weigh 0.75g sodium alginate solid in 6 conical flasks respectively, add the phosphate buffer solution of pH 6.5, heating
Dissolving, is made into the sodium alginate soln that mass fraction is 1.5%.After cooling, be separately added into 0.0140g lipase A 6,
0.0070g lipase MER and 0.0021g protease MSD, after stirring, respectively 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C,
40 DEG C, under conditions of 45 DEG C of water-baths, be added dropwise to, with No. 5 syringes, the CaCl that 50mL mass fraction is 4.0%2In solution, formed
Gel particle, standing solidification 20min.Leach gel particle and retain filtrate, gel particle is washed with deionized, sucking filtration standby
With.
Weigh 10g immobilized enzyme respectively to be put in 6 conical flasks, 6g deionized water and 10g butter is then respectively adding, is put
(35 DEG C, 150r/min) reaction 1h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, takes upper strata oil
Phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
1.4.2.6 impact of the co-immobilization time to co-immobilization enzyme activity
Weigh 0.75g sodium alginate solid in 5 conical flasks respectively, add the phosphate buffer solution of pH 6.5, heating
Dissolving, is made into the sodium alginate soln that mass fraction is 1.5%.After cooling, be separately added into 0.0140g lipase A 6,
0.0070g lipase MER and 0.0021g protease MSD, after stirring, is added dropwise to 50mL mass with No. 5 syringes and divides
Number for 4.0% CaCl2 solution in, formed gel particle, at 30 DEG C respectively standing solidification 10min, 20min, 30min,
40min、50min.Leach gel particle and retain filtrate, gel particle is washed with deionized, standby after sucking filtration.
Weigh 10g immobilized enzyme respectively to be put in 5 conical flasks, 6g deionized water and 10g butter is then respectively adding, is put
(35 DEG C, 150r/min) reaction 1h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, takes upper strata oil
Phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
1.4.3 the stability of co-immobilization enzyme
1.4.3.1 the heat stability of co-immobilization enzyme
Co-immobilization enzyme is placed in 20 DEG C of constant water bath box distinguish water-bath 0min, 30min, 60min, 90min,
120min、150min.10g immobilized enzyme being weighed after water-bath be put in conical flask, is then respectively adding 6g deionized water and 10g Huang
Oil, is placed in constant-temperature table (35 DEG C, 150r/min) reaction 2h.3000r/min centrifugation 15min after immobilized enzyme is leached, is taken
Layer oil phase, determines the acid value of butter after enzymolysis, judges the enzyme activity of immobilized enzyme.
Co-immobilization enzyme is placed in 60 DEG C of constant water bath box distinguish water-bath 0min, 10min, 20min, 30min,
40min、50min、60min.Weigh 10g immobilized enzyme after water-bath to be put in conical flask, be then respectively adding 6g deionized water and
10g butter, is placed in constant-temperature table (35 DEG C, 150r/min) reaction 2h.3000r/min centrifugation 15min after immobilized enzyme is leached,
Upper oil phase is taken, the acid value of butter after enzymolysis is determined, judges the enzyme activity of immobilized enzyme.
1.4.3.2 the operational stability of co-immobilization enzyme
Weigh 10g immobilized enzyme to be put in conical flask, 6g deionized water and 10g butter is subsequently adding, is placed in constant-temperature table
In (35 DEG C, 150r/min) reaction 2h.3000r/min centrifugation 15min after immobilized enzyme is leached, upper oil phase is taken, determines enzymolysis
The acid value of butter, judges the enzyme activity of immobilized enzyme afterwards.Immobilized enzyme after filtration is washed with deionized, and continues weight after sucking filtration
Multiple above-mentioned steps, investigate change of the co-immobilization enzyme enzyme activity with access times.
1.4.3.3 the storage-stable of co-immobilization enzyme
Co-immobilization enzyme is placed in preservation 0,2,4,6,8,10 days in 4 DEG C of refrigerator.Weigh 10g immobilized enzyme every time to be put into
In conical flask, 6g deionized water and 10g butter is subsequently adding, is placed in constant-temperature table (35 DEG C, 150r/min) reaction 2h.Leach
After immobilized enzyme, 3000r/min centrifugation 15min, takes upper oil phase, determines the acid value of butter after enzymolysis, judges the enzyme of immobilized enzyme
Vigor.
1.4.4 the preparation of milk base material
1.4.4.1 co-immobilization enzyme addition
Toward in 6 conical flasks, add 10g butter as substrate respectively, auxiliary substrate whey powder addition is 0.125g (butter
The 12.5% of quality), amount of water is 6g (the 60% of butter quality), after being placed in thermostat water bath (75 DEG C, 20min) sterilizings,
Add co-immobilization enzyme after being cooled to room temperature again respectively, the ratio of addition and butter is 0,0.5：1、1：1、1.5：1、2：1、
2.5：1, (35 DEG C, 150r/min) reaction 2h are placed in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, is taken
Upper oil phase, determines the acid value of butter after enzymolysis, and carries out sensory evaluation to the fragrance for producing.
1.4.4.2 the enzymolysis time of co-immobilization enzyme
Toward in 6 conical flasks, add 10g butter as substrate respectively, auxiliary substrate whey powder addition is 0.125g (butter
The 12.5% of quality), amount of water is 6g (the 60% of butter quality), after being placed in thermostat water bath (75 DEG C, 20min) sterilizings,
Add the co-immobilization enzyme 10g (ratio 1 of co-immobilization enzyme addition and butter quality after being cooled to room temperature again respectively：1), it is placed in
(35 DEG C, 150r/min) reaction 2h in constant-temperature table.3000r/min centrifugation 15min after immobilized enzyme is leached, upper oil phase is taken,
The acid value of butter after digesting is determined, and sensory evaluation is carried out to the fragrance for producing.
1.4.4.3 the hydrolysis temperature of co-immobilization enzyme
Toward in 6 conical flasks, add 10g butter as substrate respectively, auxiliary substrate whey powder addition is 0.125g (butter
The 12.5% of quality), amount of water is 6g (the 60% of butter quality), after being placed in thermostat water bath (75 DEG C, 20min) sterilizings,
Add the co-immobilization enzyme 10g (ratio 1 of co-immobilization enzyme addition and butter quality after being cooled to room temperature again respectively：1), respectively
It is placed in 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 150r/min reaction 2h in 55 DEG C of constant-temperature table.After leaching immobilized enzyme
3000r/min is centrifuged 15min, takes upper oil phase, determines the acid value of butter after enzymolysis, and the fragrance to producing carries out sense organ and comments
Valency.
1.5 acid value
1.5.1 reagent
(1) neutral ether alcohol mixeding liquid：By ether and ethanol according to 1：1 ratio mixing, adds 3 to drip phenolphthalein instruction
Agent, with potassium hydroxide volumetric solution to the aobvious neutrality of indicator.
(2) phenolphthalein indicator：The phenolphthalein of 10g/L, 10g is dissolved in 95% ethanol solution of 1L.
(3) potassium hydroxide-ethanol solution (c (KOH)=0.050mol/L)：4g potassium hydroxide is weighed in polyethylene can,
Plus 5ml water dissolution, with 95% ethanol dilution to 1L, closed placement 24h [17].
1.5.2 the demarcation of potassium hydroxide-ethanol solution
0.375g is accurately weighed in the Potassium Hydrogen Phthalate of 105 DEG C of -110 DEG C of dryings to constant weight, molten addition 50ml no two
The water dissolution of carbonoxide, adding 2 to drip phenolphthalein indicator, being titrated to pinkiness with potassium hydroxide-ethanol solution, while doing blank
Test.Potassium hydroxide-ethanol solution concentration is calculated by following formula.
In formula：The quality of m- Potassium Hydrogen Phthalate, g；
V1- consumes the volume of potassium hydroxide-ethanol solution, mL；
V2- blank assay consumes the volume of potassium hydroxide-ethanol solution, mL；
204.22- the molal weight of Potassium Hydrogen Phthalate
1.5.3 the measure of acid value
Accurately weigh milk base material 1.0g to be put in conical flask, add 10mL neutrality ether alcohol mixeding liquid to make oil sample molten
Solution, Deca 2 drips phenolphthalein indicator, is titrated to ethanolic potassium hydroxide standard solution and blush occurs, and color and luster change maintains 30s,
The consumption [18] of the ethanolic potassium hydroxide standard solution that record is consumed.
The acid value of milk base material is calculated by following formula：
In formula：The acid value of X- milk base material, mg/g；
The ethanolic potassium hydroxide standard solution volume that V- titration is consumed, mL；
The actual concentrations of c- ethanolic potassium hydroxide standard solution, mol/L；
M- milk base material samples quality, g；
The molal weight of 56.11- potassium hydroxide, g/mol.
1.6 enzyme activity
In same group of data, the value of experiment value peak is denoted as 100%, the ratio that the value of other experimental points is put with this
Value, is exactly the relative activity of enzyme, is represented with percent.
1.7 determining the protein quantity
Protein content during co-immobilization in filtrate is determined with dying method with coomassie brilliant blue.
1.7.1 reagent
(1) Coomassie brilliant blue reagent：Coomassie brilliant G-250 0.1g is weighed, it is 95% to be dissolved in 50mL mass fraction
In ethanol, 85% phosphatase 11 00mL is added, deionized water is diluted to 1L.
(2) standard protein solution：Using bovine serum albumin as standard protein, 10mg bovine serum albumin is accurately weighed,
It is dissolved in water, 100mL is settled to, is made into the standard protein solution of 0.1mg/mL.
1.7.2 draw bovine serum albumin standard curve
The color comparison tube of 6 dryings is taken, is numbered and add reagent toward in color comparison tube by table 3.Shake up after adding reagent, standing
After 5min, using No. 1 sample as blank, the absorbance of remaining sample is determined at 595nm.Experimental result is sat with absorbance as vertical
Mark, standard protein content draws standard curve, such as table 2 for abscissa.
The drafting of 2 bovine serum albumin standard curve of table
1.7.3 sample determination
Filtrate 1mL is taken in color comparison tube, Coomassie brilliant blue reagent 5mL is added, is shaken up, survey at 595nm after standing 5min
Determine absorbance.The protein content in filtrate is calculated according to standard curve.
1.8 fix rate
The ability (fixing rate) of carrier immobilized pheron can use following formula to be calculated.
Fixing rate=mistake！The source of quoting is not found.
In formula：M- adds the initial enzyme amount of immobilization system, mg；
The protein content of filtrate, mg/mL after c- immobilization；
The volume of filtrate, mL after V- immobilization.
1.9 sensory evaluation
Some personal composition sensory evaluation appraisal group are selected, pure by the aroma strength respectively to enzymatic hydrolysate of table 3, fragrance
Degree and lasting situation are scored, and it is 10 that appraisal result is 3 sums, total score.Standards of grading are shown in Table 3.
3 fragrance standards of grading of table
2nd, result and analysis
2.1 bovine serum albumin standard curves
Protein content during co-immobilization in filtrate is determined with dying method with coomassie brilliant blue.Under suitable conditions, examine
Mas bright blue G-250 can generate blue compound with protein, have the absorption value of maximum, protein concentration and change at 595nm
The depth degree of compound blueness is directly proportional.Dying method with coomassie brilliant blue can be stablized and rapidly measure containing for protein in enzyme liquid
Amount, bovine serum albumin standard curve is as shown in Figure 1.
2.2 fixation supports and the selection of process for fixation
Fixation support is stability and the enzyme activity for affecting immobilized enzyme, and selects suitable fixation support to be conducive to
Improve the properties of immobilized enzyme.The performance of immobilized enzyme additionally depends on fixation in addition to the carrier for being used depending on immobilization
Change method.But there were significant differences for the property such as the isoelectric point, IP, average diameter of different types of enzyme, hydrophilic and hydrophobic, does not therefore almost have
A kind of process for fixation is had to be applied to any enzyme, so suitable process for fixation will be selected according to concrete enzyme viability.
Four kinds of carriers are selected in experiment, absorption method, cross-linking method and investment is employed, respectively to lipase A 6, lipase
MER and the test of being fixed of protease MSD, determine the impact of different carriers and process for fixation to fixing rate, as a result such as
Shown in table 4.
The impact (%) of 4 different carriers of table and process for fixation to fixing rate
From table 4, with shitosan as carrier to three kinds of resolvases using embedding being fixed of method effect not
Preferable.For lipase A 6, the process for fixation effect for directly being embedded with sodium alginate is best, and fixing rate is 97.8%.Right
In lipase MER, directly wrap with macroporous resin X-5 as carrier loaded chitosan film being fixed of cross-linking method and with sodium alginate
Bury effect preferable, fixing rate is respectively 99.1% and 98.2%.And protease MSD is adopted and is directly wrapped as carrier with sodium alginate
The fixing rate highest that buries.In sum, three kinds of enzymes are adopted with sodium alginate as the direct immobilization effect for embedding of carrier more
Ideal, fixing rate is more than 97%, it may be considered that adopt the method for directly embedding as carrier with sodium alginate to enter three kinds of enzymes
Row co-immobilization.
Impact of the 2.3 co-immobilization condition optimizings to enzyme activity
2.3.1 impact of the pH value to co-immobilization enzyme activity
In enzyme immobilizatio reaction, as fixation support and enzyme are all present in buffer, and the pH of buffer can
To change the ionization state of fixation support and enzyme molecule；Additionally, enzyme is used as a kind of protein, when the pH value of system exceeds model
When enclosing, its microstructure will change, so as to cause enzyme denaturation to inactivate.Therefore pH of cushioning fluid is impact immobilized enzyme enzyme
One of key factor of vigor and fixing rate.
From figure 2 it can be seen that in below pH6.5, with the increase of immobilization system pH, fixing rate assumes increase
Trend.When pH value is 6.5, fixing rate is 98%, to reach highest；Acid value is now measured for 1.16mg/g, co-immobilization enzyme
Enzyme activity also highest.Possibly when other conditions are consistent, immobilization enzyme immobilizatio rate is higher, the immobilized enzyme of unit mass
Middle enzyme content is higher, and enzymatic hydrolysate acid value is bigger.When pH is more than 6.5, with the increase of immobilization system pH, fixing rate exists
97.5%-98% or so, change is little, and the enzyme activity change of co-immobilization enzyme is also more gentle.So, co-immobilization optimal
PH is 6.5.
2.3.2 impact of the sodium alginate concentration to co-immobilization enzyme activity
Tang Mingqiang was once studied, and sodium alginate not only affects the viscosity of solution, the roundness of micelle and mechanical strength, goes back shadow
Ring to immobilized enzyme.Sodium alginate has strong adsorptivity, can form gel with the positive bivalent cation such as copper, calcium.Sargassum
When sour na concn is too big, gel aperture is less, affects the combination of substrate and enzyme.Sodium alginate concentration too hour, gel aperture is relatively
Greatly, immobilized enzyme is easy to run off, and enzyme activity is relatively low.So sodium alginate should have an optimal concentration during co-immobilization
Value.
As Fig. 3, when immobilized enzyme is prepared, can be clearly seen that when sodium alginate concentration is less than 0.5%, formation
Gel structure is unstable, it is impossible to form gel particle.When sodium alginate concentration is 0.5%, irregular micelle is ultimately formed,
And granule is more soft, bad mechanical strength, cause the easy breaking cellular wall of sodium alginate micro ball for preparing so that most resolvase is no
Method is fixed.When sodium alginate concentration is for 1.0%-1.5%, immobilization enzyme immobilizatio rate is higher, the micelle mechanicalness of preparation
Can be good, measured acid value is higher, and immobilized enzyme is higher.The concentration for possibly suitably increasing carrier sodium alginate can make
Its ability that can carry pheron molecule strengthens, therefore fixing rate increase.Continue to increase the concentration of sodium alginate, fixing rate and
Acid value declines on the contrary.Reason is probably that the enzyme being fixed on unit mass sodium alginate is reduced when sodium alginate concentration is higher,
Fixing rate decline；The thin layer for forming immobilized enzyme micelle is too thick, and the resistance to mass tranfer between the enzyme-to-substrate being embedded increases, from
And cause immobilized enzyme decline.When sodium alginate concentration is more than 2.5%, then solidify quickly and not easy to operate, it is difficult to will
Sodium alginate soln is extruded from syringe.Therefore it is convenient to carry out co-immobilization from the sodium alginate that mass fraction is 1.5%.
2.3.3 impact of the enzyme amount to co-immobilization enzyme activity
In being fixed enzyme, when vector contg with to enzyme amount mutually suitable when can be only achieved optimal immobilization effect
Really, otherwise, will result in part resolvase to be combined with carrier when vector contg is relatively too low, cause unnecessary wave
Take, can then cause when vector contg is relatively too high carrier that its ability for combining pheron cannot be given full play to, to a certain extent
Also improve immobilization cost.Therefore, the optimum benefit for being conducive to improving immobilized enzyme to enzyme amount is selected.
As Fig. 4, when sodium alginate concentration is constant, with the increase to enzyme amount, fixing rate also increases, immobilized enzyme
Enzyme activity is in downward trend after first rising.During to enzyme amount less than 27mg/g sodium alginate, it is bright that fixing rate increases trend comparison
Aobvious, illustrate when to enzyme amount relatively hour, saturation does not occur in the immobilization of unit carrier, therefore with increasing to enzyme amount, immobilization
Rate increases and increases.And when to enzyme amount more than 27mg/g sodium alginate, fixing rate curve becomes gentle, now immobilization is described
Oneself is through being close to saturation.When to enzyme amount for 30mg/g sodium alginate, the enzyme activity of immobilized enzyme reaches maximum, now measures acid
Valency is 1.73mg/g.This is because under conditions of carrier amount is constant, when giving enzyme amount relatively fewer, the pheron of carrier embedding
Also less, it is not efficiently used carrier, therefore enzymatic activity is also very low.But when to enzyme amount relatively many when, carrier is wrapped
The pheron for burying is also more, agglomerating so as to cause pheron mutually to assemble, and the active center of enzyme molecule may be covered, affects enzyme
With Binding Capacity, the enzyme amount of although sodium alginate embedding is a lot, but enzymatic activity is still very low.So, the optimal of co-immobilization gives enzyme
Amount is 30mg/g sodium alginate.
2.3.4 CaCl2Impact of the concentration to co-immobilization enzyme activity
Ca2+Form, with sodium alginate, the significant process that calcium alginate gel is immobilized enzyme.CaCl2Determine as coagulator
Determine the mechanical strength of immobilized enzyme, also determine immobilization enzyme immobilizatio " firm " degree.
As a result as shown in figure 5, co-immobilization enzyme immobilizatio rate and enzyme activity are with CaCl2The increase of concentration and increase, when
CaCl2When concentration reaches 4.0%, co-immobilization enzyme immobilizatio rate and enzyme activity reach highest, and most of gel particle
Become well-regulated spherical.CaCl2When concentration continues to increase, fixing rate change is little, and the enzyme activity of co-immobilization enzyme is with CaCl2
The increase of concentration and decline.Possible the reason for is to work as CaCl2Concentration is too low, and when fixing altogether, embedding is incomplete, and part enzyme cannot be consolidated
Determine in gel particle, fixing rate is relatively low；The gel particle of immobilization gained is relatively soft, and mechanical strength is weak, loses during washing
More enzyme activity, the enzyme activity of immobilized enzyme is relatively low.Work as CaCl2During excessive concentration, gel particle surface can form one layer of densification
Alginic acid calcium layer, be unfavorable for the diffusion of substrate, decline the enzyme activity of immobilized enzyme.Therefore, the optimal CaCl of co-immobilization2
Concentration is 4.0%.
2.3.5 impact of the temperature to co-immobilization enzyme activity
Appropriate temperature can increase the kinetic energy of enzyme molecule, improve the contact rate between enzyme and carrier, so as to increase immobilization effect
Rate.But enzyme, used as a kind of protein, high temperature easily causes its deactivation.
From fig. 6, it can be seen that when temperature is less than 30 DEG C, immobilized temperature is affected not on co-immobilization enzyme enzyme activity
It is very big.When temperature is more than 30 DEG C, immobilized enzyme enzyme activity is on a declining curve.In the range of 20 DEG C -35 DEG C, fixing rate presents liter
Trend.Rate being fixed when 35 DEG C and reaching highest, fixing rate is 99.2%.When temperature continues to increase, fixing rate decline.Reason can
Can be that sodium alginate can solidify at low temperature when immobilization temperature is relatively low, so as to increase the difficulty of operation, fixing rate is relatively
Low；As temperature is relatively low, temperature is on the impact of immobilized enzyme enzyme activity and little.So, 30 DEG C during the optimum temperature of co-immobilization.
2.3.6 impact of the time to co-immobilization enzyme activity
The immobilization time refers to enzyme and drops onto CaCl with the mixed liquor of sodium alginate2Time of standing and reacting afterwards.
As shown in Figure 7, impact of the immobilization time to fixing rate be not obvious, with the increase of immobilization time, fixing
Rate maintains 98% or so all the time, and fixing rate is higher.And immobilized enzyme enzyme activity is as the increase of immobilization time is in first
Downward trend after rising.This is because sodium alginate and CaCl2The process for reacting and being formed gel particle needs the time.
At the co-immobilization initial stage, embedding is gradually tight, is lost in and reduces, and immobilized enzyme enzyme activity is in rising trend；Immobilized enzyme enzyme after 30min
Vigor reaches maximum, now measures acid value for 1.69mgKOH/g；But the time continues increase can make Ca2+With enzyme effect, and coagulate
Too closely, impact substrate is combined glue with enzyme, affects co-immobilization enzyme enzyme activity.Therefore the selection most suitable immobilization time is
30min.
The stability of 2.4 co-immobilization enzymes
2.4.1 the heat stability of co-immobilization enzyme
Impact of the temperature to enzyme is sufficiently complex, and temperature affects the conformation of pheron molecule, participates in enzymatic reaction functional group
Dissociated state, also affects the affinity of substrate and enzyme.
Can obtain from Fig. 8, co-immobilization enzyme still retains 81.4% enzyme activity after water-bath 150min at 20 DEG C, thermally-stabilised
Property is preferable.It is possibly due to carrier and a kind of environment is provided, protective effect has been risen to enzyme.
As can be seen from Figure 9, co-immobilization enzyme is at 60 DEG C during water-bath, and enzyme activity declines by a big margin.During 0-20min, enzyme activity
Power declines very fast；After 20min, enzyme activity change is little, and enzyme activity is maintained at 70%-75% or so.Co-immobilization enzyme exists
The reason for when 60 DEG C, enzyme activity declines rapidly is likely due to during high temperature enzyme easily inactivation and co-immobilization at relatively high temperatures
The calcium alginate of enzyme outer layer can soften because of the rising of temperature, leak out enzyme embedded therein, therefore under enzyme activity
Drop is very fast.
2.4.2 the operational stability of co-immobilization enzyme
The operational stability of immobilized enzyme is a key character for being different from resolvase, is the weight for reflecting immobilization effect
Want index.The recycling of immobilized enzyme be not only advantageous to digest after the recovery of product and purification, and be conducive to cost-effective.
From fig. 10 it can be seen that with the increase of access times, enzyme activity reduces, it may be possible to due to being stirred continuously,
Alginate carrier retentiveness changes, co-immobilization enzyme gel structure change, enzyme come off from alginate carrier and with
Substrate contact is constantly hydrolyzed.Co-immobilization enzyme is using after 4 times, and enzyme activity still keeps original 92.4%, and stability is preferable.With this
Meanwhile, co-immobilization enzyme is not had not too on sense organ using obtained milk base material after 4 times and the fragrance of primary milk base material
Big difference.
2.4.3 the storage-stable of co-immobilization enzyme
As seen from Figure 11, when co-immobilization enzyme is preserved 0-4 days, enzyme activity is maintained at more than 91.8%.Illustrate enzyme with Sargassum
After sour sodium carrier is combined, the presence of carrier prevents the interaction between pheron, it is suppressed that the degeneration of pheron and degraded,
Co-immobilization enzyme is made to have certain storage-stable.After co-immobilization enzyme is preserved 4 days, its enzyme activity declines rapidly, and the 10th
It when immobilized enzyme enzyme activity be 66.8%.The reason for enzyme activity declines is probably the protease hydrolysiss fat in sodium alginate gel
Fat enzyme, affects the enzyme activity of lipase.
Test result indicate that, the optimum condition of co-immobilization is：The pH of phosphate buffer is 6.5, the concentration of sodium alginate
For 1.5%, total enzyme concentration be:A6:MER=0.3：2：1), CaCl2Concentration is 4.0%, fixing altogether
The temperature of change is 30 DEG C, and the time of co-immobilization is 30min.Now co-immobilization enzyme immobilizatio rate is that 98.0%, the 4th makes
Original 92.4% is still kept with rear enzyme activity, after preserving 10 days, enzyme activity still keeps original 66.8%, and stability is preferable.
The preparation of 2.5 milk base materials
2.5.1 co-immobilization enzyme addition
When enzyme catalysiss generate milk base material, the addition of enzyme is larger to the flavor effect of milk base material.The addition of enzyme is relatively
When few, the amount of the flavor substance of generation is few, and milk fragrance is thin；When enzyme addition is excessive, though substantial amounts of flavor substance can be produced,
Some bad smells can be produced, while can also increase production cost.Therefore, suitable co-immobilization enzyme addition should be selected.
Organoleptic effects of the 5 co-immobilization enzyme addition of table to milk base material
From Figure 12 and Biao 5, when co-immobilization enzyme addition is less than butter quality, with the increasing of co-immobilization enzyme
Plus, the fragrant effect of product is better, and fragrance is stronger.When co-immobilization enzyme is equal to butter quality, obtained milk base material is fragrant and sweet type
Milk taste, gives off a strong fragrance, mellow and full and long times of aftertaste, now measures acid value for 1.86mg/g.When co-immobilization enzyme addition is more than Huang
During oil quality, start to produce acid smell of urine taste, and the increase with enzyme concentration, the acid value of milk base material increases comparatively fast, and acid smell of urine taste is more next
More serious.Consider, the addition of optimum co-immobilization enzyme is equal with the quality of butter.
2.5.2 the enzymolysis time of co-immobilization enzyme
In order to obtain best effect and improve production efficiency, it is also critically important to select suitable enzymolysis time.Just manage
By for, enzymolysis time is longer, and enzyme digestion reaction is more thorough.It is true that long enzymolysis time can generate more bad flavor
Material, affects the overall local flavor of perfume base.But when enzymolysis time is too short, enzyme digestion reaction is incomplete, and flavor substance generation is very few,
Local flavor is not sufficiently formed.Milk base material is wanted to reach preferable local flavor, the enzyme digestion reaction time should be controlled.
Organoleptic effects of 6 enzymolysis time of table to milk base material
From Figure 13 and Biao 6 as can be seen that the acid value of enzymatic hydrolysate increases with the increase of enzymolysis time.Work as enzymolysis time
When being 2h, it is best to produce fragrant effect, and gained milk base material gives off a strong fragrance, mellow and full, and coordinates with milk sweet taste, and fragrance remaining time is long, now
Acid value is 1.70mg/g.After enzymolysis time is more than 2h, acid value continues to rise, and starts tapinoma-odour occur, and sensory evaluation scores decline, can
Can be that the Degree of Enzymatic Hydrolysis of butter too high causes in enzymatic hydrolysate bad flavor material to generate.Consider, select enzymolysis optimum
Time be 2h.
2.5.3 the hydrolysis temperature of co-immobilization enzyme
Enzyme is more sensitive to temperature, and the difference of temperature influences whether molecular conformation distribution and the substrate butter of enzyme active center
Emulsified state, so as to have influence on the process of whole enzyme digestion reaction.When hydrolysis temperature is less than optimum temperature, temperature can inhibitory enzyme
Activity, make enzyme activity relatively low, while can also affect the viscosity of substrate, and then affect mass transfer velocity, the speed of enzyme digestion reaction becomes
Slowly.When hydrolysis temperature is higher than optimum temperature, weaken or even lose catalysis activity as high temperature can cause enzyme denaturation, Er Qie
Under higher temperature, the calcium alginate of co-immobilization enzyme outer layer can soften, and leak out enzyme embedded therein, and enzyme activity declines.
Organoleptic effects of 7 hydrolysis temperature of table to milk base material
Can see that by Figure 14 and Biao 7, when hydrolysis temperature is less than 40 DEG C, with the rising of temperature, the perfume (or spice) of milk base material
Gas is more and more stronger, and fragrance purity is increasingly coordinated, enriched, and reason is probably that the rising of temperature promotes entering for enzyme digestion reaction
OK, local flavor product is consequently increased.When temperature reaches 40 DEG C, the sensory evaluation scores highest of obtained milk base material, now
Milk base material is the milk of inclined cheese, and milk is stronger, and fragrance is purer mellow and full, and fragrance remaining time is long, and measured acid value is 1.58mg/
g.When hydrolysis temperature reaches 45 DEG C, the acid value highest of enzymatic hydrolysate, acid value is 3.58mg/g, and sensory evaluation scores reduce, it may be possible to
Because enzymolysis excessively produces bad flavor material.When hydrolysis temperature is more than 45 DEG C, it may be possible to as hydrolysis temperature is higher than most thermophilic
Degree causes enzyme denaturation and weakens or even lose catalysis activity, or because the calcium alginate of co-immobilization enzyme outer layer is because temperature
Rising and soften, leak out enzyme embedded therein, beginning to decline enzyme activity causes acid value and sensory evaluation scores to reduce.
In sum, suitable hydrolysis temperature is 40 DEG C.
2.6 resolvases together immobilized enzyme prepare milk base material comparison
Although enzyme immobilization technology solve resolvase production cost high the problems such as, due to enzyme molecule from free state become
When becoming firm state to be incorporated into carrier, the micro environment of enzyme molecule there occurs change.Therefore prepared by resolvase and co-immobilization enzyme
Still there are some difference in terms of process conditions and sensory evaluation scores in milk base material.
8 resolvase of table together immobilized enzyme prepare milk base material comparison
As shown in Table 8, although during co-immobilization during the use of resolvase and sodium alginate and co-immobilization
Production cost is generated, but as immobilized enzyme can be reused, and enzymolysis process is simpler, prepares milk using immobilized enzyme
Base material can reach the purpose for saving production cost.And milk base material prepared by resolvase and sodium alginate is all inclined cheese odor type
Milk, fragrance is purer mellow and full, and lasting situation is preferable, and sensory evaluation scores are close, but the fragrance of the milk base material of resolvase preparation
The fragrance of the milk base material for preparing than immobilized enzyme is somewhat stronger.Possibly sodium alginate gel granule is to a certain extent
Contact of the enzyme with substrate is hampered, so that some taste compounds is generated less or cannot generate, cause aroma strength slightly weak.Again may
It is because that sodium alginate gel granule hampers the diffusion of some taste compounds, makes the fragrance of obtained milk fragrance base weaker.
Test result indicate that, the optimum process condition for preparing milk base material is：With butter as substrate, amount of water is butter weight
The 60.0% of amount, substrate supplemented by whey powder, auxiliary substrate addition is the 12.5% of butter weight, the addition of co-immobilization enzyme
Mass ratio with substrate butter is 1:1, enzymolysis time is 2h, and hydrolysis temperature is 40 DEG C.The milk base material milk perfume of gained of the present invention
Taste is dense, milk is pure, pleasure degree is good, can be used to make cookiess.
Last should be noted that above example is protected only in order to technical scheme to be described rather than to the present invention
The restriction of shield scope, although being explained in detail to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (10)
1. a kind of preparation method of co-immobilization enzyme, it is characterised in that the preparation method is comprised the following steps：
(1) lipase A 6, lipase MER and protease MSD are added in sodium alginate soln, is blended after stirring
Liquid；
(2) blended liquid that step (1) is obtained is instilled CaCl2In aqueous solution, gel particle after standing solidification, is formed, obtained solidifying
Glue granule is the co-immobilization enzyme.
2. preparation method according to claim 1, it is characterised in that lipase A 6, lipase MER in step (1)
Gross weight with protease MSD is the 2.2%-3.52% of the sodium alginate weight, the lipase A 6, lipase MER and
The weight ratio of protease MSD is lipase A 6：Lipase MER：Protease MSD=2:1：0.3.
3. preparation method according to claim 1, it is characterised in that lipase A 6, lipase MER in step (1)
Gross weight with protease MSD is the 3.0% of the sodium alginate weight, the lipase A 6, lipase MER and protease
The weight ratio of MSD is lipase A 6：Lipase MER：Protease MSD=2:1：0.3.
4. preparation method according to claim 1, it is characterised in that in step (1), sodium alginate soln is that pH is
6.0～8.0, mass fraction is 1.0%～2.0% sodium alginate soln.
5. preparation method according to claim 1, it is characterised in that CaCl in step (2)2Aqueous solution is that quality is divided
Number is 3.0%～6.0% CaCl2Aqueous solution.
6. preparation method according to claim 1, it is characterised in that in step (1), sodium alginate soln is that pH is
6.5th, mass fraction is 1.5% sodium alginate soln；CaCl in step (2)2It is 4.0% that aqueous solution is mass fraction
CaCl2Aqueous solution.
7. preparation method according to claim 1, it is characterised in that in step (2) solidification temperature be 20～35 DEG C,
Hardening time is 20～40min.
8. preparation method according to claim 7, it is characterised in that in step (2) solidification temperature be 30 DEG C, solidification
Time is 30min.
9. the co-immobilization enzyme that a kind of method using as described in claim 1-8 is arbitrary is prepared from.
10. co-immobilization enzyme as claimed in claim 9 prepare milk base material in purposes.
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| CN107334135A (en) * | 2017-08-16 | 2017-11-10 | 仲恺农业工程学院 | A kind of preparation method of milk flavour of candy milk base material |
| CN107397189A (en) * | 2017-07-20 | 2017-11-28 | 浙江省海洋开发研究院 | A kind of fresh shrimp taste instant noodles seasoning powder and preparation method thereof |
| CN107960629A (en) * | 2017-07-20 | 2018-04-27 | 浙江省海洋开发研究院 | A kind of seafood shrimp taste soup jelly and preparation method thereof |
| CN108410003A (en) * | 2018-02-09 | 2018-08-17 | 江南大学 | A kind of preparation of modified polyacrylonitrile film and its method applied to immobilized enzyme |
| CN110628756A (en) * | 2019-10-15 | 2019-12-31 | 厦门理工学院 | Co-immobilized enzyme and preparation method and application thereof |
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| CN107397189A (en) * | 2017-07-20 | 2017-11-28 | 浙江省海洋开发研究院 | A kind of fresh shrimp taste instant noodles seasoning powder and preparation method thereof |
| CN107960629A (en) * | 2017-07-20 | 2018-04-27 | 浙江省海洋开发研究院 | A kind of seafood shrimp taste soup jelly and preparation method thereof |
| CN107960629B (en) * | 2017-07-20 | 2021-01-15 | 浙江省海洋开发研究院 | Seafood and shrimp-flavor soup jelly and preparation method thereof |
| CN107334135A (en) * | 2017-08-16 | 2017-11-10 | 仲恺农业工程学院 | A kind of preparation method of milk flavour of candy milk base material |
| CN108410003A (en) * | 2018-02-09 | 2018-08-17 | 江南大学 | A kind of preparation of modified polyacrylonitrile film and its method applied to immobilized enzyme |
| CN108410003B (en) * | 2018-02-09 | 2020-11-06 | 江南大学 | Preparation of polyacrylonitrile modified membrane and method for applying polyacrylonitrile modified membrane to immobilized enzyme |
| CN110628756A (en) * | 2019-10-15 | 2019-12-31 | 厦门理工学院 | Co-immobilized enzyme and preparation method and application thereof |
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