CN106434622B

CN106434622B - Preparation method of co-immobilized enzyme

Info

Publication number: CN106434622B
Application number: CN201610736261.9A
Authority: CN
Inventors: 汪薇; 任文彬; 白卫东; 于立梅
Original assignee: Zhongkai University of Agriculture and Engineering
Current assignee: Zhongkai University of Agriculture and Engineering
Priority date: 2016-08-26
Filing date: 2016-08-26
Publication date: 2020-12-15
Anticipated expiration: 2036-08-26
Also published as: CN106434622A

Abstract

The invention provides a preparation method of co-immobilized enzyme, which comprises the following steps: (1) adding lipase A6, lipase MER and protease MSD into sodium alginate solution, and stirring to obtain a blended solution; (2) dripping the blending liquid obtained in the step (1) into CaCl₂And standing and curing the mixture in the aqueous solution to form gel particles, wherein the prepared gel particles are the co-immobilized enzyme. The co-immobilized enzyme prepared by the method has high immobilization rate, and the enzyme activity operation stability and storage stability of the co-immobilized enzyme are good.

Description

Preparation method of co-immobilized enzyme

Technical Field

The invention relates to a preparation method of an enzyme, in particular to a preparation method of a co-immobilized enzyme.

Background

The enzyme concept was first proposed in 1878 by william-zui of the german physiologist. Enzymes, commonly known as enzymes, are a special class of proteins produced synthetically by plants, animals, microorganisms and human cells. Enzymes are often referred to as biocatalysts because of their catalytic ability to enhance the rate and quality of biochemical reactions. Compared with the traditional catalyst, the enzyme has the advantages of high efficiency, specificity, diversity, mild reaction condition and the like, and is widely applied to various fields. However, the enzyme protein is easily denatured under the conditions of acid, alkali, heat, organic solution and the like, so that the enzyme activity is reduced or lost, the enzyme reaction is mostly carried out in the solution, the recovery is not easy after the reaction, the separation and purification of the reaction product are difficult, and the industrial continuous and automatic production is difficult to realize, so the application development of the enzyme engineering is greatly limited.

The immobilized enzyme is prepared by treating enzyme chemically or physically, so that the enzyme with strong water solubility is combined with solid water-insoluble carrier or embedded by the carrier to form a unified whole.

After immobilization, immobilized enzymes have more advantages than free enzymes:

(1) the immobilized enzyme is easy to separate from a substrate and a product, the immobilized enzyme can be recovered by simple operations such as centrifugation or filtration after the reaction is finished, the enzyme activity is reduced less, the immobilized enzyme can be used repeatedly in multiple batches, and the production cost is reduced;

(2) after the immobilization treatment, the general stability is greatly improved, the pH stability, the thermal stability and the like are improved, and the sensitivity to an inhibitor is reduced.

(3) The immobilized enzyme is suitable for automatic and continuous production, is easy to control the catalytic process, can not bring enzyme protein into the product to cause enzyme residue, simplifies the later purification process, improves the utilization efficiency of the enzyme and reduces the production cost.

Of course, immobilized enzymes have disadvantages:

(1) part of enzyme activity is lost during immobilization;

(2) the immobilization of the enzyme requires equipment and a carrier, so that the production cost is increased;

(3) immobilized enzymes are only suitable when the substrate is soluble and are not well suited for macromolecular substrates;

(4) the intracellular enzymes secreted by the microorganisms can only be immobilized after isolation.

With the continuous and intensive research on immobilized enzymes, the immobilization method is slowly transited to the combination of various methods. However, few immobilization techniques have been developed so far, which are suitable for any enzyme, and thus an appropriate immobilization method is selected depending on the characteristics and the intended use of a specific enzyme.

The immobilization methods of enzymes can be classified into 4 types, adsorption, covalent bonding, crosslinking, and entrapment.

The adsorption method is one of the most used methods for immobilizing enzymes, and the adsorption method is an immobilization method for immobilizing enzymes on a carrier by using an acting force such as van der waals force, ionic bond, hydrogen bond, physical adsorption, or the like. Generally, physical adsorption and ion adsorption can be classified.

The Luyuman uses an adsorption method to fix the lipase on the macroporous anion resin D201, and the obtained immobilized enzyme has better operation stability. The prepared immobilized enzyme is used for catalyzing the natural butter to carry out hydrolysis reaction, and the hydrolysate has soft and pure fragrance.

The covalent bonding method is an immobilization method in which an unnecessary functional group on an enzyme is bonded to a functional group on the surface of a carrier by a covalent bonding method.

The chemical crosslinking method is to crosslink the zymoprotein molecules with the help of a bifunctional or multifunctional reagent to form covalent bonds between the zymoprotein molecules and the bifunctional or multifunctional reagent, so as to obtain a three-dimensional crosslinked network structure. In this process, cross-linking occurs between enzyme molecules, and at the same time, there is some degree of cross-linking within the molecules. Chemical crosslinking methods can be divided into crosslinked enzyme crystals and crosslinked enzyme aggregates.

The entrapment method is a method in which an enzyme is mixed with a carrier solution, and then, polymerization is performed by means of an initiator, and the enzyme is physically confined in a network structure of the carrier, thereby realizing immobilization of the enzyme. The method has the advantages of low immobilization cost, large immobilized enzyme quantity, simple and easy operation, mild immobilization conditions, high enzyme molecule stability and the like, but because the enzyme is immobilized in the internal space of the carrier, a reaction substrate can reach the active center of the enzyme to perform catalytic reaction only through diffusion due to the influence of mass transfer resistance. At the same time, the violent enzymatic reactions may damage the polymeric microcapsule membrane or network, resulting in enzyme leakage. In terms of the selection of carrier materials, polyacrylamide, gelatin, carrageenan, chitosan, sodium alginate and the like are common choices.

The research of Gaoshanxin and the like takes sodium alginate as a carrier and CaCl₂Chitosan solution is used as coagulant, and bromelain is immobilized by adopting an embedding method. Tests show that the thermal stability and the optimum temperature of the immobilized enzyme prepared by the method are improved, the reaction pH shifts to alkalinity, and the enzymological properties are improved. Meanwhile, the immobilized enzyme also has good operability, the relative enzyme activity is still kept by more than 80 percent after the immobilized enzyme is repeatedly used for 4 times, and the immobilization effect is good.

Sodium alginate such as Luyuchira as carrier, CaCl₂Solutions ofAs a coagulant, lipase was immobilized by an embedding method. Tests show that the thermal stability of the immobilized enzyme prepared by the method is good, and the enzyme activity of the immobilized enzyme is only reduced by 30% after the immobilized enzyme is heated for 1.5 hours at the temperature of 60 ℃. Moreover, the method also has good operation stability, the enzyme activity of the immobilized enzyme is still kept above 95 percent after the immobilized enzyme is continuously and repeatedly used for 10 times.

Co-immobilized enzymes began in the 80's of the 20 th century and are a technique for immobilizing two or more enzymes on the same carrier to form a co-immobilized system. The co-immobilized enzyme is based on the immobilization of a single enzyme, and the optimum immobilization method of the co-immobilized enzyme is considered comprehensively. Compared with common immobilized enzymes, the co-immobilized enzymes can fully exert the characteristics of different enzymes, and simultaneously combine the catalytic properties of the co-immobilized enzymes, so that the synergistic effect is fully embodied, and the catalytic efficiency is improved. Meanwhile, the reaction time is shortened, the reaction steps are reduced, continuous production is realized, and the product quality can be better controlled.

However, at present, there are few reports on co-immobilized enzymes at home and abroad, and the studies are mainly focused on saccharifying enzymes, and the studies on co-immobilized proteases and lipases are few. The lipase and the protease are two most extensive enzymes in industrial production and application, and have wide development prospect. If the lipase and the protease are co-immobilized on the same carrier and are used as an enzyme reactor, the industrial production can be realized, the process is simplified, and the cost is reduced.

The activity of the method is improved by 25 percent compared with single enzyme immobilization by using chitosan as a carrier and glutaraldehyde as a cross-linking agent to co-immobilize papain and trypsin on the carrier, and the method is widely applied to the production of turbidity resistance, clarification and the like of wines.

The research of Liu Yuan Qin discusses the influence of different proteases on the lipase activity, and the result shows that the trypsin has the effect of improving the activity of the lipase Palatase 20000L. Meanwhile, by researching a co-immobilization method, the protease and the lipase are successfully co-immobilized on the macroporous resin, so that the activity of the protease can be effectively improved, and the loss of the activity of the lipase is less.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a preparation method of a co-immobilized enzyme, and also provides the co-immobilized enzyme prepared by the method.

In order to realize the purpose, the technical scheme is as follows: a preparation method of a co-immobilized enzyme, comprising the following steps:

(1) adding lipase A6, lipase MER and protease MSD into sodium alginate solution, and stirring to obtain a blended solution;

(2) dripping the blending liquid obtained in the step (1) into CaCl₂And standing and curing the mixture in the aqueous solution to form gel particles, wherein the prepared gel particles are the co-immobilized enzyme.

Preferably, the total weight of the lipase A6, the lipase MER and the protease MSD in the step (1) is 2.2-3.52% of the weight of the sodium alginate, and the weight ratio of the lipase A6, the lipase MER and the protease MSD is lipase A6: lipase MER: protease MSD ═ 2:1: 0.3.

preferably, the total weight of the lipase A6, the lipase MER and the protease MSD in the step (1) is 3.0 percent of the weight of the sodium alginate, and the weight ratio of the lipase A6, the lipase MER and the protease MSD is lipase A6: lipase MER: protease MSD ═ 2:1: 0.3.

preferably, the sodium alginate solution in the step (1) is a sodium alginate solution with pH of 6.0-8.0 and mass fraction of 1.0% -2.0%, and CaCl in the step (2)₂The water solution is CaCl with the mass fraction of 3.0-6.0%₂An aqueous solution.

Preferably, the sodium alginate solution in the step (1) is a sodium alginate solution with pH of 6.5 and mass fraction of 1.5%; CaCl in the step (2)₂The aqueous solution is CaCl with the mass fraction of 4.0 percent₂An aqueous solution. The sodium alginate solution is prepared by the following method: adding the sodium alginate solid into a phosphoric acid buffer solution with pH of 6.5, heating and dissolving to prepare a sodium alginate solution with the mass fraction of 1.5%.

Preferably, the curing temperature in the step (2) is 20-35 ℃, and the curing time is 20-40 min.

Preferably, the curing temperature in the step (2) is 30 ℃, and the curing time is 30 min.

The invention provides a co-immobilized enzyme prepared by the method.

The invention provides application of the co-immobilized enzyme in preparing a milk-flavor base material.

The invention has the beneficial effects that: the invention provides a preparation method of co-immobilized enzyme, and the co-immobilized enzyme prepared by the method has high immobilization rate and good enzyme activity operation stability and storage stability.

Drawings

FIG. 1 is a graph of the standard curve of bovine serum albumin in example 1 of the present invention;

FIG. 2 is a line graph showing the effect of pH on co-immobilized enzyme activity and immobilization efficiency in example 1 of the present invention;

FIG. 3 is a line graph showing the effect of sodium alginate concentration on co-immobilization enzyme activity and immobilization efficiency in example 1 of the present invention;

FIG. 4 is a line graph showing the influence of the amount of enzyme on the activity and immobilization efficiency of a co-immobilized enzyme in example 1 of the present invention;

FIG. 5 is a line graph showing the effect of CaCl2 concentration on co-immobilized enzyme activity and immobilization efficiency in example 1 of the present invention;

FIG. 6 is a line graph showing the effect of co-immobilization temperature on co-immobilization enzyme activity and immobilization efficiency in example 1 of the present invention;

FIG. 7 is a line graph showing the effect of co-immobilization time on co-immobilization enzyme activity and immobilization efficiency in example 1 of the present invention;

FIG. 8 is a line graph showing the thermal stability at 20 ℃ of the co-immobilized enzyme in example 1 of the present invention;

FIG. 9 is a line graph showing the thermal stability at 60 ℃ of the co-immobilized enzyme in example 1 of the present invention;

FIG. 10 is a line graph showing the operational stability of the co-immobilized enzyme in example 1 of the present invention;

FIG. 11 is a line graph showing the storage stability of a co-immobilized enzyme in example 1 of the present invention;

FIG. 12 is a line graph showing the influence of the amount of co-immobilized enzyme added on the acid value and sensory score in example 1 of the present invention;

FIG. 13 is a line graph showing the effect of enzymatic hydrolysis time on acid number and sensory score of the co-immobilized enzyme in example 1 of the present invention;

FIG. 14 is a line graph showing the effect of enzymatic hydrolysis temperature of co-immobilized enzymes on acid number and sensory score in example 1 of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

The method takes sodium alginate as a carrier, adopts an embedding method to carry out co-immobilization on protease MSD, lipase A6 and lipase MER, takes immobilization rate and enzyme activity of co-immobilized enzyme as main indexes, optimizes co-immobilization conditions, uses the co-immobilized enzyme obtained after optimization to prepare the milk flavor base material, takes acid value and sensory evaluation as main indexes, and optimizes the preparation process of the milk flavor base material.

1. Materials and methods

1.1 test materials

(1) The butter is commercially available Duomei fresh salt-free butter.

(2) Whey powder (lactose 60%, protein 9%, fat 15%) was purchased from guangzhou city herd ltd.

(3) Lipase A "Tianye" 6(Lipase A "Amano"6), abbreviated Lipase A6, was distributed by Amano wild enzymes preparation commercial (Shanghai) Co., Ltd.

(4) Lipase MER "sky" (Lipase MER "Amano"), abbreviated Lipase MER, was distributed by Amano sky enzyme preparation commerce (shanghai) ltd.

(5) Protease M "tianye" SD (Protease M "Amano" SD), abbreviated Protease MSD, was given by Amano tianase preparation merchandize (shanghai) ltd.

1.2 test reagents

Diethyl ether, absolute ethyl alcohol, KOH, potassium hydrogen phthalate, phenolphthalein indicator and the like are all in analytical purity on the market.

1.3 test main equipment

(1) Main test equipment

TABLE 1 instruments used mainly in the experiments

(2) Other apparatus or devices

Alkaline burette, erlenmeyer flask, graduated flask, beaker, brandreth platform, medicine spoon, glass stick, pipette, weighing paper etc..

1.4 test methods

1.4.1 selection of immobilization Carrier and method of immobilization

1.4.1.1 macroporous resin chitosan film crosslinking method

0.1g of chitosan is weighed and dissolved in 100mL of acetic acid with the mass fraction of 1 percent to prepare 10mg/mL of chitosan-acetic acid solution. Weighing 10g of macroporous resin and 50mL of chitosan-acetic acid solution, fully mixing, carrying out rotary vacuum pumping on the mixture in a water bath at 50 ℃ for 10min under the condition of 1300Pa, and washing the mixture to be neutral by using deionized water. 30mL of 0.125% glutaraldehyde solution was added and left to crosslink at 20 ℃ for 2 hours. Residual glutaraldehyde is removed by washing with deionized water until the absorbance of the wash solution at 280nm is less than 0.01.

Accurately weighing 1g of the pretreated macroporous resin into a conical flask, adding 10mL of deionized water to wet the resin, accurately weighing 10mg of the free enzyme, fully mixing with the resin, placing the mixture in a 35 ℃ constant temperature shaking table at 150r/min for oscillation adsorption for 1h, filtering and retaining the filtrate. The resin was washed with deionized water several times, filtered and stored at 4 ℃.

1.4.1.2 macroporous resin adsorption method

Weighing 10g of resin, placing the resin in a conical flask, adding 95% ethanol into the conical flask, soaking for 24 hours, carrying out suction filtration, and washing with deionized water. The resin was soaked in 25mL of 5% HC1 solution for 4h, filtered, and washed neutral with deionized water. Then, the mixture is soaked in 25mL of 5% NaOH solution for 4 hours, filtered, and washed to be neutral by deionized water. Pumping and filtering, and storing at room temperature for later use.

1.4.1.3 sodium alginate embedding method

Accurately weighing 1g of sodium alginate solid, adding deionized water, heating and dissolving to prepare a 1% sodium alginate solution. After cooling to 45 ℃, adding 10mg of free enzyme, fully and uniformly stirring, and dripping 100mL of CaCl with the mass fraction of 3% by using a No. 5 syringe₂In the solution, gel particles are formed and are kept stand and solidified for 1h at the temperature of 35 ℃. Filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and storing at 4 ℃ after suction filtration.

1.4.1.4 Chitosan embedding method

Accurately weighing 1g of chitosan solid, adding 1% acetic acid solution, stirring and dissolving to prepare 1% chitosan acetic acid solution. After cooling to 45 ℃, adding 10mg of free enzyme, fully and uniformly stirring, dripping into 100mL of NaOH solution with the mass fraction of 1% by using a No. 5 syringe to form gel particles, and standing and solidifying for 1h at 35 ℃. Filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and storing at 4 ℃ after suction filtration.

1.4.2 Effect of Co-immobilization Condition optimization on enzyme Activity

1.4.2.1 Effect of pH on Co-immobilized enzyme Activity

0.50g of sodium alginate solid is respectively weighed and added into 7 conical flasks, phosphoric acid buffer solutions with the pH values of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 are respectively added, and the mixture is heated and dissolved to prepare 1.0 percent sodium alginate solution. After cooling, 0.0080g of lipase A6, 0.0040g of lipase MER and 0.0012g of protease MSD are respectively added, after fully and uniformly stirring, a No. 5 syringe is used for dripping into 50mL of CaCl2 solution with the mass fraction of 3.0% to form gel particles, and standing and curing are carried out for 20min at the temperature of 35 ℃. And filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and performing suction filtration for later use.

Respectively weighing 10g of immobilized enzyme, putting the immobilized enzyme into 7 conical flasks, respectively adding 6g of deionized water and 10g of butter, and putting the flasks in a constant-temperature shaking table (35 ℃, 150r/min) for reaction for 1 h. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme.

1.4.2.2 Effect of sodium alginate concentration on Co-immobilized enzyme Activity

0.25g, 0.50g, 0.75g, 1.0g and 1.25g of sodium alginate solid are respectively weighed, added with phosphoric acid buffer solution with pH of 6.5 and heated to be dissolved to prepare sodium alginate solution with mass fractions of 0.5%, 1.0%, 1.5%, 2.0% and 2.5%. After cooling, 0.0080g of lipase A6, 0.0040g of lipase MER and 0.0012g of protease MSD are respectively added, after fully stirring uniformly, 50mL of CaCl with the mass fraction of 3.0% is dripped into a No. 5 syringe₂In the solution, gel particles are formed, and the mixture is kept stand and solidified for 20min at the temperature of 35 ℃. And filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and performing suction filtration for later use.

Respectively weighing 10g of immobilized enzyme, putting the immobilized enzyme into 5 conical flasks, respectively adding 6g of deionized water and 10g of butter, and placing the mixture in a constant-temperature shaking table (35 ℃, 150r/min) for reaction for 1 h. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme.

1.4.2.3 Effect of enzyme amount on Co-immobilized enzyme Activity

Weighing 0.75g of sodium alginate solid in 6 conical flasks respectively, adding a phosphoric acid buffer solution with pH of 6.5, heating and dissolving to prepare a sodium alginate solution with the mass fraction of 1.5%. Cooling, and respectively adding lipase A6 in amounts of 0.080%, 0.100%, 0.120%, 0.140%, 0.160% and 0.180% of butter; respectively adding lipase MER with enzyme amount of 0.040%, 0.050%, 0.060%, 0.070%, 0.080% and 0.090%; protease MSD was added in amounts of 0.012%, 0.015%, 0.018%, 0.021%, 0.027%, 0.03%, respectively. After fully stirring evenly, use 5The syringe is dropped into 50mL CaCl with the mass fraction of 3.0%₂In the solution, gel particles are formed, and the mixture is kept stand and solidified for 20min at the temperature of 35 ℃. And filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and performing suction filtration for later use.

Respectively weighing 10g of immobilized enzyme, putting the immobilized enzyme into 6 conical flasks, respectively adding 6g of deionized water and 10g of butter, and putting the flasks in a constant-temperature shaking table (35 ℃, 150r/min) for reaction for 1 h. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme.

1.4.2.4CaCl₂Effect of concentration on Co-immobilized enzyme Activity

Weighing 0.75g of sodium alginate solid in 6 conical flasks respectively, adding a phosphoric acid buffer solution with pH of 6.5, heating and dissolving to prepare a sodium alginate solution with the mass fraction of 1.5%. Cooling, adding 0.0140g of lipase A6, 0.0070g of lipase MER and 0.0021g of protease MSD, stirring, and dripping into 50mL of CaCl with mass fraction of 1.0%, 2.0%, 3.0%, 4.0%, 5.0% and 6.0% with No. 5 syringe₂In the solution, gel particles are formed, and the mixture is kept stand and solidified for 20min at the temperature of 35 ℃. And filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and performing suction filtration for later use.

1.4.2.5 Effect of Co-immobilization temperature on Co-immobilization enzyme Activity

Weighing 0.75g of sodium alginate solid in 6 conical flasks respectively, adding a phosphoric acid buffer solution with pH of 6.5, heating and dissolving to prepare a sodium alginate solution with the mass fraction of 1.5%. After cooling, 0.0140g of lipase A6, 0.0070g of lipase MER and 0.0021g of protease MSD were added, respectively, and after stirring well, 50mL of the mixture was dropped into a volume fraction of 4 with a No. 5 syringe in a water bath at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃.0% of CaCl₂Forming gel particles in the solution, standing and solidifying for 20 min. And filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and performing suction filtration for later use.

1.4.2.6 Effect of Co-immobilization time on Co-immobilization enzyme Activity

Weighing 0.75g of sodium alginate solid in 5 conical flasks respectively, adding a phosphoric acid buffer solution with pH of 6.5, heating and dissolving to prepare a sodium alginate solution with the mass fraction of 1.5%. Cooling, adding 0.0140g of lipase A6, 0.0070g of lipase MER and 0.0021g of protease MSD, stirring, dripping into 50mL of CaCl2 solution with mass fraction of 4.0% by No. 5 syringe to form gel particles, standing and solidifying at 30 deg.C for 10min, 20min, 30min, 40min and 50 min. And filtering out gel particles, reserving filtrate, washing the gel particles with deionized water, and performing suction filtration for later use.

1.4.3 stability of Co-immobilized enzymes

1.4.3.1 thermal stability of Co-immobilized enzymes

And putting the co-immobilized enzyme in a constant-temperature water bath box at 20 ℃ for water bath for 0min, 30min, 60min, 90min, 120min and 150min respectively. After water bath, 10g of immobilized enzyme is weighed and placed into a conical flask, then 6g of deionized water and 10g of butter are respectively added, and the mixture is placed into a constant temperature shaking table (35 ℃, 150r/min) for reaction for 2 h. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme.

And putting the co-immobilized enzyme in a constant-temperature water bath box at 60 ℃ for water bath for 0min, 10min, 20min, 30min, 40min, 50min and 60min respectively. After water bath, 10g of immobilized enzyme is weighed and placed into a conical flask, then 6g of deionized water and 10g of butter are respectively added, and the mixture is placed into a constant temperature shaking table (35 ℃, 150r/min) for reaction for 2 h. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme.

1.4.3.2 operational stability of Co-immobilized enzymes

Weighing 10g of immobilized enzyme, putting the immobilized enzyme into a conical flask, adding 6g of deionized water and 10g of butter, and placing the mixture into a constant-temperature shaking table (35 ℃, 150r/min) for reaction for 2 h. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme. Washing the filtered immobilized enzyme with deionized water, continuously repeating the steps after suction filtration, and investigating the change of the enzyme activity of the co-immobilized enzyme along with the use times.

1.4.3.3 storage stability of Co-immobilized enzymes

The co-immobilized enzyme was stored in a refrigerator at 4 ℃ for 0, 2, 4, 6, 8, 10 days. 10g of immobilized enzyme is weighed each time and put into a conical flask, then 6g of deionized water and 10g of butter are added, and the mixture is put into a constant temperature shaking table (35 ℃, 150r/min) for reaction for 2 hours. And (4) filtering out the immobilized enzyme, centrifuging for 15min at 3000r/min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and judging the enzyme activity of the immobilized enzyme.

1.4.4 preparation of milk-flavored base

1.4.4.1 amount of co-immobilized enzyme

Respectively adding 10g of butter as a substrate into 6 conical flasks, adding 0.125g of auxiliary substrate whey powder (12.5% of the mass of the butter) and 6g of water (60% of the mass of the butter), placing the conical flasks in a constant-temperature water bath kettle (75 ℃, 20min), sterilizing, cooling to room temperature, and respectively adding co-immobilized enzymes, wherein the ratio of the addition amount to the butter is 0, 0.5: 1. 1: 1. 1.5: 1. 2: 1. 2.5: 1, placing the mixture in a constant temperature shaking table (35 ℃, 150r/min) for reaction for 2 hours. And (3) filtering out the immobilized enzyme, centrifuging at 3000r/min for 15min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and performing sensory evaluation on the generated fragrance.

1.4.4.2 enzymolysis time of co-immobilized enzyme

10g of butter is respectively added into 6 conical flasks as a substrate, the addition amount of the auxiliary substrate whey powder is 0.125g (12.5 percent of the mass of the butter), the addition amount of water is 6g (60 percent of the mass of the butter), the conical flasks are placed in a constant-temperature water bath kettle (75 ℃ and 20min) for sterilization, after cooling to room temperature, 10g of co-immobilized enzyme is respectively added (the ratio of the addition amount of the co-immobilized enzyme to the mass of the butter is 1: 1), and the conical flasks are placed in a constant-temperature shaking table (35 ℃ and 150r/min) for reaction for 2 h. And (3) filtering out the immobilized enzyme, centrifuging at 3000r/min for 15min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and performing sensory evaluation on the generated fragrance.

1.4.4.3 enzymolysis temperature of co-immobilized enzyme

10g of butter is respectively added into 6 conical flasks as a substrate, the addition amount of the cosubstrate whey powder is 0.125g (12.5 percent of the mass of the butter), the addition amount of water is 6g (60 percent of the mass of the butter), the conical flasks are placed in a constant-temperature water bath kettle (75 ℃ and 20min) for sterilization, then 10g of co-immobilized enzyme is respectively added after cooling to the room temperature (the ratio of the addition amount of the co-immobilized enzyme to the mass of the butter is 1: 1), and the conical flasks are respectively placed in a constant-temperature shaking table with the temperature of 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ for reaction for 2h at the. And (3) filtering out the immobilized enzyme, centrifuging at 3000r/min for 15min, taking the upper oil phase, measuring the acid value of the butter subjected to enzymolysis, and performing sensory evaluation on the generated fragrance.

1.5 acid value

1.5.1 reagents

(1) Neutral ether ethanol mixture: ethyl ether and ethanol were mixed as 1:1, adding 3 drops of phenolphthalein indicator, and titrating the solution with potassium hydroxide until the indicator is neutral.

(2) Phenolphthalein indicator: 10g/L, 10g phenolphthalein was dissolved in 1L of 95% ethanol solution.

(3) Potassium hydroxide ethanol solution (c (koh) ═ 0.050 mol/L): weighing 4g potassium hydroxide in a polyethylene container, adding 5ml water to dissolve, diluting to 1L with 95% ethanol, sealing and standing for 24h [17 ].

1.5.2 calibration of Potassium hydroxide ethanol solution

Accurately weighing 0.375g of potassium hydrogen phthalate which is dried at 105-110 ℃ to constant weight, dissolving the potassium hydrogen phthalate in 50ml of carbon dioxide-free water, adding 2 drops of phenolphthalein indicator, titrating the mixture with potassium hydroxide ethanol solution until the mixture is pink, and simultaneously carrying out a blank test. The concentration of the potassium hydroxide ethanol solution was calculated by the following formula.

In the formula: m-mass of potassium hydrogen phthalate, g;

v1-volume of potassium hydroxide-depleted ethanol solution, mL;

v2-volume of potassium hydroxide ethanol solution consumed in blank test, mL;

204.22 molar mass of Potassium Hydrogen phthalate

1.5.3 determination of the acid value

Accurately weighing 1.0g of milk flavor base material, placing the milk flavor base material into a conical flask, adding 10mL of neutral ether ethanol mixed solution to dissolve an oil sample, dropwise adding 2 drops of phenolphthalein indicator, titrating with a potassium hydroxide ethanol standard solution until reddish, maintaining the color change for 30s, and recording the consumption amount [18] of the consumed potassium hydroxide ethanol standard solution.

The acid value of the milk-flavored base is calculated according to the following formula:

in the formula: x-acid value of milk base, mg/g;

v, titrating the volume of the consumed potassium hydroxide ethanol standard solution, mL;

c-actual concentration of the potassium hydroxide ethanol standard solution, mol/L;

m-milk flavor base material sample mass, g;

56.11 molar mass of potassium hydroxide, g/mol.

1.6 relative enzyme Activity

The highest value of the experimental values in the same set of data was taken as 100%, and the ratio of the values of the other experimental points to this point is the relative activity of the enzyme, expressed as a percentage.

1.7 protein content determination

The content of the enzyme protein in the filtrate during co-immobilization was determined by Coomassie blue staining.

1.7.1 reagents

(1) Coomassie brilliant blue reagent: weighing Coomassie brilliant blue G-2500.1G, dissolving in 50mL ethanol with mass fraction of 95%, adding 85% phosphoric acid 100mL, and diluting to 1L with deionized water.

(2) Standard protein solution: taking bovine serum albumin as standard protein, accurately weighing 10mg of bovine serum albumin, adding water for dissolving, and fixing the volume to 100mL to prepare 0.1mg/mL standard protein solution.

1.7.2 drawing bovine serum albumin standard curve

6 dry cuvettes were numbered and reagents were added to the cuvettes as in Table 3. Adding reagent, shaking, standing for 5min, and measuring absorbance of the rest samples at 595nm with sample No. 1 as blank control. The results of the experiment were plotted as a standard curve with absorbance as the ordinate and standard protein content as the abscissa, as shown in table 2.

TABLE 2 bovine serum albumin standard curve plotting

1.7.3 sample determination

And (3) putting 1mL of the filtrate into a colorimetric tube, adding 5mL of Coomassie brilliant blue reagent, shaking uniformly, standing for 5min, and then measuring the absorbance at 595 nm. The protein content of the filtrate was calculated according to the standard curve.

1.8 immobilization Rate

The ability of the carrier to immobilize the enzyme protein (immobilization rate) can be calculated by the following formula.

Immobilization rate (m-cV)/mx 100%

In the formula: m-adding the initial enzyme amount of the immobilization system, mg;

c-the content of the enzyme protein in the immobilized filtrate is mg/mL;

v-volume of filtrate after immobilization, mL.

1.9 sensory evaluation

Selecting a plurality of individuals to form a sensory evaluation group, and respectively scoring the aroma intensity, the aroma purity and the aroma retention condition of the enzymolysis products according to the table 3, wherein the total score is 10, and the scoring result is the sum of 3 persons. The scoring criteria are shown in table 3.

TABLE 3 Scent criteria for fragrance

2. Results and analysis

2.1 bovine serum albumin standard curve

The content of the enzyme protein in the filtrate during co-immobilization was determined by Coomassie blue staining. Under appropriate conditions, Coomassie brilliant blue G-250 can generate blue compounds with protein, the absorption value is maximum at 595nm, and the protein concentration is proportional to the shade of blue of the compounds. The Coomassie brilliant blue staining method can stably and rapidly measure the content of protein in the enzyme solution, and a bovine serum albumin standard curve is shown in figure 1.

2.2 selection of immobilization Carrier and immobilization method

The immobilized carrier influences the stability and enzyme activity of the immobilized enzyme, and the selection of the proper immobilized carrier is beneficial to improving various performances of the immobilized enzyme. The properties of the immobilized enzyme depend on the immobilization method in addition to the support used for immobilization. However, since different kinds of enzymes have significant differences in properties such as isoelectric point, average diameter, and hydrophilicity and hydrophobicity, almost none of the immobilization methods is suitable for any enzyme, and thus an appropriate immobilization method is selected according to the characteristics of a specific enzyme.

In the experiment, four carriers were selected, and an adsorption method, a cross-linking method and an embedding method were used to perform immobilization tests on lipase a6, lipase MER and protease MSD, respectively, to determine the effect of different carriers and immobilization methods on immobilization rates, and the results are shown in table 4.

TABLE 4 Effect of different carriers and immobilization methods on immobilization (%)

As can be seen from Table 4, the effect of immobilizing three free enzymes by embedding with chitosan as carrier is not ideal. For lipase A6, the immobilization method by direct embedding with sodium alginate has the best effect, and the immobilization rate is 97.8%. For lipase MER, the cross-linking method of loading chitosan membrane with macroporous resin X-5 as carrier has good immobilization effect and direct embedding effect with sodium alginate, and the immobilization rate is 99.1% and 98.2%, respectively. And the protease MSD has the highest immobilization rate by directly embedding with sodium alginate as a carrier. In conclusion, the immobilization effect of the three enzymes directly embedded by taking sodium alginate as a carrier is ideal, the immobilization rate is more than 97%, and the method of directly embedding by taking sodium alginate as a carrier can be considered to perform co-immobilization on the three enzymes.

2.3 Effect of optimization of Co-immobilization conditions on enzyme Activity

2.3.1 Effect of pH on Co-immobilized enzyme Activity

In the enzyme immobilization reaction, because the immobilized carrier and the enzyme are both present in the buffer solution, and the pH of the buffer solution can change the ionization state of the immobilized carrier and the enzyme molecules; in addition, the enzyme is a protein, and when the pH value of the system is out of the range, the microstructure of the enzyme is changed, so that the enzyme is denatured and inactivated. Therefore, the pH value of the buffer solution is one of the important factors influencing the enzyme activity and the immobilization rate of the immobilized enzyme.

As can be seen from FIG. 2, at pH6.5 or less, the immobilization rate tends to increase as the pH of the immobilization system increases. When the pH value is 6.5, the immobilization rate is 98 percent, and the highest immobilization rate is achieved; at this time, the acid value is 1.16mg/g, and the enzyme activity of the co-immobilized enzyme is also highest. Probably, when other conditions are consistent, the higher the immobilization rate of the immobilized enzyme, the higher the enzyme content in the immobilized enzyme per unit mass, and the larger the acid value of the enzymolysis product. When the pH value is more than 6.5, the immobilization rate is about 97.5-98% along with the increase of the pH value of the immobilization system, the change is not large, and the change of the enzyme activity of the co-immobilized enzyme is relatively smooth. Therefore, the optimum pH for co-immobilization was 6.5.

2.3.2 Effect of sodium alginate concentration on Co-immobilized enzyme Activity

The Tang Ming dynasty researches that sodium alginate not only influences the viscosity of a solution, the roundness and the mechanical strength of colloidal particles, but also influences the activity of immobilized enzyme. Sodium alginate has strong adsorbability, and can form gel with positive divalent cations such as copper and calcium. When the concentration of sodium alginate is too large, the pore size of the gel is small, and the combination of the substrate and the enzyme is influenced. When the concentration of the sodium alginate is too small, the pore diameter of the gel is larger, the immobilized enzyme is easy to lose, and the enzyme activity is lower. Therefore, the concentration of sodium alginate during the co-immobilization should be an optimal value.

As shown in fig. 3, when the immobilized enzyme was prepared, it was clearly seen that when the sodium alginate concentration was less than 0.5%, the gel structure formed was unstable and no gel particles could be formed. When the concentration of the sodium alginate is 0.5%, irregular colloidal particles are finally formed, the particles are soft, the mechanical strength is poor, the prepared sodium alginate microspheres are easy to break the walls, and most of free enzymes cannot be fixed. When the concentration of the sodium alginate is 1.0-1.5%, the immobilization rate of the immobilized enzyme is high, the prepared colloidal particles have good mechanical properties, the measured acid value is high, and the activity of the immobilized enzyme is high. It is possible that the immobilization rate is increased by appropriately increasing the concentration of the carrier sodium alginate so that the ability to carry the enzyme protein molecules is increased. The immobilization rate and the acid value are reduced on the contrary by continuously increasing the concentration of the sodium alginate. The reason may be that when the concentration of sodium alginate is high, the enzyme immobilized on a unit mass of sodium alginate decreases, and the immobilization rate decreases; the thin layer forming the colloidal particles of the immobilized enzyme is too thick, and the mass transfer resistance between the embedded enzyme and the substrate is increased, so that the activity of the immobilized enzyme is reduced. When the concentration of the sodium alginate is more than 2.5%, the sodium alginate is solidified quickly and is difficult to operate, and the sodium alginate solution is difficult to extrude from a syringe. Therefore, sodium alginate with the mass fraction of 1.5 percent is preferably selected for co-immobilization.

2.3.3 Effect of enzyme amount on Co-immobilized enzyme Activity

When the immobilized enzyme is carried out, the optimal immobilization effect can be achieved when the carrier content is appropriate to the enzyme supply amount, otherwise, when the carrier content is relatively too low, part of free enzyme cannot be combined with the carrier, unnecessary waste is caused, when the carrier content is relatively too high, the carrier cannot fully exert the enzyme protein combining capacity, and the immobilization cost is also improved to a certain extent. Therefore, the optimum enzyme supply amount is selected to improve the efficiency of the immobilized enzyme.

As shown in fig. 4, when the concentration of sodium alginate was constant, the immobilization rate increased with the increase in the amount of enzyme supplied, and the enzyme activity of the immobilized enzyme tended to increase first and then decrease. When the enzyme dosage is less than 27mg/g sodium alginate, the increase trend of the immobilization rate is obvious, which shows that when the enzyme dosage is smaller, the immobilization of the unit carrier does not saturate, so that the immobilization rate increases with the increase of the enzyme dosage. When the enzyme amount is more than 27mg/g sodium alginate, the immobilization rate curve becomes flat, which indicates that the immobilization is close to saturation. When the enzyme dosage is 30mg/g sodium alginate, the enzyme activity of the immobilized enzyme reaches the maximum value, and the acid value is measured to be 1.73 mg/g. This is because, in the case where the amount of the carrier is not changed, when the amount of the enzyme to be administered is relatively small, the amount of the enzyme protein entrapped in the carrier is also small, so that the carrier is not sufficiently utilized, and thus the enzyme activity is also low. However, when the enzyme is administered in a relatively large amount, the amount of the enzyme embedded in the carrier is also large, so that the enzyme proteins are aggregated into a cluster, the active center of the enzyme molecule may be masked, and the binding of the enzyme to the substrate is affected, and the enzyme activity is low although the amount of the enzyme embedded in sodium alginate is large. Therefore, the optimum enzyme dosage for co-immobilization is 30mg/g sodium alginate.

2.3.4CaCl₂Effect of concentration on Co-immobilized enzyme Activity

Ca²⁺The formation of calcium alginate gel with sodium alginate is an important process for immobilizing enzymes. CaCl₂The mechanical strength of the immobilized enzyme as a coagulant is determined, and the degree of "firmness" of immobilization of the immobilized enzyme is also determined.

The results are shown in FIG. 5, where the immobilization rate and enzyme activity of the co-immobilized enzyme are determined by CaCl₂Increase in concentration when CaCl is increased₂When the concentration reaches 4.0 percent, the immobilization rate and the enzyme activity of the co-immobilized enzyme reachTo the highest, and most of the gel particles are in a regular spherical shape. CaCl₂When the concentration is continuously increased, the immobilization rate is not changed greatly, and the enzyme activity of the co-immobilized enzyme is along with CaCl₂The concentration increases and decreases. The possible reason is when CaCl is present₂The concentration is too low, the embedding is incomplete during co-immobilization, partial enzyme cannot be immobilized in gel particles, and the immobilization rate is low; the gel particles obtained by immobilization are softer, the mechanical strength is weak, more enzyme activity is lost during washing, and the enzyme activity of the immobilized enzyme is lower. When CaCl₂When the concentration is too high, a compact calcium alginate layer is formed on the surface of the gel particles, which is not beneficial to the diffusion of a substrate and reduces the enzyme activity of the immobilized enzyme. Therefore, co-immobilized optimum CaCl₂The concentration was 4.0%.

2.3.5 Effect of temperature on Co-immobilized enzyme Activity

The proper temperature can increase the kinetic energy of enzyme molecules and improve the contact rate between the enzyme and the carrier, thereby increasing the immobilization efficiency. However, as a protein, the enzyme is easily denatured and inactivated by high temperature.

As can be seen from FIG. 6, the temperature of immobilization did not greatly affect the enzyme activity of the co-immobilized enzyme at a temperature not exceeding 30 ℃. When the temperature exceeds 30 ℃, the enzyme activity of the immobilized enzyme is in a descending trend. The immobilization rate is in an increasing trend within the range of 20-35 ℃. The immobilization rate reaches the highest at 35 ℃, and the immobilization rate is 99.2%. As the temperature continues to increase, the immobilization rate decreases. The reason is probably that when the immobilization temperature is lower, the sodium alginate can be solidified at a low temperature, so that the operation difficulty is increased, and the immobilization rate is lower; because the temperature is lower, the temperature has little influence on the enzyme activity of the immobilized enzyme. Therefore, the optimum temperature for co-immobilization was 30 ℃.

2.3.6 Effect of time on Co-immobilized enzyme Activity

The immobilization time refers to that the mixed liquid of the enzyme and the sodium alginate drops on CaCl₂And standing for reaction time.

As is clear from FIG. 7, the influence of the immobilization time on the immobilization rate was insignificant, and the immobilization rate was always maintained at about 98% with the increase in immobilization time, and the immobilization rate was high. While immobilizing the enzymeThe enzyme activity is in a trend of increasing first and then decreasing along with the increase of the immobilization time. This is because sodium alginate is bound to CaCl₂The process of reacting and forming the gel particles is time consuming. At the initial stage of co-immobilization, embedding is gradually compact, loss is reduced, and the enzyme activity of the immobilized enzyme is in an increasing trend; after 30min, the enzyme activity of the immobilized enzyme reaches the maximum, and the acid value is measured to be 1.69 mgKOH/g; however, the time increases continuously to cause Ca²⁺And the gel is too tight, thus affecting the combination of the substrate and the enzyme activity of the co-immobilized enzyme. Therefore, the optimum immobilization time was selected to be 30 min.

2.4 stability of the Co-immobilized enzymes

2.4.1 thermal stability of the Co-immobilized enzymes

The influence of temperature on enzymes is complex, temperature affecting the conformation of the enzyme protein molecules, the dissociation state of the functional groups involved in the enzymatic reaction, and also the affinity of the substrate for the enzyme.

As can be seen from FIG. 8, the co-immobilized enzyme still retains 81.4% of enzyme activity after being subjected to water bath at 20 ℃ for 150min, and has good thermal stability. Probably because the vector provides an environment that protects the enzyme.

As can be seen from FIG. 9, the enzyme activity of the co-immobilized enzyme was greatly reduced in a water bath at 60 ℃. When the time is 0-20min, the enzyme activity is reduced rapidly; after 20min, the enzyme activity is not changed greatly, and the relative enzyme activity is kept at about 70-75%. The reason that the enzyme activity of the co-immobilized enzyme is rapidly reduced at 60 ℃ is probably because the enzyme is easily inactivated at high temperature and the calcium alginate on the outer layer of the co-immobilized enzyme is softened due to the increase of the temperature, so that the enzyme embedded in the calcium alginate leaks out, and the enzyme activity is rapidly reduced.

2.4.2 operating stability of the Co-immobilized enzymes

The operational stability of the immobilized enzyme is an important characteristic different from that of the free enzyme, and is an important index reflecting the immobilization effect. The reuse of the immobilized enzyme is not only beneficial to the recovery and purification of the product after enzymolysis, but also beneficial to saving the cost.

As can be seen from FIG. 10, the relative enzyme activity decreases with the increase of the number of times of use, probably because the water-holding capacity of the sodium alginate carrier changes due to continuous stirring, the gel structure of the co-immobilized enzyme changes, and the enzyme falls off from the sodium alginate carrier and is continuously hydrolyzed by contacting with the substrate. After the co-immobilized enzyme is used for 4 times, the enzyme activity is still kept at 92.4 percent of the original enzyme activity, and the stability is better. Meanwhile, the fragrance of the milk-flavor base prepared after the co-immobilized enzyme is used for 4 times is not greatly different from that of the first milk-flavor base in sense.

2.4.3 storage stability of Co-immobilized enzymes

As can be seen from FIG. 11, the enzyme activity was maintained at 91.8% or more when the co-immobilized enzyme was stored for 0 to 4 days. After the enzyme is combined with the sodium alginate carrier, the existence of the carrier prevents the interaction between enzyme and protein, inhibits the denaturation and degradation of the enzyme and protein, and ensures that the co-immobilized enzyme has certain storage stability. After the co-immobilized enzyme is stored for 4 days, the enzyme activity of the co-immobilized enzyme is rapidly reduced, and the enzyme activity of the co-immobilized enzyme is 66.8% at the 10 th day. The reason for the reduction of the enzyme activity is probably that the protease in the sodium alginate gel hydrolyzes the lipase and influences the enzyme activity of the lipase.

The experimental results show that the optimal conditions for co-immobilization are as follows: the pH of the phosphate buffer was 6.5, the sodium alginate concentration was 1.5%, and the total enzyme addition was 30mg/g sodium alginate (MSD: A6: MER ═ 0.3: 2: 1), CaCl₂The concentration is 4.0%, the temperature of co-immobilization is 30 ℃, and the time of co-immobilization is 30 min. At the moment, the immobilization rate of the co-immobilized enzyme is 98.0%, the enzyme activity is still kept at 92.4% after the co-immobilized enzyme is used for the 4 th time, the enzyme activity is still kept at 66.8% after the co-immobilized enzyme is stored for 10 days, and the stability is good.

2.5 preparation of milk-flavored base

2.5.1 Co-immobilized enzyme addition amount

When the milk-flavor base material is generated by enzyme catalysis, the addition amount of the enzyme has great influence on the flavor of the milk-flavor base material. When the addition amount of the enzyme is small, the amount of the generated flavor substance is small, and the milk flavor is light; when the amount of the enzyme is too large, although a large amount of flavor substances are generated, some unpleasant odor is generated, and the production cost is also increased. Therefore, the amount of the co-immobilized enzyme to be added should be selected appropriately.

TABLE 5 sensory impact of Co-immobilized enzyme addition on milk flavor base

As can be seen from fig. 12 and table 5, when the amount of co-immobilized enzyme added is less than the mass of butter, the more excellent the flavor-producing effect and the stronger the flavor as the co-immobilized enzyme increases. When the co-immobilized enzyme is equal to the mass of the butter, the obtained milk-flavor base material is sweet and fragrant milk flavor, has strong fragrance, is mellow and smooth and has long aftertaste, and the acid value is measured to be 1.86 mg/g. When the addition amount of the co-immobilized enzyme is larger than the mass of the butter, the sour odor begins to be generated, and the sour value of the milk fragrance base material is increased rapidly and the sour odor is more and more serious along with the increase of the addition amount of the enzyme. Considering comprehensively, the most suitable co-immobilized enzyme is added in an amount equal to the quality of the butter.

2.5.2 enzymatic hydrolysis time of Co-immobilized enzymes

It is also important to select an appropriate time for the enzymatic hydrolysis in order to obtain the best results and to improve the production efficiency. Theoretically, the longer the enzymatic hydrolysis time, the more complete the enzymatic reaction. In fact, too long an enzymatic hydrolysis time can generate more undesirable flavor substances, affecting the overall flavor of the base. However, when the time for the enzymatic hydrolysis is too short, the enzymatic reaction is incomplete, the generation of flavor substances is too little, and the flavor is not sufficiently developed. The time of the enzymolysis reaction should be controlled well for the milk-flavor base material to achieve the ideal flavor.

TABLE 6 organoleptic impact of enzymatic hydrolysis time on milk flavor base

As can be seen from fig. 13 and table 6, the acid value of the enzymatic hydrolysis product increases with the increase of the enzymatic hydrolysis time. When the enzymolysis time is 2 hours, the fragrance production effect is best, the obtained milk fragrance base material has strong fragrance, is mellow and smooth, is coordinated with the milk sweet taste, and has long fragrance retention time, and the acid value is 1.70 mg/g. When the enzymolysis time exceeds 2 hours, the acid value continues to rise, rancid taste begins to appear, sensory score is reduced, and the generation of bad flavor substances in the enzymolysis products is probably caused by the excessively high enzymolysis degree of the butter. Comprehensively considering, the optimum time for enzymolysis is selected to be 2 h.

2.5.3 temperature of enzymatic hydrolysis of Co-immobilized enzymes

The enzyme is sensitive to temperature, and the difference of the temperature can affect the molecular conformation distribution of the enzyme activity center and the emulsification state of the substrate butter, thereby affecting the whole enzymolysis reaction process. When the enzymolysis temperature is lower than the optimal temperature, the temperature can inhibit the activity of the enzyme, so that the activity of the enzyme is lower, and simultaneously, the viscosity of a substrate can be influenced, thereby influencing the mass transfer speed and slowing down the speed of the enzymolysis reaction. When the enzymolysis temperature is higher than the optimum temperature, the enzyme denaturation is caused by high temperature to weaken or even lose the catalytic activity, and the calcium alginate on the outer layer of the co-immobilized enzyme is softened at higher temperature, so that the enzyme embedded in the co-immobilized enzyme leaks out, and the enzyme activity is reduced.

TABLE 7 organoleptic impact of enzymatic hydrolysis temperature on milk savory base

As can be seen from fig. 14 and table 7, when the enzymolysis temperature is lower than 40 ℃, the aroma of the milk-flavor base material is stronger and the aroma purity is more harmonious and richer as the temperature is increased, probably because the temperature is increased to promote the enzymolysis reaction, and the flavor products are increased. When the temperature reaches 40 ℃, the sensory score of the obtained milk flavor base material is the highest, the milk flavor base material is the milk flavor of partial cheese, the milk flavor is strong, the flavor is pure and mellow, the flavor retention time is long, and the measured acid value is 1.58 mg/g. When the enzymolysis temperature reaches 45 ℃, the acid value of the enzymolysis product is the highest, the acid value is 3.58mg/g, the sensory score is reduced, and probably because the enzymolysis excessively generates poor flavor substances. When the enzymolysis temperature exceeds 45 ℃, the enzyme denaturation is possibly caused by the enzymolysis temperature being higher than the optimal temperature, so that the catalytic activity is weakened or even lost, or the calcium alginate on the outer layer of the co-immobilized enzyme is softened due to the temperature rise, so that the enzyme embedded in the co-immobilized enzyme is leaked out, the enzyme activity begins to be reduced, and the acid value and the sensory score are reduced. In summary, the suitable temperature for enzymatic hydrolysis is 40 ℃.

2.6 comparison of free enzyme with Co-immobilized enzyme for preparing milk-flavored base stocks

Although the immobilized enzyme technology solves the problems of high production cost of free enzyme, the micro-environment of enzyme molecules changes when the enzyme molecules are changed from a free state to a firm state and are bonded to a carrier. Therefore, there are some differences in the process conditions and sensory scores between the milk flavor bases prepared with free enzyme and co-immobilized enzyme.

TABLE 8 comparison of free enzyme with Co-immobilized enzyme for making milk flavor base

As can be seen from Table 8, although the use of free enzyme and sodium alginate in the co-immobilization process and the co-immobilization process generate production costs, the immobilized enzyme can be reused and the enzymolysis process is simpler, so that the milk flavor base material prepared by using the immobilized enzyme can achieve the purpose of saving the production costs. The milk-flavor base materials prepared by the free enzyme and the sodium alginate are cheese-flavor milk-flavor, the fragrance is pure and mellow, the fragrance retaining condition is better, the sensory scores are similar, but the fragrance of the milk-flavor base materials prepared by the free enzyme is slightly stronger than that of the milk-flavor base materials prepared by the immobilized enzyme. Probably, the sodium alginate gel particles prevent the contact of enzyme and substrate to a certain extent, so that some flavor development substances are generated less or cannot be generated, and the fragrance intensity is slightly weak. And probably because the sodium alginate gel particles prevent the diffusion of certain flavor development substances, so that the prepared milk-flavor base has weaker fragrance.

The experimental result shows that the optimal process conditions for preparing the milk-flavor base material are as follows: the butter is used as a substrate, the added water amount is 60.0 percent of the weight of the butter, the whey powder is used as an auxiliary substrate, the added amount of the auxiliary substrate is 12.5 percent of the weight of the butter, the mass ratio of the added amount of the co-immobilized enzyme to the substrate butter is 1:1, the enzymolysis time is 2 hours, and the enzymolysis temperature is 40 ℃. The milk-flavor base material obtained by the invention has the advantages of thick milk flavor, pure milk flavor and good joyfulness, and can be used for making biscuits.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of co-immobilized enzyme is characterized by comprising the following steps:

(1) adding lipase A6, lipase MER and protease MSD into a sodium alginate solution with the pH of 6.0-8.0 and the mass fraction of 1.0-2.0%, and uniformly stirring to obtain a blended solution, wherein the total weight of the lipase A6, the lipase MER and the protease MSD is 2.2-3.52% of the weight of the sodium alginate, and the weight ratio of the lipase A6 to the protease MER to the protease MSD is lipase A6: lipase MER: protease MSD ═ 2:1: 0.3;

(2) dripping the blended liquid obtained in the step (1) into CaCl with the mass fraction of 3.0-6.0%₂And standing and curing the mixture in an aqueous solution at the temperature of between 20 and 35 ℃ for 20 to 40min to form gel particles, wherein the prepared gel particles are the co-immobilized enzyme.

2. The method of claim 1, wherein the total weight of lipase A6, lipase MER and protease MSD in step (1) is 3.0% of the weight of sodium alginate.

3. The process according to claim 1, wherein the sodium alginate solution in the step (1) has a pH of 6.5 and a mass fraction of 1.5%The sodium alginate solution of (4); CaCl in the step (2)₂The aqueous solution is CaCl with the mass fraction of 4.0 percent₂An aqueous solution.

4. The production method according to claim 1, wherein the curing temperature in the step (2) is 30 ℃ and the curing time is 30 min.

5. A co-immobilized enzyme prepared by the method of any one of claims 1-4.

6. Use of a co-immobilized enzyme according to claim 5 in the preparation of a milk-based base.