Application of Cationic Surfactant and Mesoporous Silica in Synergistically Inhibiting Polyphenol Oxidase Activity

Abstract

The present invention discloses the application of a cationic surfactant and mesoporous silica in synergistically inhibiting the activity of polyphenol oxidase. In the present invention, the cationic surfactant DTAB or [C 12 mim]Br is incubated with mesoporous silica nanoparticles together with polyphenol oxidase, which can cause more than 90% loss of the activity of polyphenol oxidase, and the usage amounts of the cationic surfactant DTAB and [C 12 mim]Br are significantly lower than the usage amounts when DTAB or [C 12 mim]Br is used alone.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioenzymes, and relates to the application of cationic surfactants and mesoporous silica in synergistically inhibiting the activity of polyphenol oxidase. Background Art

[0002] Polyphenol oxidase (PPO) is a class of copper-containing proteins that are widely distributed in plant cells. The oxidation of phenolic substrates by PPO is considered to be the main cause of browning in many fruits and vegetables during ripening, transportation, storage, and processing. Browning affects the appearance and taste of food, reduces consumer acceptance, and causes huge economic losses to the food industry. In addition, the enzymatic reaction product - quinone compounds can bind to plant proteins, reducing the digestibility of proteins and the nutritional value of plants. Therefore, how to inhibit the activity of polyphenol oxidase in plants is a concern for researchers.

[0003] Currently, the relatively mature method for inhibiting PPO enzyme activity is heat inactivation. Baking and heating Yalong fruits at 100 - 150 °C for 30 - 120 minutes can inactivate PPO in Yalong fruits and prevent browning. However, high-temperature treatment will affect the flavor and appearance of fruits and easily cause losses of vitamins and minerals.

[0004] As an amphiphilic compound, a surfactant can self-assemble in a solution, which provides a rich variety of microenvironments for proteins. Research shows that protein activity can be regulated by surfactants. Chinese Patent CN105734025A discloses that traditional quaternary ammonium salt cationic surfactants such as cetyltrimethylammonium bromide (CTAB) and dodecyltrimethylammonium bromide (DTAB) can enhance the activity of crude PPO extracted from apples. The literature (Effect of ionic and non-ionic surfactants on the activity and stability of mushroom tyrosinase (DOI: 10.1016 / j.molcatb.2007.03.001)) uses the cationic surfactant CTAB (20 mM), the anionic surfactant AOT (1.5 mM), and the non-ionic surfactant Brij 52 (1.2 mM) to regulate the activity of polyphenol oxidase extracted and purified from mushrooms. The results show that: the anionic surfactant AOT can significantly increase the PPO enzymatic reaction rate v max ; the non-ionic surfactant Brij 52 can increase the PPO enzyme activity by reducing K m ; while the cationic surfactant CTAB will not reduce the enzymatic reaction rate v max , but can inhibit the enzyme activity by increasing the Michaelis constant K m of the enzyme.

[0005] In summary, the existing methods for inhibiting PPO enzyme activity all have different problems. High-temperature treatment causes significant loss of food value; CTAB has limited inhibitory effect on the activity of polyphenol oxidase extracted and purified from mushrooms, and it does not reduce the enzyme reaction rate v max , which is achieved by increasing the Michaelis constant K of the enzyme m to inhibit the activity of this PPO enzyme, and the CTAB concentration used reaches 20 mM, which has relatively high biological toxicity and limits its further application in food. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of cationic surfactants and mesoporous silica nanoparticles (MSNs) in synergistically inhibiting polyphenol oxidase activity.

[0007] The technical solution to achieve the purpose of the present invention is as follows:

[0008] The application of cationic surfactants and mesoporous silica in synergistically inhibiting polyphenol oxidase activity, wherein the cationic surfactant is dodecyltrimethylammonium bromide (DTAB) or 1-dodecyl-3-methylimidazolium bromide ([C 12 mim]Br).

[0009] The structural formula of DTAB described in the present invention is

[0010] The [C 12 mim]Br described in the present invention has the following structural formula

[0011] Furthermore, the specific application method is: adding DTAB and mesoporous silica to a solution containing polyphenol oxidase, or adding [C 12 mim]Br and mesoporous silica to a solution containing polyphenol oxidase.

[0012] In the present invention, the mesoporous silica is flower-shaped mesoporous silica or spherical mesoporous silica.

[0013] Preferably, to further inhibit the activity of polyphenol oxidase, the pH of the solution containing polyphenol oxidase is adjusted to the appropriate pH of polyphenol oxidase, that is, pH is 6 - 7.5, more preferably the optimal pH of polyphenol oxidase, that is, pH is 7.0; the temperature of the solution containing polyphenol oxidase is adjusted to the appropriate temperature of polyphenol oxidase, that is, the temperature is 20 - 40 °C, preferably the optimal temperature of polyphenol oxidase, that is, 30 °C.

[0014] Preferably, to further inhibit the activity of polyphenol oxidase, the addition amount of DTAB is 0.5 mM or more, more preferably 3 - 5 mM.

[0015] Preferably, to further inhibit the activity of polyphenol oxidase, 12 the addition amount of [C

[0016] mim]Br is above 0.5 mM, more preferably 1 - 3 mM.

[0017] Preferably, to further inhibit the activity of polyphenol oxidase, the mass ratio of mesoporous silica to polyphenol oxidase is 10:1.

[0018] Preferably, to further inhibit the activity of polyphenol oxidase, the addition amount of mesoporous silica is 1 - 3.6 mg / mL.

[0019] Preferably, to further inhibit the activity of polyphenol oxidase, the concentration of polyphenol oxidase is 0.36 - 1 mg / mL.

[0020] In the present invention, the solution containing polyphenol oxidase is prepared by dissolving polyphenol oxidase in a pH 7.0, 0.1 M Na2HPO4 - NaH2PO4 buffer solution.

[0021] In the present invention, the addition method of the cationic surfactant and mesoporous silica can be to mix the cationic surfactant and mesoporous silica first and then add them to the solution containing polyphenol oxidase, or to mix the solution containing polyphenol oxidase with the cationic surfactant first and then add mesoporous silica, or to mix the solution containing polyphenol oxidase with mesoporous silica first and then add the cationic surfactant. The mixing order during incubation has little effect on the inhibitory effect on the activity of polyphenol oxidase.

[0022] The present invention discovers that under the condition of 30 °C without high temperature, after co - incubating the cationic surfactant DTAB or [C 12 mim]Br, mesoporous silica nanoparticles and polyphenol oxidase, the activity of polyphenol oxidase can be lost by more than 90%, and the usage amounts of the cationic surfactant DTAB and [C 12 mim]Br are significantly lower than those when using DTAB or [C 12 mim]Br alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a graph showing the effect of DTAB concentration on PPO enzyme activity in the DTAB / MSNs / PPO system under the conditions of T = 30 °C and pH = 7;

[0024] Figure 2 It is a graph showing the effect of [C 12 mim]Br concentration on PPO enzyme activity in the [C 12 mim]Br / MSNs / PPO system under the conditions of T = 30 °C and pH = 7;

[0025] Figure 3 Graph of the loading rate of PPO enzyme by MSNs at different DTAB concentrations under the conditions of T = 30 °C and pH = 7;

[0026] Figure 4 For different [C 12 Graph of the loading rate of PPO enzyme by MSNs at different [C

[0027] Figure 5 Graph of the effect of the sample addition sequence on the activity of PPO enzyme in the DTAB / MSNs / PPO system under the conditions of T = 30 °C and pH = 7;

[0028] Figure 6 For different [C 12 Graph of the effect of the sample addition sequence on the activity of PPO enzyme in the [C

[0029] Figure 7 Infrared spectrum of PPO immobilized by MSNs / DTAB;

[0030] Figure 8 Graph of the effect of substrate concentration on the activity of PPO in the DTAB / MSNs / PPO system under the conditions of T = 30 °C and pH = 7;

[0031] Table 1 shows the effects of DTAB and MSNs on the Michaelis constant K m and the maximum reaction rate v max of PPO under the conditions of T = 30 °C and pH = 7. Specific embodiments

[0032] The present invention will be further described in detail below in conjunction with examples and drawings.

[0033] Example 1

[0034] Experiment on the synergistic inhibition of polyphenol oxidase activity by DTAB and mesoporous silica:

[0035] (1) Preparation of polyphenol oxidase solution

[0036] The commercial PPO enzyme was purchased from Beijing Solarbio Science & Technology Co., Ltd. (CAS: 9002 - 10 - 2), and the enzyme was extracted from mushrooms. Using a Na2HPO4 - NaH2PO4 buffer solution (0.1 M, pH 7.0) as the solvent, a 20 mg / mL enzyme mother liquor was prepared.

[0037] (2) Preparation of mesoporous silica dispersion

[0038] Preparation references for mesoporous silica (DOI: 10.1039 / C7GC02139A). Specifically, the double-template method is used, with cationic surfactant CTAB and anionic surfactant SDS as template agents, tetraethyl orthosilicate as the silicon source, and the one-pot method is used to react to obtain silica nanospheres. Then, the template agents are removed by calcination to obtain mesoporous silica nanospheres. Among them, the template agent used for preparing flower-shaped silica is [CTAB]:[SDS]=42 mM:6 mM, and the template agent used for preparing spherical silica is [CTAB]:[SDS]=42 mM:2 mM.

[0039] Using Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) as the dispersant, ultrasonic dispersion (100 W, 20 min) is carried out to obtain a 20 mg / mL mesoporous silica dispersion.

[0040] (3) Preparation of surfactant solution

[0041] Using Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) as the solvent, a 20 mM DTAB stock solution is prepared.

[0042] (4) Preparation of substrate solution

[0043] Using Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) as the solvent, a catechol stock solution with a concentration of 75 mM is prepared.

[0044] (5) Enzyme pretreatment

[0045] A certain amount of DTAB stock solution and mesoporous silica dispersion are added to the PPO stock solution, and incubated with shaking at 30 °C for 1 h to obtain the enzyme solution to be tested. In the incubation system, the concentration of mesoporous silica is 3.6 mg / mL, and the concentration of PPO is 0.36 mg / mL.

[0046] (6) Enzyme activity determination (UV-visible spectrophotometry)

[0047] Using catechol as the substrate, the enzyme activity of PPO is tested. The substrate solution in (4) is mixed with the enzyme solution to be tested in (5), and the absorbance value of the reaction system at 420 nm within 10 s is recorded. In the test system, the concentration of catechol is 4 mM. The absorbance value (A) at 420 nm is plotted against time (t), and through the initial slope of this kinetic curve, combined with the molar extinction coefficient of the product o-benzoquinone (1100 M -1 cm -1 ), the initial reaction rate ν (mM·s -1 ) of PPO catalyzing the oxidation of catechol is obtained. The enzyme activity without adding any additives is defined as 100%.

[0048] Figure 1 Effect of DTAB concentration on PPO enzyme activity in the DTAB / MSNs / PPO system under the conditions of T = 30 °C and pH = 7. As can be seen from the figure, when only DTAB is added to the system, when the DTAB concentration does not exceed 3 mM, the PPO enzyme activity remains almost unchanged; when the DTAB concentration reaches 3 mM, the PPO activity begins to decrease: when the DTAB concentration is 5 mM, the PPO activity is 68%; when the DTAB concentration reaches 10 mM, the PPO is completely inactivated. When both DTAB and flower-like mesoporous silica are present, 0.5 mM of DTAB can reduce the PPO enzyme activity to 77%, and 3 mM of DTAB can reduce the PPO enzyme activity to 22%; when the DTAB concentration reaches 5 mM, the PPO activity is reduced to less than 10%. When both DTAB and spherical mesoporous silica are present, 0.5 mM of DTAB can reduce the PPO enzyme activity to 70%, and 3 mM of DTAB can reduce the PPO enzyme activity to 14%; when the DTAB concentration reaches 5 mM, the PPO activity is reduced to less than 10%.

[0049] Example 2

[0050] [C 12 mim]Br and mesoporous silica synergistic inhibition of polyphenol oxidase activity experiment:

[0051] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0052] (2) Preparation of mesoporous silica dispersion: The same as in Example 1.

[0053] (3) Preparation of surfactant solution

[0054] Using Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) as the solvent, prepare a 20 mM [C 12 mim]Br stock solution.

[0055] (4) Preparation of substrate solution: The same as in Example 1.

[0056] (5) Enzyme pretreatment

[0057] Add a certain amount of [C 12 mim]Br stock solution and mesoporous silica dispersion to the PPO stock solution, incubate at 30 °C with shaking for 1 h to obtain the enzyme solution to be measured. In the incubation system, the mesoporous silica concentration is 3.6 mg / mL and the PPO concentration is 0.36 mg / mL.

[0058] (6) Enzyme activity determination (UV-visible spectrophotometry)

[0059] Using catechol as the substrate, the enzyme activity of PPO was tested. The substrate solution in (4) was mixed with the enzyme solution to be tested in (5), and the absorbance value of the reaction system at 420 nm within 10 s was recorded. In the test system, the concentration of catechol was 4 mM. The absorbance value (A) at 420 nm was plotted against time (t), and through the initial slope of this kinetic curve, combined with the molar extinction coefficient of the product o-benzoquinone (1100 M -1 cm -1 ), the initial reaction rate ν (mM·s -1 ) of PPO-catalyzed oxidation of catechol was obtained. The enzyme activity without adding any additives was defined as 100%.

[0060] Figure 2 For the [C 12 mim]Br / MSNs / PPO system at T = 30 °C and pH = 7, it is a graph showing the effect of the concentration of [C 12 mim]Br on the PPO enzyme activity. As can be seen from the figure, when only [C 12 mim]Br was added to the system, 1 mM of [C 12 mim]Br had little effect on the PPO enzyme activity, 3 mM of [C 12 mim]Br could reduce the PPO enzyme activity to 29%, and 5 mM of [C 12 mim]Br was required to completely inactivate the PPO enzyme. When [C 12 mim]Br and flower-like mesoporous silica were added simultaneously, 0.5 mM of [C 12 mim]Br could reduce the PPO activity to 61%, 1 mM of [C 12 mim]Br could reduce the PPO enzyme activity to 39%, and 3 mM of [C 12 mim]Br could completely inactivate the PPO enzyme. When [C 12 mim]Br and spherical mesoporous silica were added simultaneously, 0.5 mM of [C 12 mim]Br could reduce the PPO activity to 50%, 1 mM of [C 12 mim]Br could reduce the PPO enzyme activity to 43%, and 3 mM of [C 12 mim]Br could reduce the PPO enzyme activity to 18%.

[0061] Example 3

[0062] Experiment on the enzyme loading rate of MSNs for PPO in the presence of different concentrations of DTAB:

[0063] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0064] (2) Preparation of mesoporous silica dispersion: The same as in Example 1.

[0065] (3) Preparation of surfactant solution: The same as in Example 1.

[0066] (4) Loading experiment of PPO by MSNs in the presence of DTAB

[0067] Add a certain amount of DTAB stock solution and mesoporous silica dispersion to the PPO stock solution, incubate with shaking at 30 °C for 1 h, centrifuge at 30 °C and 1000 rpm for 20 min, and take the supernatant for measurement. In the incubation system, the concentration of mesoporous silica is 1 mg / mL and the concentration of PPO is 1 mg / mL.

[0068] (5) Determination of PPO loading rate (UV-visible spectrophotometry)

[0069] Measure the absorbance value of 1 mg / mL PPO solution, denoted as A0, measure the absorbance value at 280 nm of the supernatant in (4), denoted as A1, and calculate the PPO loading rate according to the following formula:

[0070] PPO loading rate = (A0 - A1) / A0 × 100%

[0071] Figure 3 It is the graph of the loading rate of PPO by MSNs at different concentrations of DTAB under the conditions of T = 30 °C and pH = 7. It can be seen from the graph that mesoporous silica hardly adsorbs PPO. As the content of DTAB in the system increases, the loading rate of PPO by mesoporous silica increases. When the amount of DTAB used is 1 mM, the loading rate of PPO by flower-like mesoporous silica reaches 22%, and the loading rate of PPO by spherical mesoporous silica reaches 19%.

[0072] Example 4

[0073] Loading rate experiment of PPO by MSNs in the presence of different concentrations of [C 12 mim]Br:

[0074] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0075] (2) Preparation of mesoporous silica dispersion: The same as in Example 1.

[0076] (3) Preparation of surfactant solution: The same as in Example 2.

[0077] (4) Loading experiment of PPO by MSNs in the presence of [C 12 mim]Br:

[0078] Add a certain amount of [C 12The mim]Br mother liquor and the mesoporous silica dispersion were added to the PPO mother liquor, incubated with shaking at 30 °C for 1 h, centrifuged at 30 °C and 1000 rpm for 20 min, and the supernatant was taken for testing. In the incubation system, the concentration of mesoporous silica was 1 mg / mL and the concentration of PPO was 1 mg / mL.

[0079] (5) Determination of PPO loading rate (UV-visible spectrophotometry)

[0080] Measure the absorbance value of the 1 mg / mL PPO solution, denoted as A0, and measure the absorbance value of the supernatant in (4) at 280 nm, denoted as A1. Calculate the PPO loading rate using the following formula:

[0081] PPO loading rate = (A0 - A1) / A0 × 100%.

[0082] Figure 4 For T = 30 °C and pH = 7, at different concentrations of [C 12 mim]Br, the loading rate of PPO by MSNs is shown in the figure. It can be seen from the figure that mesoporous silica hardly adsorbs PPO. As the content of [C 12 mim]Br in the system increases, the loading rate of PPO by mesoporous silica increases. When the usage amount of [C 12 mim]Br is 1 mM, the loading rate of PPO by flower-like mesoporous silica reaches 34%, and the loading rate of PPO by spherical mesoporous silica reaches 30%.

[0083] Example 5

[0084] Experiment on the effect of the sample addition sequence on the activity of PPO enzyme in the DTAB / MSNs / PPO system:

[0085] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0086] (2) Preparation of mesoporous silica dispersion:

[0087] Mesoporous silica was prepared by an existing method, referring to the reference (DOI: 10.1039 / C7GC02139A). Specifically, the double-template method was used, with the cationic surfactant CTAB (42 mM) and the anionic surfactant SDS (2 mM) as template agents, and tetraethyl orthosilicate as the silicon source. Silica nanospheres were obtained by a one-pot reaction, and then the template agents were removed by calcination to obtain spherical mesoporous silica nanospheres.

[0088] Using a Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) as the dispersant, ultrasonic dispersion (100 W, 20 min) was carried out to obtain a 20 mg / mL mesoporous silica dispersion.

[0089] (3) Preparation of surfactant solution: The same as in Example 1.

[0090] (4) Preparation of substrate solution: The same as in Example 1.

[0091] (5) Enzyme and substrate pretreatment

[0092] a. Mix DTAB with the enzyme and MSNs with the substrate

[0093] Add a certain amount of DTAB mother liquor to the PPO mother liquor and incubate with shaking at 30 °C for 1 h to obtain the enzyme solution to be measured. Add a certain amount of mesoporous silica dispersion to the catechol mother liquor and incubate with shaking in the dark at 30 °C for 1 h to obtain the substrate solution to be used. In the incubation system, the concentration of mesoporous silica is 3.624 mg / mL, the concentration of PPO is 0.3624 mg / mL, and the concentration of DTAB is 1 mM.

[0094] b. Mix MSNs with the enzyme and DTAB with the substrate

[0095] Add a certain amount of mesoporous silica dispersion to the PPO mother liquor and incubate with shaking at 30 °C for 1 h to obtain the enzyme solution to be measured. Add a certain amount of DTAB mother liquor to the catechol mother liquor and incubate with shaking in the dark at 30 °C for 1 h to obtain the substrate solution to be used. In the incubation system, the concentration of mesoporous silica is 3.624 mg / mL, the concentration of PPO is 0.3624 mg / mL, and the concentration of DTAB is 1 mM.

[0096] c. Mix DTAB and MSNs together with the substrate

[0097] Incubate the PPO mother liquor with shaking at 30 °C for 1 h to obtain the enzyme solution to be measured. Add a certain amount of DTAB mother liquor and mesoporous silica dispersion to the catechol mother liquor and incubate with shaking in the dark at 30 °C for 1 h to obtain the substrate solution to be used. In the incubation system, the concentration of mesoporous silica is 3.624 mg / mL, the concentration of PPO is 0.3624 mg / mL, and the concentration of DTAB is 1 mM.

[0098] (6) Enzyme activity assay (UV-visible spectrophotometry)

[0099] Using catechol as the substrate, the enzyme activity of PPO was tested. Mix the substrate solution to be used in (5) with the enzyme solution to be measured, and record the absorbance value of the reaction system at 420 nm within 10 s. In the test system, the concentration of catechol is 4 mM. Plot the absorbance value (A) at 420 nm against time (t), and through the initial slope of this kinetic curve, combined with the molar extinction coefficient of the product o-benzoquinone (1100 M -1 cm -1 ), the initial reaction rate ν (mM·s -1)。The enzyme activity without any additives is defined as 100%.

[0100] Figure 5 Figure showing the effect of the sample addition sequence on the PPO enzyme activity in the DTAB / MSNs / PPO system at T = 30 °C and pH = 7. As can be seen from the figure, only when DTAB and mesoporous silica are mixed with the enzyme simultaneously does DTAB / MSNs have an obvious inhibitory effect on the PPO enzyme activity, while the other three sample addition sequences (DTAB mixed with the enzyme, MSNs mixed with the substrate; MSNs mixed with the enzyme, DTAB mixed with the substrate; DTAB and mesoporous silica mixed with the substrate simultaneously) have no obvious effect on the PPO enzyme activity.

[0101] Example 6

[0102] Effect of sample addition sequence on the PPO enzyme activity in the 12 [C

[0103] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0104] (2) Preparation of mesoporous silica dispersion: The same as in Example 5.

[0105] (3) Preparation of surfactant solution: The same as in Example 2.

[0106] (4) Preparation of substrate solution: The same as in Example 1.

[0107] (5) Pretreatment of enzyme and substrate

[0108] a. [C 12 mim]Br mixed with the enzyme, MSNs mixed with the substrate

[0109] Add a certain amount of [C 12 mim]Br mother liquor to the PPO mother liquor, incubate at 30 °C with shaking for 1 h to obtain the enzyme solution to be measured. Add a certain amount of mesoporous silica dispersion to the catechol mother liquor, incubate at 30 °C in the dark with shaking for 1 h to obtain the substrate solution to be used. In the incubation system, the concentration of mesoporous silica is 3.624 mg / mL, the concentration of PPO is 0.3624 mg / mL, and the concentration of [C 12 mim]Br is 1 mM.

[0110] b. MSNs mixed with the enzyme, [C 12 mim]Br mixed with the substrate

[0111] Add a certain amount of mesoporous silica dispersion to the PPO mother liquor, incubate at 30 °C with shaking for 1 h to obtain the enzyme solution to be measured. Add a certain amount of [C 12The [mim]Br mother liquor was added to the catechol mother liquor, and incubated with shaking in the dark at 30 °C for 1 h to obtain the substrate solution to be used. In the incubation system, the concentration of mesoporous silica was 3.624 mg / mL, the concentration of PPO was 0.3624 mg / mL, and the [C 12 concentration of [mim]Br was 1 mM.

[0112] c. [C 12 [mim]Br and MSNs were mixed with the substrate together

[0113] The PPO mother liquor was incubated with shaking at 30 °C for 1 h to obtain the enzyme solution to be measured. A certain amount of [C 12 The [mim]Br mother liquor and the mesoporous silica dispersion were added to the catechol mother liquor, and incubated with shaking in the dark at 30 °C for 1 h to obtain the substrate solution to be used. In the incubation system, the concentration of mesoporous silica was 3.624 mg / mL, the concentration of PPO was 0.3624 mg / mL, and the [C 12 concentration of [mim]Br was 1 mM.

[0114] (6) Enzyme activity assay (UV-visible spectrophotometry)

[0115] Using catechol as the substrate, the enzyme activity of PPO was tested. The substrate solution to be used in (5) was mixed with the enzyme solution to be measured, and the absorbance value of the reaction system at 420 nm within 10 s was recorded. In the test system, the concentration of catechol was 4 mM. The absorbance value (A) at 420 nm was plotted against time (t), and through the initial slope of this kinetic curve, combined with the molar extinction coefficient of the product o-benzoquinone (1100 M -1 cm -1 ), the initial reaction rate ν (mM·s -1 ) of PPO catalyzing the oxidation of catechol was obtained. The enzyme activity without adding any additives was defined as 100%.

[0116] Figure 6 For the addition sequence in the [mim]Br / MSNs / PPO system under the conditions of T = 30 °C and pH = 7, the influence diagram of the PPO enzyme activity. It can be seen from the figure that only when [C 12 [mim]Br and mesoporous silica are mixed with the enzyme at the same time, [C 12 [mim]Br / MSNs has an obvious inhibitory effect on the PPO enzyme activity, while the other three addition sequences ([C 12 [mim]Br is mixed with the enzyme and MSNs is mixed with the substrate; MSNs is mixed with the enzyme and [C 12 [mim]Br is mixed with the substrate; [C 12 [mim]Br and mesoporous silica are mixed with the substrate at the same time) have little influence on the PPO enzyme activity. 12 ​

[0117] Example 7

[0118] Infrared spectrum experiment of PPO immobilized on MSNs / DTAB:

[0119] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0120] (2) Preparation of mesoporous silica dispersion: The same as in Example 5.

[0121] (3) Preparation of surfactant solution: The same as in Example 1.

[0122] (4) Immobilization of PPO on MSNs / DTAB

[0123] Add a certain amount of DTAB mother liquor or / and PPO mother liquor to the mesoporous silica dispersion, incubate with shaking at 30 °C for 1 h, centrifuge at 30 °C and 1000 rpm for 20 min, remove the supernatant, wash the precipitate with Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0), and freeze-dry to obtain the solid to be tested. In the incubation system, the concentration of mesoporous silica is 1 mg / mL, the concentration of PPO is 1 mg / mL, and the concentration of DTAB is 1 mM.

[0124] (5) Infrared spectrum measurement

[0125] Mix the solid powder to be tested with potassium bromide at a mass ratio of 1:100, press into tablets, and record the infrared absorption spectrum in the wavenumber range of 4000 - 400 cm -1 Wave number range.

[0126] Figure 7 is the infrared spectrum of PPO immobilized on MSNs / DTAB. The sharp peak at 1099 cm -1 is attributed to the Si-O stretching vibration of mesoporous silica. After immobilizing PPO, the characteristic absorption peak of PPO located in the amide I band (1600 - 1700 cm -1 ) can be observed. The peak at 3400 - 3440 cm -1 can be attributed to the N-H stretching vibration of PPO, while the peaks at 2926 cm -1 and 2856 cm -1 can be attributed to the C-H stretching vibration of DTAB. The infrared spectrum proves the formation of the complex of DTAB, MSNs, and PPO.

[0127] Example 8

[0128] Effect experiment of DTAB and MSNs on the Michaelis constant (K m ) and maximum reaction rate (v max ) of PPO enzymatic reaction:

[0129] (1) Preparation of polyphenol oxidase solution: The same as in Example 1.

[0130] (2) Preparation of mesoporous silica dispersion: The same as in Example 5.

[0131] (3) Preparation of surfactant solution: The same as in Example 1.

[0132] (4) Preparation of substrate solution

[0133] Using Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) as the solvent, prepare a catechol stock solution with a concentration of 75 mM. Use Na2HPO4-NaH2PO4 buffer solution (0.1 M, pH 7.0) and catechol stock solution (75 mM) to prepare catechol solutions with concentrations ranging from 0 to 8 mM.

[0134] (5) Enzyme pretreatment

[0135] Add a certain amount of DTAB stock solution and mesoporous silica dispersion to the PPO stock solution, and incubate with shaking at 30 °C for 1 h to obtain the enzyme solution to be tested. In the incubation system, the concentration of mesoporous silica is 3.6 mg / mL, the concentration of PPO is 0.36 mg / mL, and the concentration of DTAB is 1 mM.

[0136] (6) Enzyme activity assay (UV-visible spectrophotometry)

[0137] Using catechol as the substrate, test the enzyme activity of PPO. In the test system, the concentration of catechol ranges from 0 to 8 mM, and the test wavelength is 420 nm. Plot the absorbance value (A) at 420 nm against time (t), and through the initial slope of this kinetic curve, combined with the molar extinction coefficient of the product o-benzoquinone (1100 M -1 cm -1 ), obtain the initial reaction rate ν (μM·s -1 ) of PPO-catalyzed oxidation of catechol. Define the enzyme activity without adding any additives as 100%.

[0138] (7) Calculate the Michaelis constant (K m ) and maximum reaction rate (v max ) of the PPO-catalyzed reaction

[0139] Plot the PPO enzyme activity against the substrate concentration, and read the maximum reaction rate v max of the PPO-catalyzed reaction from the graph. Then, according to the definition of the Michaelis constant, K m is the substrate concentration when v = v max / 2.

[0140] Figure 8Table 1 is the effect of substrate concentration on PPO activity in the DTAB / MSNs / PPO system under the conditions of T = 30 ° C and pH = 7. Figure 8 K of DTAB and MSNs for PPO obtained from data sorting m 、v max As shown in the table, the cationic surfactant DTAB alone increases the Michaelis constant of PPO, and the mesoporous silica alone has little effect on the Michaelis constant of PPO, while the synergistic effect of DTAB and MSNs can reduce the Michaelis constant of PPO. This shows that the synergistic inhibition of DTAB and MSNs on the enzyme activity of PPO is an anti-competitive inhibition.

[0141] Table 1 Effect of DTAB and mesoporous silica on the Michaelis constant K of PPO m and the maximum reaction rate v max Impact

[0142]

[0143]

[0144] Based on the above results, the following conclusions can be drawn: by introducing mesoporous silica nanoparticles while using cationic surfactants, the effect of effectively inhibiting the activity of polyphenol oxidase can be achieved, while the amount of cationic surfactant used can be significantly reduced, and the hydrogen bonding provided by imidazole cationic surfactants can further reduce the amount of cationic surfactant used. In the presence of cationic surfactants, the increase in the PPO loading rate of mesoporous silica indicates that the interaction between MSNs and PPO is enhanced in the presence of cationic surfactants. Changing the mixing order of surfactants, MSNs and PPO enzymes does not affect the inhibitory effect of surfactants / MSNs on PPO enzyme activity, but if surfactants and MSNs are mixed with PPO and substrates respectively, or surfactants and MSNs are mixed with substrates, the inhibitory effect of surfactants / MSNs on PPO enzyme activity is limited, indicating that only cationic surfactants, mesoporous silica and PPO enzymes can be incubated together to significantly inhibit PPO enzyme activity, indicating that the reduction in PPO enzyme activity is caused by the synergistic effect of cationic surfactants and mesoporous silica. The reduction in PPO Michaelis constant indicates that the synergistic inhibition of PPO enzyme activity by cationic surfactants and mesoporous silica is anti-competitive inhibition.

Claims

1. Application of a cationic surfactant and mesoporous silica in synergistically inhibiting polyphenol oxidase activity, characterized in that, The specific application method is as follows: Add dodecyltrimethylammonium bromide and mesoporous silica to the solution containing polyphenol oxidase, or add 1-dodecyl-3-methylimidazolium bromide and mesoporous silica to the solution containing polyphenol oxidase. The polyphenol oxidase is polyphenol oxidase extracted from mushrooms. The addition amount of dodecyltrimethylammonium bromide or 1-dodecyl-3-methylimidazolium bromide is above 0.5 mM. The mass ratio of mesoporous silica to polyphenol oxidase is 1:1 to 10:

1. The addition amount of mesoporous silica is 1 to 3.6 mg / mL, and the concentration of polyphenol oxidase is 0.36 to 1 mg / mL.

2. The application according to claim 1, characterized in that, The mesoporous silica described is flower-shaped mesoporous silica or spherical mesoporous silica.

3. The application according to claim 1, characterized in that, The pH value of the solution containing polyphenol oxidase is 6 to 7.5, and the temperature is 20 to 40 °C.

4. The application according to claim 1, characterized in that The pH value of the solution containing polyphenol oxidase is 7.0, and the temperature is 30 °C.

5. The application according to claim 1, characterized in that, The addition amount of dodecyltrimethylammonium bromide is 3 to 5 mM, and the addition amount of 1-dodecyl-3-methylimidazolium bromide is 1 to 3 mM.

6. The application according to claim 1, wherein The solution containing polyphenol oxidase is prepared by dissolving polyphenol oxidase in a 0.1 M Na2HPO4-NaH2PO4 buffer solution with a pH of 7.

0.

7. The application according to claim 1, wherein The addition method of the cationic surfactant and mesoporous silica is to first mix the cationic surfactant and mesoporous silica and then add them to the solution containing polyphenol oxidase, or first mix the solution containing polyphenol oxidase with the cationic surfactant and then add mesoporous silica, or first mix the solution containing polyphenol oxidase with mesoporous silica and then add the cationic surfactant.

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