A method for detecting amylase activity in a zanthoxylum bungeanum essential oil emulsion

Abstract

The present application relates to a kind of zanthoxylum bungeanum essential oil emulsion starch enzyme activity detection method, which is by using centrifugal demulsification method combined with accurate chemical analysis method, the starch enzyme activity in zanthoxylum bungeanum essential oil emulsion is quantitatively analyzed, specifically including the following steps: S1, instrument and reagent preparation;S2, solution preparation;S3, sample processing;S4, demulsification treatment and DNS method detects absorbance and calculates starch enzyme activity;The present application can accurately quantify the reducing sugar level in essential oil emulsion, the accuracy compared with direct detection method is significantly improved, and the method is suitable for the starch enzyme activity detection in other essential oil emulsion.

Description

Technical Field

[0001] This invention belongs to the field of amylase activity detection technology, specifically relating to a method for detecting amylase activity in Sichuan pepper essential oil emulsion. Background Technology

[0002] Sichuan pepper essential oil, as a natural plant extract, has broad application prospects due to its multiple excellent biological activities, including antibacterial, anti-inflammatory, and antioxidant properties. However, the low water solubility of Sichuan pepper essential oil prevents it from effectively contacting enzymes in aqueous solutions, thus affecting the determination of its activity.

[0003] To address the aforementioned water solubility challenge, some studies have employed emulsion preparation techniques. By using emulsifiers to disperse Sichuan pepper essential oil into tiny droplets, the contact area with enzymes is increased, providing a basis for the binding of active components in the essential oil to enzymes. In amylase activity detection, the 3,5-dinitrosalicylic acid (DNS) method is currently the most widely used method for quantifying reducing sugars. Its principle is based on the colorimetric reaction between DNS and reducing sugars, with the reducing sugar content calculated based on the absorbance value, thus determining enzyme activity. However, in actual testing, it was found that the milky white turbidity of Sichuan pepper essential oil emulsions severely interferes with the colorimetric reaction of the DNS method, leading to an inflated absorbance value and failing to accurately reflect the true amount of reducing sugars produced. Ultimately, this causes the calculated amylase inhibition rate of Sichuan pepper essential oil to deviate from the actual value.

[0004] Based on this, this application was developed. Summary of the Invention

[0005] To address the above problems, the present invention aims to overcome the shortcomings of existing methods for detecting reducing sugars in essential oil emulsions due to color interference from colorimetric reactions, and provides a method for detecting reducing sugars in Sichuan pepper essential oil emulsions. The results of reducing sugar content determination in the Sichuan pepper essential oil emulsion treated by this invention show no significant difference from the true values, and the reliability is significantly higher than that of the untreated group. This invention has significant advantages in detecting reducing sugar levels in essential oil emulsions and can be used for the detection of amylase activity in essential oil emulsions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for detecting amylase activity in Sichuan pepper essential oil emulsion, which involves preparing Sichuan pepper essential oil nanoemulsion using Tween-80 and combining it with precise chemical analysis methods to quantitatively analyze the reducing sugar level in the Sichuan pepper essential oil emulsion, specifically including the following steps: S1. Instrument and reagent preparation; Prepare the instruments and reagents required for the test; S2. Solution preparation: Prepare DNS solution, 1% starch solution, 1 U / mL amylase solution, 1 mol / L NaOH solution, and phosphate buffer solution for later use; S3. Preparation of Sichuan pepper essential oil emulsions with different concentrations; S4. The amylase activity in Sichuan pepper essential oil emulsion was determined by a modified 3,5-dinitrosalicylic acid method; blank group, control group and sample group were set up. Blank group: Add 1 mL of phosphate buffer and 1 mL of 1% starch solution to centrifuge tubes; Control group: 0.5 mL phosphate buffer, 0.5 mL amylase solution and 1 mL 1% starch solution were added to centrifuge tubes; Sample group: Add 0.5 mL of Sichuan pepper essential oil emulsion, 0.5 mL of amylase solution, and 1 mL of 1% starch solution to centrifuge tubes; Then, each group was incubated at 37±1℃ for 8-12 min, and the emulsion was broken by centrifugation. An appropriate amount of supernatant was mixed with an equal volume of DNS solution and then boiled in a water bath for 3 min. Then, 0.5 mL of the reaction solution after boiling in a water bath was mixed with an equal volume of distilled water, and the absorbance was detected at 540 nm. The amylase activity was calculated according to Formula 1. Formula 1: Amylase activity (%) = [A (sample) - A (blank)] / [A (control) - A (blank)] × 100%.

[0007] Specifically, the instruments and reagents used in step S1 are as follows: The instruments include: ultraviolet spectrophotometer, low-temperature high-speed centrifuge, electronic balance, digital display constant temperature water bath, electric furnace, pH meter, high-speed homogenizer, ultrasonic cleaner, and ultrasonic cell disruptor. The reagents include: 3,5-dinitrosalicylic acid (DNS), sodium hydroxide, potassium sodium tartrate, phenol, sodium metabisulfite, starch, and distilled water. Further, the DNS reagent consists of: 6.3 g DNS, 21 g NaOH, 182.0 g potassium sodium tartrate, 5.0 g phenol, and 5.0 g sodium metabisulfite; 1% starch solution; 1 U / mL amylase solution; 1 mol / L NaOH solution; phosphate buffer (pH=8.0); and 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL glucose standard solutions.

[0008] Specifically, the DNS solution preparation method in step S2 is as follows: (1) Weigh 6.3 g of DNS, add 200 mL of distilled water and stir to dissolve to obtain DNS solution; (2) Weigh 21.0 g of sodium hydroxide and add it to 100 mL of distilled water and stir to dissolve. Slowly add the dissolved NaOH solution to the DNS solution; (3) Weigh 182.0 g of potassium sodium tartrate, dissolve it in 200 mL of distilled water and mix it with the solution obtained in step (2); (4) Add 5.0 g of phenol and 5.0 g of sodium metabisulfite to the solution obtained in step (3) and stir until the solution is completely clear. Dilute to 1000 mL.

[0009] Specifically, the preparation method for a 1% starch solution is as follows: Weigh 10.0 g of starch and add it to 200 mL of distilled water. Stir to dissolve the starch, then slowly add it to 500 mL of boiling water to fully gelatinize it. Make up the volume to 1000 mL.

[0010] Further, the phosphate buffer solution is prepared as follows: Weigh 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate and dissolve them in 800 mL of distilled water. Add 1 mol / L sodium hydroxide solution dropwise until the solution pH=8.0, and then bring the volume to 1000 mL.

[0011] Further, in step S2, the 1 U / mL amylase solution is prepared by weighing 10 mg of amylase and dissolving it in 100 mL of phosphate buffer. After centrifugation (3000 rpm, 10 min), the supernatant is collected to obtain a 1 U / mL lipase solution.

[0012] Specifically, in step S3, different concentrations of Sichuan pepper essential oil emulsions were prepared by using 10% Tween 80 solution to prepare different concentrations of Sichuan pepper essential oil, which were then homogenized and ultrasonically crushed to obtain Sichuan pepper essential oil emulsions; the specific steps are as follows: (1) Preparation of 10% Tween 80 solution: 100 mL of Tween 80 was added to 900 mL of distilled water, stirred and ultrasonically dissolved for later use (power 700-900 w, time 8-15 min); (2) Preparation of essential oil emulsions of different concentrations: 0.50 mL, 1.25 mL and 2.50 mL of Sichuan pepper essential oil were added to 49.5 mL, 48.75 mL and 47.5 mL of 10% Tween 80 solution respectively to obtain 1%, 2.5% and 5% Sichuan pepper essential oil; after homogenization and ultrasonication, Sichuan pepper essential oil emulsions of 1%, 2.5% and 5% were obtained.

[0013] Furthermore, the Sichuan pepper essential oil was homogenized using a high-speed homogenizer at 9000-11000 rpm for 2-5 minutes; then ultrasonicated using an ultrasonic cell disruptor at 1500-2500 W for 20-40 seconds to obtain 1%, 2.5%, and 5% Sichuan pepper essential oil emulsions. Preferably, the homogenization conditions were: 10000 rpm for 3 minutes; the ultrasonic disruption conditions were: 2000 W for 30 seconds.

[0014] Furthermore, the centrifugal demulsification conditions in step S4 are: rotation speed 9000 rpm / min, time 15 min, and temperature 5℃.

[0015] Compared with the prior art, the advantages and beneficial effects of the method of the present invention are as follows: This invention confirms the accuracy of the method for detecting reducing sugars by measuring the results of glucose standard solutions. The reducing sugar content in different concentrations of Sichuan pepper essential oil emulsions was determined using a demulsification-centrifugation method to calculate the amylase activity inhibition rate. The measured reducing sugar content in the Sichuan pepper essential oil emulsion treated with this invention showed no significant difference from the true value, and its reliability was significantly higher than that of the untreated group. This invention has significant advantages in detecting reducing sugar levels in essential oil emulsions and can be used for the detection of amylase activity in essential oil emulsions. Attached Figure Description

[0016] Figure 1 This is the standard curve for glucose. Figure 2 The optimal conditions for centrifugation were used to detect amylase activity; in the figure: EO: essential oil emulsion, UF: not centrifuged. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention shall all fall within the scope of the present invention.

[0018] In the following examples, all raw materials used are common commercially available products that can be purchased directly, or can be prepared using conventional techniques in the art.

[0019] Room temperature refers to 25±5℃.

[0020] In the following examples, the reagents or solutions used were prepared as follows.

[0021] The method for preparing DNS solution is as follows: (1) Weigh 6.3 g of DNS, add 200 mL of distilled water and stir to dissolve to obtain DNS solution; (2) Weigh 21.0 g of sodium hydroxide and add it to 100 mL of distilled water and stir to dissolve. Slowly add the dissolved NaOH solution to the DNS solution; (3) Weigh 182.0 g of potassium sodium tartrate, dissolve it in 200 mL of distilled water and mix it with the solution obtained in step (2); (4) Add 5.0 g of phenol and 5.0 g of sodium metabisulfite to the solution obtained in step (3) and stir until the solution is completely clear. Dilute to 1000 mL.

[0022] The method for preparing a 1% starch solution is as follows: Weigh 10.0 g of starch and add it to 200 mL of distilled water. Stir to dissolve the starch, then slowly add it to 500 mL of boiling water to fully gelatinize it. Make up the volume to 1000 mL.

[0023] The phosphate buffer solution is prepared as follows: Weigh 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate and dissolve them in 800 mL of distilled water. Add 1 mol / L sodium hydroxide solution dropwise until the solution pH=8.0, and then bring the volume to 1000 mL.

[0024] Preparation of 1 U / mL amylase solution: Weigh 10 mg of amylase and dissolve it in 100 mL of phosphate buffer. After centrifugation (3000 rpm, 10 min), take the supernatant to obtain 1 U / mL lipase solution. Example 1

[0025] Instrument precision test.

[0026] Preparation of glucose standard solutions: Weigh 1 mg, 2 mg, 4 mg, 6 mg, 8 mg and 10 mg respectively, add to 8 mL of distilled water to dissolve and make up to 10 mL to obtain glucose standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg / mL.

[0027] Precisely prepared glucose standard solutions with different concentration gradients (0.1, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL) were used. A glucose standard curve was prepared using the DNS method. The specific steps were as follows: 1.5 mL of phosphate buffer and 0.5 mL of glucose standard solution were added to a centrifuge tube and incubated at 37±1℃ for 10 min. 0.5 mL of the reaction solution was mixed thoroughly with 0.5 mL of DNS solution and boiled in a water bath for 3 min. After natural cooling, 0.5 mL of the mixture was mixed with an equal volume of distilled water, and the absorbance was measured at 540 nm. A glucose standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis. The results are shown below. Figure 1 See Table 1.

[0028] Table 1 Preparation of glucose standard curve

[0029] The results are as follows Figure 1 As shown in Table 1, the RSD range of the absorbance of glucose solutions with different concentration gradients was between 0.011% and 0.022%, indicating good instrument precision. The equation for the glucose standard curve fitting is y = 1.2185x - 0.0246, R... 2 =0.9983, indicating a linear correlation between glucose concentration and absorbance. Example 2

[0030] The effect of centrifugation conditions on absorbance detection results in emulsion systems.

[0031] The preparation of Sichuan pepper essential oil emulsions of different concentrations is as follows: (1) Preparation of 10% Tween 80 solution: Take 100 mL of Tween 80 and add it to 900 mL of distilled water to dissolve and set aside; (2) Preparation of essential oil emulsions of different concentrations: Take 0.50 mL, 1.25 mL and 2.50 mL of Sichuan pepper essential oil respectively and add them to 49.5 mL, 48.75 mL and 47.5 mL of 10% Tween 80 solution to obtain 1%, 2.5% and 5% Sichuan pepper essential oil; use a high-speed homogenizer to homogenize the Sichuan pepper essential oil at 10000 rpm for 3 min; then use an ultrasonic cell disruptor to sonicate at 2000w for 30 s to obtain 1%, 2.5% and 5% Sichuan pepper essential oil emulsions.

[0032] This example investigates the effects of different centrifugation conditions and the color of the Sichuan pepper essential oil emulsion itself on absorbance detection in the DNS method by adding an equal volume of 0.5 mg / mL glucose solution to a 5% Sichuan pepper essential oil emulsion.

[0033] In the experiment, a control group was prepared by mixing 0.5 ml of distilled water with an equal volume of 0.5 mg / mL glucose solution, and a sample group was prepared by mixing 0.5 ml of 5% Sichuan pepper essential oil emulsion with an equal volume of 0.5 mg / mL glucose solution. The mixture was incubated at 37℃ for 10 min, and then the resulting product was centrifuged to break the emulsion. An orthogonal experiment was conducted using three factors: rotation speed, time, and temperature. Three levels were selected for each of the three factors (temperature, rotation speed, and time), and an L9(3) experiment was performed. 3 An orthogonal experiment was conducted, using the deviation rate of the detected values ​​from the control group as the evaluation index, to optimize the centrifugation process. The results of the orthogonal experiment and the analysis of variance are shown in Tables 2 and 3.

[0034] After centrifugation and demulsification, 0.5 mL of the supernatant was mixed with an equal volume of DNS solution and then boiled in a water bath for 3 min. Then, 0.5 mL of the reaction solution after boiling in a water bath was mixed with an equal volume of distilled water, and the absorbance was measured at 540 nm and the absorbance deviation rate was calculated.

[0035] Table 2. Results of the orthogonal experiment

[0036] Table 3. Analysis of Variance of Orthogonal Experiments

[0037] Note: * indicates p < 0.05, and the deviation rate is calculated as (experimental value - true value) / true value * 100%.

[0038] The range analysis in Tables 2 and 3 shows that the order of importance of the three factors affecting the accuracy of absorbance detection is: rotation speed > temperature > time. Furthermore, factor A is most suitable with A3, factor B with B3, and factor C with C3. Therefore, the optimal experimental combination is A3B3C3, i.e., rotation speed 9000 rpm / min, time 15 min, and temperature 5℃. The variance analysis results show that rotation speed has a significant impact on the detection results. Among the nine experimental groups of this invention, experiment 9 has the smallest deviation in absorbance detection results, at 3%. Example 3

[0039] The accuracy of centrifugation demulsification method in detecting amylase activity in Sichuan pepper essential oil emulsion.

[0040] α-Amylase hydrolyzes α-(1,4) glycosidic bonds in starch to produce reducing sugars. The DNS method is a common method for detecting reducing sugar levels in solution systems. Reducing sugars contain free aldehyde (-CHO) or ketone (-C=O) groups in their molecular structure. Under boiling water bath conditions, the reducing groups of the reducing sugar reduce the nitro (-NO2) group in the DNS molecule to an amino group (-NH2), exhibiting a characteristic absorption peak around 540 nm. Detecting reducing sugars in essential oil emulsion systems can be distorted by the color of the emulsion system.

[0041] This example compares the accuracy of optimal centrifugation conditions in detecting reducing sugar levels in essential oil emulsions of different concentrations and calculates amylase activity, as follows: blank group, control group and sample group were set up; Blank group: Add 1 mL of phosphate buffer and 1 mL of 1% starch solution to centrifuge tubes; Control group: 0.5 mL phosphate buffer, 0.5 mL amylase solution and 1 mL 1% starch solution were added to centrifuge tubes; Sample group: Add 0.5 mL of Sichuan pepper essential oil emulsion (concentration of 1%, 2.5% or 5%, preparation method as described in Example 2), 0.5 mL of amylase solution and 1 mL of 1% starch solution to centrifuge tubes; Then, each group was incubated at 37℃ for 10 min, followed by demulsification by centrifugation (9000 rpm / min, 15 min, 5℃). 0.5 mL of the supernatant was mixed with an equal volume of DNS solution and then boiled in a water bath for 3 min. Next, 0.5 mL of the reaction solution after the water bath was mixed with an equal volume of distilled water, and the absorbance was measured at 540 nm. Amylase activity was calculated according to Formula 1. A control group without demulsification by centrifugation was used. Results are shown below. Figure 2 .

[0042] Formula 1: Amylase activity (%) = [A (sample) - A (blank)] / [A (control) - A (blank)] × 100%.

[0043] Test results as follows Figure 2 As shown, the amylase activity in the essential oil emulsions without centrifugation-demulsification treatment was significantly higher than that in the centrifugation-demulsification treatment group. This indicates that the color of the essential oil emulsion significantly affects the detection of reducing sugar levels, leading to inflated results. However, the results after centrifugation-demulsification treatment showed that 1% Sichuan pepper essential oil emulsion did not affect amylase activity, while 2.5% and 5% Sichuan pepper essential oil emulsions significantly inhibited amylase activity. Furthermore, the RSD values ​​of amylase activity measured by the centrifugation-demulsification method were between 0.015% and 0.026%, while the RSD values ​​of amylase activity measured by the direct detection method without centrifugation-demulsification treatment were between 0.045% and 0.059%. Therefore, this invention can effectively avoid the deviation of reducing sugar levels from actual values ​​caused by the color of the essential oil emulsion system itself, thus accurately reflecting amylase activity.

Claims

1. A method for detecting amylase activity in Sichuan pepper essential oil emulsion, characterized in that, Includes the following steps: S1. Instrument and reagent preparation; Prepare the instruments and reagents required for the test; S2. Solution preparation: Prepare DNS solution, 1% starch solution, 1 U / mL amylase solution, 1 mol / L NaOH solution, and phosphate buffer solution for later use; S3. Preparation of Sichuan pepper essential oil emulsions with different concentrations; S4. The amylase activity in Sichuan pepper essential oil emulsion was determined by a modified 3,5-dinitrosalicylic acid method. Blank group: Add 1 mL of phosphate buffer and 1 mL of 1% starch solution to centrifuge tubes; Control group: 0.5 mL phosphate buffer, 0.5 mL amylase solution and 1 mL 1% starch solution were added to centrifuge tubes; Sample group: Add 0.5 mL of Sichuan pepper essential oil emulsion, 0.5 mL of amylase solution, and 1 mL of 1% starch solution to centrifuge tubes; Then incubate at 37±1℃ for 8-12 min, break the emulsion by centrifugation, take an appropriate amount of supernatant and mix it with an equal volume of DNS solution, then boil in water for 3 min, then take 0.5 mL and mix it with an equal volume of distilled water, and detect the absorbance at 540 nm. Calculate the amylase activity according to Formula 1. Formula 1: Amylase activity (%) = [A (sample) - A (blank)] / [A (control) - A (blank)] × 100%.

2. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 1, characterized in that, The DNS solution preparation method in step S2 is as follows: (1) Weigh 6.3 g of DNS, add 200 mL of distilled water and stir to dissolve to obtain DNS solution; (2) Weigh 21.0 g of sodium hydroxide and add it to 100 mL of distilled water and stir to dissolve, then add the dissolved NaOH solution to the DNS solution; (3) Weigh 182.0 g of potassium sodium tartrate, dissolve it in 200 mL of distilled water and mix it with the solution obtained in step (2); (4) Add 5.0 g of phenol and 5.0 g of sodium metabisulfite to the solution obtained in step (3) and stir until the solution is completely clear, then dilute to 1000 mL.

3. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 1, characterized in that, In step S2, the 1 U / mL amylase solution is prepared by weighing 10 mg of amylase and dissolving it in 100 mL of phosphate buffer. After centrifugation, the supernatant is collected to obtain a 1 U / mL lipase solution.

4. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 1, characterized in that, The method for preparing the 1% starch solution in step S2 is as follows: Weigh 10.0 g of starch, add it to 200 mL of distilled water and stir to dissolve. Then add it to 500 mL of boiling water to gelatinize and bring the volume up to 1000 mL.

5. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 1, characterized in that, The phosphate buffer solution in step S2 is prepared as follows: Weigh 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate and dissolve them in 800 mL of distilled water. Add 1 mol / L sodium hydroxide solution dropwise until the solution pH=8.0, and then bring the volume to 1000 mL.

6. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 1, characterized in that, The preparation of different concentrations of Sichuan pepper essential oil emulsion in step S3 is as follows: (1) Preparation of 10% Tween 80 solution: Take 100 mL of Tween 80 and add it to 900 mL of distilled water to dissolve and set aside; (2) Preparation of different concentrations of essential oil emulsion: Take 0.50 mL, 1.25 mL and 2.50 mL of Sichuan pepper essential oil respectively and add them to 49.5 mL, 48.75 mL and 47.5 mL of 10% Tween 80 solution to obtain 1%, 2.5% and 5% Sichuan pepper essential oil; after homogenization and sonication, obtain 1%, 2.5% and 5% Sichuan pepper essential oil emulsion.

7. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 6, characterized in that, The Sichuan pepper essential oil was homogenized using a high-speed homogenizer at 9000-11000 rpm for 2-5 minutes; then it was sonicated using an ultrasonic cell disruptor at 1500-2500W for 20-40 seconds.

8. The method for detecting amylase activity in Sichuan pepper essential oil emulsion according to claim 1, characterized in that, The centrifugation demulsification conditions in step S4 are: rotation speed 9000 rpm / min, time 15 min, and temperature 5℃.

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