High-heat-resistance glucose oxidase emulsion and preparation method thereof

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a high-heat-resistance glucose oxidase emulsion and a preparation method thereof.The high-heat-resistance glucose oxidase emulsion is prepared from glucose oxidase serving as a core material, olive oil serving as an oil phase, gelatin and sodium carboxymethyl cellulose serving as outer water phase wall materials, Span 80 serving as a lipophilic emulsifier, Tween 80 serving as a hydrophilic emulsifier and a water phase emulsifier. The preparation method comprises the following steps: dissolving, adding a lipophilic emulsifier, shearing, adding a hydrophilic emulsifier, mixing, secondarily shearing, adjusting the pH value and cooling to obtain the high-heat-resistance glucose oxidase emulsion. According to the method disclosed by the invention, by combining favorable conditions of local enterprises and through double emulsion embedding treatment, not only can the enzyme activity of the glucose oxidase be effectively maintained, but also the heat resistance of the glucose oxidase can be improved, and the production requirements are met.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and particularly to a highly heat-resistant glucose oxidase emulsion and a preparation method thereof. Background Art

[0002] Glucose oxidase is widely used in the fields of biomedicine, food industry and feed industry. Its availability in different fields mainly depends on its activity, thermal stability, acid and alkali resistance, etc. Glucose oxidase is often used as a new enzyme preparation or feed additive to improve the quality of target products, and has the characteristics of being green, safe, pollution-free, high catalytic efficiency, etc. For example: replacing antibiotics added to feed to improve the meat quality of livestock products such as broilers, maintaining the health of their intestines, improving the immunity of animal bodies, reducing the incidence of various diseases, and improving feed utilization rate. In addition, glucose oxidase, as a green and harmless enzyme preparation, is mostly used in the baking industry to improve the dough and bread-making performance and act as a substitute for potassium bromate. Although glucose oxidase is widely used in the fields of food, feed, etc., it still has certain limitations. As an enzyme, although glucose oxidase has the characteristics of trace high efficiency and specificity, its stability is low and it is easily restricted by environmental conditions. Too high or too low environmental temperature and pH limit its function. Double emulsion encapsulation is to encapsulate glucose oxidase as the core material into the oil phase and the outer aqueous phase, adding two barriers, which can retain the enzyme activity of glucose oxidase to a large extent and can also improve the heat resistance of glucose oxidase.

[0003] Double emulsion is a hollow core-porous structure composed of an inner aqueous phase dispersed in an oil phase. This structure can significantly improve the physical and chemical stability of the core material and has been widely used in the food industry. Foreign scholars have successfully encapsulated grape seed polyphenol-rich extract into double emulsion, improving its storage stability and emulsion stability. At the same time, some domestic scholars have encapsulated salidroside in double emulsion, significantly improving its stability. In addition, double emulsion encapsulation of probiotics can avoid the influence of the gastrointestinal barrier and improve its survival rate. However, the influence of double emulsion encapsulation on the physicochemical stability of glucose oxidase has not been studied yet. Summary of the Invention

[0004] The present invention discloses a preparation method of a highly heat-resistant glucose oxidase emulsion, belonging to the technical field of food processing. In this method, glucose oxidase is used as the core material, olive oil as the oil phase, gelatin and sodium carboxymethylcellulose as the wall material, Span 80 as the lipophilic emulsifier, and Tween 80 as the hydrophilic emulsifier. Through dissolution, addition of the lipophilic emulsifier, shearing, addition of the hydrophilic emulsifier, mixing, secondary shearing, pH adjustment, and cooling, the highly heat-resistant glucose oxidase emulsion is obtained. Combining the favorable conditions of local enterprises, through double emulsion embedding treatment, the present invention can not only effectively maintain the enzyme activity of glucose oxidase, but also improve the heat resistance of glucose oxidase, meeting the production requirements.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention discloses a highly heat-resistant glucose oxidase emulsion, comprising the following components:

[0007]

[0008]

[0009] Preferably, the volume fraction of the glucose oxidase is 3% (w / v), the total concentration of the outer aqueous phase wall material solution is 4% (w / v), and the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5.

[0010] Preferably, the pH is 5.5.

[0011] Furthermore, the present invention provides a preparation method of a highly heat-resistant glucose oxidase emulsion, comprising the following steps:

[0012] Using glucose oxidase as the core material, olive oil as the oil phase, gelatin and sodium carboxymethylcellulose as the outer aqueous phase wall material, Span80 as the lipophilic emulsifier, and Tween 80 as the hydrophilic emulsifier, through dissolution, addition of the lipophilic emulsifier, shearing, addition of the hydrophilic emulsifier, mixing, secondary shearing, pH adjustment, and cooling, the highly heat-resistant glucose oxidase emulsion is obtained.

[0013] Furthermore, for the preparation method of the highly heat-resistant glucose oxidase emulsion, it is characterized in that the conditions for the dissolution are: temperature 25°C, the volume fraction of the glucose oxidase is 3% (w / v), the total concentration of the outer aqueous phase wall material solution is 4% (w / v), and the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5.

[0014] Since glucose oxidase is easily inactivated by environmental factors, strict preservation methods are required during its use. Retaining a high activity of commercial glucose oxidase is an important prerequisite for preparing double emulsions of heat-resistant glucose oxidase. Temperature, mass fraction, solution concentration, and the mass ratio of wall material will all affect the embedding effect of double emulsions.

[0015] By controlling conditions such as temperature and volume fraction, the influence of the environment on the enzyme activity of glucose oxidase can be reduced, and the stability of double emulsions can be better guaranteed.

[0016] Furthermore, the method for preparing the high heat-resistant glucose oxidase emulsion is characterized in that the condition for adding the lipophilic emulsifier is: adding Span 80 with a volume fraction of 10% (w / w) to the oil phase.

[0017] Furthermore, the method for preparing the high heat-resistant glucose oxidase emulsion is characterized in that the shearing condition is: adding the inner aqueous phase solution to the oil phase at a ratio of 2:1 (v / v), with a shearing rate of 10000 rpm and a shearing time of 5 min to obtain a primary emulsion.

[0018] The shearing rate and shearing time can affect the stability of double emulsions. If the shearing rate is too low, the glucose oxidase solution cannot be completely dispersed in the double structure, resulting in a decrease in stability; if the shearing rate is too high, the viscosity of the emulsion gradually decreases, and the droplets are too small and tend to coalesce into large droplets, resulting in a decrease in stability.

[0019] Furthermore, the method for preparing the high heat-resistant glucose oxidase emulsion is characterized in that the condition for adding the hydrophilic emulsifier is: adding Tween 80 with a volume fraction of 2% (w / w) to the outer aqueous phase.

[0020] Furthermore, the method for preparing the high heat-resistant glucose oxidase emulsion is characterized in that the mixing includes: pouring the primary emulsion described in claim 4 into the outer aqueous phase solution at a ratio of 1:1 (v / v).

[0021] Furthermore, the method for preparing the high heat-resistant glucose oxidase emulsion is characterized in that the condition for the secondary shearing is: a shearing rate of 8000 rpm and a shearing time of 3 min.

[0022] The second shearing rate and shearing time should be lower than the first shearing because if the second shearing rate is too high and the time is too long, the glucose oxidase that has been encapsulated by the oil phase will be destroyed and released back into the solution, resulting in a decrease in the stability of the emulsion.

[0023] Further, the preparation method of the highly heat-resistant glucose oxidase emulsion is characterized in that the pH adjustment includes: adjusting the pH value to 5.5 with 1 mol / L HCl and NaOH.

[0024] Adjusting the pH is an important step to promote the electrostatic binding of the wall material gelatin and sodium carboxymethylcellulose in the outer aqueous phase. When the pH value is 5.5, gelatin is positively charged and sodium carboxymethylcellulose is negatively charged, which promotes the electrostatic binding between the two, forming a network structure on the outside of the emulsion and improving the stability of the glucose oxidase double emulsion.

[0025] Further, the preparation method of the highly heat-resistant glucose oxidase emulsion is characterized in that the cooling includes: cooling the prepared glucose oxidase double emulsion for 20 min under the cooling condition of 4°C.

[0026] The cooling time is an important factor affecting the stability of the double emulsion. If the cooling time is short, the biological macromolecules in the emulsion cannot effectively aggregate, resulting in reduced stability; if the cooling time is long, the small droplets in the emulsion stick to each other, causing the outer wall to rupture and reducing the stability.

[0027] The present invention also provides a preparation method of the above-mentioned highly heat-resistant glucose oxidase emulsion.

[0028] The present invention also provides a product processed from the above-mentioned highly heat-resistant glucose oxidase emulsion, which is characterized in that the product can effectively maintain the enzyme activity of glucose oxidase, and can also improve the heat resistance of glucose oxidase to meet the production requirements.

[0029] Glucose oxidase has obvious advantages in industries such as food, feed, and biomedicine. However, its disadvantages such as low stability and easy inactivation limit its further application in the industry. Therefore, by combining the favorable conditions of local enterprises, highly active glucose oxidase is selected. After being processed by the present invention and made into an emulsion through double emulsion embedding treatment of glucose oxidase, it can meet the perennial needs of enterprises.

[0030] The present invention discloses the following technical effects:

[0031] In view of the lack of production process standards for highly heat-resistant glucose oxidase and the quality standard system for related products in enterprises, and the lack of comprehensive mastery of the production technology of highly heat-resistant glucose oxidase, resulting in unstable product quality, which seriously affects the product quality. By the method of double emulsion embedding glucose oxidase, it aims to improve the heat resistance and stability of glucose oxidase. The double emulsion of glucose oxidase prepared by the present invention has a lower cumulative release efficiency compared with the double emulsion of glucoamylase. Under the condition of the same enzyme amount, the action time of glucose oxidase is longer and the sustained release effect is better. In addition, the retained enzyme activity rate and enzyme loading amount are both higher than those of the double emulsion of glucoamylase, which is beneficial to improving the use efficiency of the double emulsion and reducing the production cost. Finally, the double emulsion of glucose oxidase has high tolerance to ethanol and metal ions, and the loss of enzyme activity is less, which improves the stability of glucose oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 It is a diagram of a double emulsion sample. In the figure, the left bottle is a glucose oxidase solution, the middle bottle is a water-in-oil emulsion, and the right bottle is a double emulsion.

[0034] Figure 2 It is the verification of the heat resistance performance of the double emulsion. Among them: Figure A is 40 °C; Figure B is 50 °C; Figure C is 60 °C; Figure D is 70 °C; Figure E is 80 °C; Figure F is 90 °C.

[0035] Figure 3 It is a comparative line chart of the cumulative release efficiency of glucose oxidase and glucoamylase in different types of double emulsions. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing specific implementation embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded within the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0040] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] A preparation method of a high heat-resistant glucose oxidase emulsion, comprising the following steps:

[0043] (1) Dissolution: temperature 25°C, volume fraction of glucose oxidase 3% (w / v), total concentration of the outer aqueous phase wall material solution 4% (w / v), mass ratio of gelatin to sodium carboxymethylcellulose 1:5.

[0044] (2) Adding lipophilic emulsifier: Add Span 80 with a volume fraction of 10% (w / w) to the oil phase olive oil.

[0045] (3) Shearing: Add the inner aqueous phase solution into the oil phase at a ratio of 2:1 (v / v), shearing rate 10000 rpm, shearing time 5 min, to obtain a primary emulsion.

[0046] (4) Adding hydrophilic emulsifier: Add Tween 80 with a volume fraction of 2% (w / w) to the outer aqueous phase.

[0047] (5) Mixing: Pour the primary emulsion described in step (3) into the outer aqueous phase solution at a ratio of 1:1 (v / v).

[0048] (6) Secondary shearing: Shearing rate 8000 rpm, shearing time 3 min.

[0049] (7) Adjusting pH: Adjust the pH value to 5.5 with 1 mol / L HCl and NaOH.

[0050] (8) Cooling: Cool the prepared double emulsion of glucose oxidase for 20 min under the condition of cooling at 4°C.

[0051] (9) Heat resistance verification: Place the prepared double emulsion of glucose oxidase, glucose oxidase solution, and primary emulsion in a water bath at 60°C for heat preservation for 20 min. Take samples every 5 min, cool immediately, and then measure the activity of glucose oxidase according to the group standard T / CAAA083-2022.

[0052] Example 2

[0053] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, and the mass ratio of gelatin to sodium carboxymethylcellulose is 1:1.

[0054] Example 3

[0055] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, and the mass ratio of gelatin to sodium carboxymethylcellulose is 1:3.

[0056] Example 4

[0057] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, and the mass ratio of gelatin to sodium carboxymethylcellulose is 1:7.

[0058] Example 5

[0059] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, and the mass ratio of gelatin to sodium carboxymethylcellulose is 1:10.

[0060] Example 6

[0061] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5, and the total concentration of the outer aqueous phase wall material solution is 3% (w / v).

[0062] Example 7

[0063] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5, and the total concentration of the outer aqueous phase wall material solution is 5% (w / v).

[0064] Example 8

[0065] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5, and the total concentration of the outer aqueous phase wall material solution is 6% (w / v).

[0066] Example 9

[0067] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5, and the total concentration of the outer aqueous phase wall material solution is 7% (w / v).

[0068] Example 10

[0069] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5, and the total concentration of the outer aqueous phase wall material solution is 8% (w / v).

[0070] Example 11

[0071] The difference from Example 1 is only that during the dissolution process in step (1), the temperature is controlled at 25°C, the mass ratio of gelatin to sodium carboxymethylcellulose is 1:5, and the total concentration of the outer aqueous phase wall material solution is 9% (w / v).

[0072] Example 12

[0073] The difference from Example 1 is only that the ratio in step (3) is changed to 3:1.

[0074] Example 13

[0075] The difference from Example 1 is only that the ratio in step (3) is changed to 1:1.

[0076] Example 14

[0077] The difference from Example 1 is only that the ratio in step (3) is changed to 1:2.

[0078] Example 15

[0079] The difference from Example 1 is only that the ratio in step (5) is changed to 2:1.

[0080] Example 16

[0081] The difference from Example 1 is only that the ratio in step (5) is changed to 1:2.

[0082] Example 17

[0083] The difference from Example 1 is only that the ratio in step (5) is changed to 1:3.

[0084] Example 18

[0085] The difference from Example 1 is only that the pH value in step (7) is changed to 3.0.

[0086] Example 19

[0087] It is only different from Example 1 in that the pH value in step (7) is changed to 3.5.

[0088] Example 20

[0089] It is only different from Example 1 in that the pH value in step (7) is changed to 4.0.

[0090] Example 21

[0091] It is only different from Example 1 in that the pH value in step (7) is changed to 4.5.

[0092] Example 22

[0093] It is only different from Example 1 in that the pH value in step (7) is changed to 5.0.

[0094] Example 23

[0095] It is only different from Example 1 in that the pH value in step (7) is changed to 6.0.

[0096] Example 24

[0097] It is only different from Example 1 in that the cooling time in step (8) is changed to 10 min.

[0098] Example 25

[0099] It is only different from Example 1 in that the cooling time in step (8) is changed to 30 min.

[0100] Example 26

[0101] It is only different from Example 1 in that the cooling time in step (8) is changed to 40 min.

[0102] Example 27

[0103] It is only different from Example 1 in that the cooling time in step (8) is changed to 50 min.

[0104] Example 28

[0105] It is only different from Example 1 in that the water bath temperature in step (9) is changed to 40 °C.

[0106] Example 29

[0107] It is only different from Example 1 in that the water bath temperature in step (9) is changed to 40 °C.

[0108] Example 30

[0109] It is only different from Example 1 in that the water bath temperature in step (9) is changed to 50°C.

[0110] Example 31

[0111] It is only different from Example 1 in that the water bath temperature in step (9) is changed to 70°C.

[0112] Example 32

[0113] It is only different from Example 1 in that the water bath temperature in step (9) is changed to 80°C.

[0114] Example 33

[0115] It is only different from Example 1 in that the water bath temperature in step (9) is changed to 90°C.

[0116] Example 34

[0117] It is only different from Example 1 in that the glucose oxidase in step (1) is changed to glucoamylase.

[0118] Example 35

[0119] It is only different from Example 1 in that the outer aqueous phase wall material in step (1) is changed to chitosan and arabic gum, with a mass ratio of 1:5.

[0120] Example 36

[0121] It is only different from Example 1 in that the outer aqueous phase wall material in step (1) is changed to chitosan and sodium alginate, with a mass ratio of 1:5.

[0122] Example 37

[0123] It is only different from Example 1 in that the gelatin in the outer aqueous phase wall material in step (1) is changed to chitosan.

[0124] Example 38

[0125] It is only different from Example 1 in that the sodium carboxymethyl cellulose in the outer aqueous phase wall material in step (1) is changed to arabic gum.

[0126] Example 39

[0127] It is only different from Example 1 in that the sodium carboxymethyl cellulose in the outer aqueous phase wall material in step (1) is changed to sodium alginate.

[0128] Example 40

[0129] It is only different from Example 1 in that the olive oil in step (2) is changed to soybean oil.

[0130] Example 41

[0131] The difference from Example 1 is only that the olive oil in step (2) is replaced with salad oil.

[0132] Test Example 1

[0133] Performance verification experiments were carried out on Examples 1 - 41 (labeled as Samples 1 - 41), and the specific process is as follows:

[0134] 1.1 Determination of the mass ratio of gelatin to sodium carboxymethylcellulose

[0135] Prepare 1% (w / v) gelatin aqueous solution and 1%, 3%, 5%, 7%, 10% (w / v) sodium carboxymethylcellulose aqueous solutions respectively for standby. Mix the gelatin aqueous solution and the sodium carboxymethylcellulose aqueous solution in equal proportion, heat to 60 °C, and stir for 30 min under constant temperature magnetic stirring to promote the mixing of the two wall materials. After complete mixing, slowly add 1 mol / L HCl or NaOH dropwise while stirring to different pH values, centrifuge to separate the upper equilibrium phase and the lower condensed phase, and measure the absorbance value A of the equilibrium phase at 600 nm 600 ; The condensed phase is dried to constant weight at 90 °C, and its yield Y (%) is measured. The transmittance T (%) and the yield Y (%) are obtained according to formula (1 - 1) and formula (1 - 2), and the results are shown in Table 1.

[0136]

[0137] Table 1 Effects of the mass ratio of gelatin / sodium carboxymethylcellulose on the transmittance of the equilibrium phase and the yield of the condensed phase

[0138] Sample number Transmittance T of equilibrium phase (%) Yield Y of condensed phase (%) Sample 2 97.33±0.58 9.75±0.27 Sample 3 95.91±0.11 15.75±0.40 Sample 1 93.59±0.48 19.08±0.10 Sample 4 91.62±0.43 16.75±0.29 Sample 5 85.57±0.43 12.62±0.32

[0139] 1.2 Effects of the total concentration of wall materials on the double emulsion encapsulation rate

[0140] Fix the mass ratio of gelatin to sodium carboxymethylcellulose at 1:5, and change the total concentration of wall materials for the test. The results are shown in Table 2.

[0141] Table 2 Effects of the total concentration of wall materials on the double emulsion encapsulation rate

[0142] Sample number Sample 6 Sample 1 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Entrapment efficiency (%) 32.23±1.08 36.63±1.20 31.42±0.78 25.45±1.18 22.74±0.83 17.53±1.22 23.72±1.11

[0143] 1.3 Effects of the water - oil ratio of the primary emulsion on the double emulsion encapsulation rate

[0144] Mix the glucose oxidase aqueous solution as the internal aqueous phase with the oil phase in different proportions for the test. The results are shown in Table 3.

[0145] Table 3 Effects of the water - oil ratio of the primary emulsion on the double emulsion encapsulation rate

[0146] Sample number Sample 12 Sample 1 Sample 13 Sample 14 Entrapment efficiency (%) 51.26±1.81 75.32±2.67 49.38±3.59 48.75±2.94

[0147] 1. Influence of core - wall ratio on the encapsulation efficiency of double emulsions

[0148] The primary emulsion was mixed with the external aqueous phase at different ratios for experiments, and the results are shown in Table 4.

[0149] Table 4 Influence of core - wall ratio on the encapsulation efficiency of double emulsions

[0150] Sample number Sample 15 Sample 1 Sample 16 Sample 17 Entrapment efficiency (%) 44.66±1.78 58.10±1.59 45.48±2.13 21.05±1.02

[0151] 1.5 Determination of the pH value of the complex coacervation system

[0152] The emulsion was adjusted to different pH values for experiments, and the results are shown in Table 5.

[0153] Table 5 Influence of pH value on the light transmittance of the equilibrium phase and the yield of the coacervate phase

[0154] Sample number Transmittance T of equilibrium phase (%) Yield Y of condensed phase (%) Sample 18 50.45±0.39 1.12±0.10 Sample 19 41.40±0.40 1.17±0.08 Sample 20 93.73±0.27 1.27±0.01 Sample 21 96.28±0.18 6.97±0.03 Sample 22 97.35±0.28 6.92±0.03 Sample 1 97.22±0.11 7.39±0.05 Sample 23 95.31±0.14 6.91±0.02

[0155] 1.6 Influence of cooling time on the encapsulation efficiency of double emulsions

[0156] The emulsion was placed at 4℃ and cooled for different times for experiments, and the results are shown in Table 6.

[0157] Table 6 Influence of cooling time on the encapsulation efficiency of double emulsions

[0158] Sample number Sample 24 Sample 1 Sample 25 Sample 26 Sample 27 Entrapment efficiency (%) 24.26±2.04 49.21±1.92 19.56±2.18 16.94±2.02 13.90±2.02

[0159] 1.7 Verification of the heat - resistant performance of glucose oxidase double emulsions

[0160] The emulsion was placed in a water bath at different temperature conditions (40, 50, 60, 70, 80, 90℃) and incubated for 20 min. Samples were taken every 5 min, immediately cooled, and then the glucose oxidase activity was determined according to the group standard T / CAAA083 - 2022. The results are as Figure 2 .

[0161] 1.8 Verification of the performance of glucose oxidase double emulsions

[0162] (1) Determination of the enzyme activity retention rate

[0163] The W1 / O / W2 double emulsion was diluted in distilled water, and the glucose oxidase activity and glucoamylase activity were determined. The results are shown in Table 7.

[0164] (2) Determination of the enzyme - loading amount

[0165] According to the determination method of the encapsulation efficiency of double emulsions, the enzyme - loading amount was calculated by formula 1 - 1. The results are shown in Table 7.

[0166]

[0167] Where: TEC represents the total enzyme content in the system, mL; SEC represents the enzyme content free on the surface of the emulsion;

[0168] V represents the volume of the W1 / O / W2 double emulsion, mL.

[0169] (3) Liquid sustained-release analysis

[0170] Weigh a certain amount of the W1 / O / W2 double emulsion and free enzyme, disperse them in 10 mL of acetate buffer (pH 4.5), put them into a cellulose dialysis bag with a cut-off molecular weight of 1 million, place it in a capped glass bottle containing 100 mL of acetate buffer, and place it at room temperature. Use an ultraviolet spectrophotometer to measure the enzyme content and enzyme activity respectively, and calculate the cumulative release efficiency of glucose oxidase and glucoamylase according to Formula 1-2. The results are as Figure 3 .

[0171]

[0172] Where: C r represents the enzyme content released cumulatively; C t represents the encapsulated enzyme content.

[0173] Table 7 Comparison of the performance of different types of double emulsions

[0174] Sample number Retained enzyme activity rate (%) Enzyme loading (mL / mL) Sample 1 82.19±1.13 0.0975±0.0004 Sample 34 75.52±0.94 0.0921±0.0021

[0175] (4) Verification of the tolerance to ethanol and metal ions

[0176] Place the emulsion in a water bath at a constant temperature of different concentrations of ethanol solutions (0%, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) for 20 min, cool it immediately, and measure the enzyme activity. The results are shown in Table 8.

[0177] Place the emulsion in a water bath at a constant temperature of different metal ion solutions (K + , Na + , Mg 2+ , Ca 2+ , Zn 2+ , Cu 2+ , Fe 3+ ) for 20 min, cool it immediately, and measure the enzyme activity. The results are shown in Table 9.

[0178] Table 8 Relative enzyme activity of the double emulsion after treatment with different ethanol concentrations for 20 min

[0179]

[0180]

[0181] Table 9 Relative enzyme activity of double emulsions after treatment with different metal ions for 20 min

[0182]

[0183] 1.9 Comparison of properties of different wall material combinations

[0184] Two wall material substances, chitosan and gelatin, which are positively charged in an acidic environment, and three wall material substances, sodium alginate, sodium carboxymethylcellulose, and arabic gum, which are negatively charged in an acidic environment, are compounded in pairs. Their cyst formation ability, emulsion stability, and solubility are compared, and the superior wall material combination is selected as the outer aqueous phase wall material for the complex coacervation reaction of W1 / O / W2 double emulsions for experiments. The results are shown in Table 10.

[0185] Table 10 Comparison of properties of different wall material combinations

[0186]

[0187] 1.10 Influence of different oil phases on the encapsulation rate of double emulsions

[0188] Double emulsions are prepared using olive oil, soybean oil, and salad oil as the oil phase respectively to explore the influence of different oil phases on the encapsulation rate of double emulsions. The results are shown in Table 11.

[0189] Table 11 Influence of different types of oil on the encapsulation rate of double emulsions

[0190] Sample number Sample 40 Sample 41 Sample 1 Entrapment efficiency (%) 70.06±1.32 68.39±2.17 73.44±3.24

[0191] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A highly thermostable glucose oxidase emulsion, characterized in that: Including components:

2. The emulsion according to claim 1, characterized in that The glucose oxidase volume fraction is 3% (w / v), the total concentration of the external aqueous phase wall material solution is 4% (w / v), and the mass ratio of gelatin to sodium carboxymethyl cellulose is 1:

5.

3. A method for preparing a highly thermostable glucose oxidase emulsion, characterized in that: The following steps are involved: The high heat-resistant glucose oxidase emulsion is obtained by taking glucose oxidase as the core material, olive oil as the oil phase, gelatin and sodium carboxymethyl cellulose as the external water phase wall materials, Span80 as the lipophilic emulsifier, Tween 80 as the hydrophilic emulsifier, dissolving, adding the lipophilic emulsifier, shearing, adding the hydrophilic emulsifier, mixing, secondary shearing, adjusting the pH value and cooling.

4. The method for preparing the highly thermostable glucose oxidase emulsion according to claim 3, characterized in that: The dissolution conditions are: temperature 25° C., glucose oxidase volume fraction 3% (w / v), total concentration of external aqueous phase wall material solution 4% (w / v), wherein the mass ratio of gelatin to sodium carboxymethyl cellulose is 1:

5.

5. The method for preparing the highly thermostable glucose oxidase emulsion according to claim 3, characterized in that: The condition for adding the lipophilic emulsifier is: adding Span 80 with a volume fraction of 10% (w / w) to the oil phase.

6. The method for preparing the highly thermostable glucose oxidase emulsion according to claim 3, characterized in that: The shearing conditions are: adding the inner aqueous phase solution into the oil phase at a ratio of 2:1 (v / v), with a shear rate of 10000 rpm and a shear time of 5 min to obtain a primary emulsion; the secondary shearing conditions are: a shear rate of 8000 rpm and a shear time of 3 min; the conditions for adding a hydrophilic emulsifier are: adding Tween 80 with a volume fraction of 2% (w / w) to the outer aqueous phase; the mixing comprises: pouring the primary emulsion according to claim 4 into the outer aqueous phase solution at a ratio of 1:1 (v / v).

7. The method for preparing the highly thermostable glucose oxidase emulsion according to claim 3, characterized in that: The pH adjustment includes: adjusting the pH value to 5.5 using 1 mol / L HCl and NaOH.

8. The method for preparing the highly thermostable glucose oxidase emulsion according to claim 3, characterized in that: The cooling comprises: cooling the prepared glucose oxidase double emulsion at 4° C. for 20 minutes.

9. A highly thermostable glucose oxidase produced by the method for preparing the highly thermostable glucose oxidase emulsion according to any one of claims 3 to 8.

10. A product processed by the highly thermostable glucose oxidase emulsion according to claim 9, characterized in that: The product can effectively maintain the enzymatic activity of glucose oxidase and also improve the heat resistance of glucose oxidase to meet production needs.

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