Nano-enzyme gel microspheres, preparation method thereof and application of nano-enzyme gel microspheres in edible oil detection

Abstract

The invention provides nano-enzyme gel microspheres and a preparation method and application thereof in edible oil detection, the preparation method of the nano-enzyme gel microspheres comprises the following steps: 1) weighing ferric chloride and ferrous chloride, ultrasonically dissolving the ferric chloride and ferrous chloride in ultrapure water, and sequentially adding a sodium hydroxide solution and citric acid for reaction under the conditions of nitrogen protection and the like; (2) after the reaction is finished, cooling, washing to be neutral by using ultrapure water through magnetic attraction separation, and concentrating to obtain a Fe3O4 (at) COOH nano-enzyme solution; 3) adding the nano-enzyme solution into a sodium alginate solution, performing ultrasonic treatment, and stirring to obtain a mixed solution; and sucking the mixed solution by using an injector, dropwise adding the mixed solution into a calcium chloride solution at a constant speed, curing, washing and drying to obtain the Fe3O4 (at) COOH nano-enzyme gel microspheres. The invention belongs to the technical field of biology, and provides Fe3O4 (at) COOH nano-enzyme gel microspheres which have the advantages of being good in stability, high in catalytic rate, convenient to recycle and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a nano-enzyme gel microsphere, a preparation method thereof and application thereof in edible oil detection. BACKGROUND

[0002] The unsaturated fatty acids contained in plant edible oil are prone to oxidation and deterioration under the influence of oxygen, light, heat and other factors. Edible oil with excessive oxidation degree will not only destroy the taste, smell and nutrition of food, but also seriously endanger human health. Therefore, peroxide value (POV) as an important indicator for judging the oxidation degree of edible oil plays an important role in the detection of oil quality. According to the GB 5009.227-2023 "National Food Safety Standard Determination of Peroxide Value in Food" of China, the peroxide value of edible plant oil should not be higher than 0.25g / 100g.

[0003] With the pursuit of a better life and the pursuit of high-quality food, it is of great significance to detect the peroxide value of edible oil simply, quickly and efficiently to protect the quality of edible oil and the safety of human life and health. At present, the methods for detecting the POV value of edible oil mainly include iodometric method, spectroscopic method and chromatographic method. The iodometric method is simple and easy to operate, but it is easily affected by human factors, resulting in unsatisfactory accuracy and repeatability of the detection results. The spectroscopic method and the chromatographic method rely on expensive large instruments and require professional technicians to operate and use a large amount of organic reagents, which greatly limits the routine needs and simple use of people in daily life. The paper entitled "Fe3O4@COOH Nano-enzyme Colorimetric Detection of Peroxide Value of Edible Pure Plant Oil" published by the applicant in the journal "Food Science" discloses a colorimetric method for detecting the peroxide value of edible plant oil by using Fe3O4@COOH nano-enzyme, which has the advantages of simplicity, rapidness, sensitivity and high efficiency, and provides a new idea for the detection of the peroxide value of edible plant oil. SUMMARY

[0004] To solve the problems in the prior art, the present inventors have made further research on the basis of the previous paper results and found that Fe3O4@COOH nano-enzyme is prone to aggregation due to its extremely small particle size and its own magnetism, which leads to stability problems and further reduces the affinity of Fe3O4@COOH nano-enzyme to the substrate and easily affects the colorimetric value. On the basis of this discovery, the inventors have made a large number of experimental explorations and optimizations and provided a Fe3O4@COOH nano-enzyme gel microsphere, which has the advantages of good stability, high catalytic rate and easy recovery. By establishing a related colorimetric method, the application of the Fe3O4@COOH nano-enzyme gel microsphere in the detection of the peroxide value of edible oil is realized, the shortcomings of the magnetic nano-enzyme, such as weak affinity to the substrate due to easy aggregation and easy influence on the colorimetric value due to dispersion of the nano-particles in the oil, are overcome, and the dependence on expensive large instruments is avoided.

[0005] The object of the present application will be further illustrated by the following detailed description. Preparation

[0006] In one aspect, the present application provides a nanoscale enzyme gel microsphere, and a preparation method thereof, comprising the following steps:

[0007] 1) Weigh ferric chloride or ferric chloride hydrate, ferrous chloride or ferrous chloride hydrate, and ultrasonically dissolve them in ultrapure water. Under the conditions of nitrogen protection, 78-82℃ water bath and stirring, sodium hydroxide solution, citric acid or citric acid hydrate are sequentially added to react for 55-65 min;

[0008] 2) After the reaction is completed, cool, separate by magnetic attraction, and wash with ultrapure water until neutral. Concentrate to obtain a Fe3O4@COOH nanoscale enzyme solution;

[0009] 3) Add the Fe3O4@COOH nanoscale enzyme solution to a sodium alginate solution, and simultaneously perform ultrasonic treatment and stirring to obtain a mixed solution. Then, use a syringe to draw the mixed solution and drop it into a calcium chloride solution at a constant speed. After solidification, wash with ultrapure water and dry to obtain a Fe3O4@COOH nanoscale enzyme gel microsphere.

[0010] By using the above technical solution, the nanoscale enzyme gel microsphere with peroxidase-like activity is prepared by using a chemical co-precipitation method combined with an ion cross-linking method, and has the advantages of good stability, high catalytic rate, and easy recovery.

[0011] Preferably, the solidification time is 28-35 min; and the average particle size of the Fe3O4@COOH nanoscale enzyme gel microsphere is 2.8-3.2 mm.

[0012] Preferably, the solidification time is 29.5-30.5 min; and the average particle size of the Fe3O4@COOH nanoscale enzyme gel microsphere is 2.9-3.1 mm.

[0013] Preferably, the concentration of the sodium alginate solution is 25.5-26.5 g / L; and the concentration of the calcium chloride solution is 7.2-7.8 g / L.

[0014] Preferably, the concentration of the sodium alginate solution is 25.8-26.2 g / L; and the concentration of the calcium chloride solution is 7.4-7.6 g / L.

[0015] In another aspect, the present application provides a preparation method of a nanoscale enzyme gel microsphere, comprising the following steps:

[0016] 1) Weighing ferric chloride or ferric chloride hydrate, ferrous chloride or ferrous chloride hydrate, ultrasonic dissolution in ultrapure water, under the conditions of nitrogen protection, 78-82℃ water bath and stirring, adding sodium hydroxide solution, citric acid or citric acid hydrate in turn, and reacting for 55-65min;

[0017] 2) After the reaction is completed, cooling, separation by magnetic attraction and washing with ultrapure water until neutral, and concentration to obtain Fe3O4@COOH nanoscale enzyme solution;

[0018] 3) The Fe3O4@COOH nanoscale enzyme solution is added to a sodium alginate solution, ultrasonic treatment is carried out at the same time, stirring is carried out to obtain a mixed solution; then the mixed solution is taken by a syringe and is dropped into a calcium chloride solution at a constant speed, after solidification, washing with ultrapure water and drying, Fe3O4@COOH nanoscale enzyme gel microspheres are obtained.

[0019] In another aspect, the application also provides the application of the nanoscale enzyme gel microspheres in edible oil detection, and the detection method adopts an enzyme-labeled instrument colorimetric method or a smart phone digital picture colorimetric method; the index of the edible oil detection is a peroxide value.

[0020] Preferably, the edible oil includes rapeseed oil, peanut oil, corn oil, soybean oil and sunflower seed oil.

[0021] In another aspect, the application also provides an enzyme-labeled instrument colorimetric method for detecting the peroxide value of edible oil by using the nanoscale enzyme gel microspheres, which comprises the following steps: the Fe3O4@COOH nanoscale enzyme gel microspheres, HAc-NaAc buffer, a series of edible oil with specific peroxide values or edible oil samples and 3,3',5,5'-tetramethylbenzidine are added into a centrifugal tube, mixed uniformly, reacted for 5.5-7.5min, the nanoscale enzyme gel microspheres are separated by magnetic attraction, the supernatant is taken to detect the absorbance value at 652nm in an enzyme-labeled instrument hole plate, a standard curve for detecting the peroxide value of edible oil is established according to the absorbance values of the series of edible oil, and the peroxide value of the edible oil sample is calculated.

[0022] In another aspect, the application also provides a smart phone digital picture colorimetric method for detecting the peroxide value of edible oil by using the nanoscale enzyme gel microspheres, which comprises the following steps: the Fe3O4@COOH nanoscale enzyme gel microspheres, HAc-NaAc buffer, a series of edible oil with specific peroxide values or edible oil samples and 3,3',5,5'-tetramethylbenzidine are added into a centrifugal tube, mixed uniformly, reacted for 5.5-7.5min, the nanoscale enzyme gel microspheres are separated by magnetic attraction, the supernatant is taken to detect the absorbance value at 652nm in an enzyme-labeled instrument hole plate, a standard curve for detecting the peroxide value of edible oil is established according to the absorbance values of the series of edible oil, and the peroxide value of the edible oil sample is calculated.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] (1) The present application is based on the previous paper results, and through in-depth research, it is found that Fe3O4@COOH nanoscale enzyme is easy to agglomerate, which causes stability problems, and further reduces the substrate affinity of Fe3O4@COOH nanoscale enzyme and easily affects the colorimetric value. Then, a nanoscale enzyme gel microsphere with peroxidase-like activity is prepared by chemical co-precipitation combined with ion cross-linking technology, the preparation conditions of the nanoscale enzyme gel microsphere are optimized, and the color development effect is optimized. The average particle size of Fe3O4@COOH nanoscale enzyme gel microsphere is about 3mm, and the POV value of edible oil is detected in the form of microsphere, which overcomes the shortcomings of weak substrate affinity caused by the easy agglomeration of magnetic nanoscale enzyme, and shows better stability and separation characteristics. At the same time, due to the increase of more charged charges and functional groups, Fe3O4@COOH nanoscale gel microspheres show higher catalytic rate. The Km value of Fe3O4@COOH nanoscale gel microspheres for H2O2 and TMB is 1.76mmol / L and 281mmol / L, respectively, and the Vmax value is 1.641×10 -8 Ms -1 , 2.422×10 -8 Ms -1 ; The Km values are all less than HPR (horseradish peroxidase), which indicates that the microspheres have higher affinity for H2O2 and TMB. The reason is that the microspheres have a porous network structure, a large specific surface area, and active substances can be more dispersed on the surface and inside the microspheres, thereby providing more active sites. Due to the large volume of the gel microspheres, the Km value is greater than that of Fe3O4NPs (H2O2: 1.54mmol / L, TMB: 0.098mmol / L) and Fe3O4@COOH (H2O2: 0.315mmol / L, TMB: 0.702mmol / ) in the form of nanoparticles, but due to the fact that the microspheres overcome the agglomeration problem of nanoscale enzyme particles, and the surface has more charged charges and rich functional groups, the Vmax of the microspheres is higher than that of Fe3O4@COOH (H2O2: 0.629×10 -8 Ms -1 , TMB: 0.208×10 -8 Ms -1 ). Fe3O4NPs are Fe3O4 nanoparticles without carboxyl modification. The Fe3O4@COOH nanoscale gel microspheres provided by the present application can exhibit faster detection rate in the detection of actual samples.

[0025] (2) The application provides application of Fe3O4@COOH nanoscale enzyme gel microspheres in detection of edible oil, realizes direct detection of edible oil samples, and overcomes the shortcomings of weak affinity of magnetic nanoscale enzyme to substrates and influence of dispersion of nanoparticles in oil on colorimetric value, is convenient to take and use, is easy to recycle and reuse, and has stable reusability.

[0026] (3) The application also provides an enzyme-labeled instrument colorimetric method and a smart phone digital picture colorimetric method for detecting the peroxide value of edible oil by using the Fe3O4@COOH nanoscale enzyme gel microspheres, avoids dependence on expensive large instruments, is simple to operate, and is fast in detection, and has good universality and practicability, and is conducive to guaranteeing food safety. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A condition investigation result graph of preparation of nanoscale enzyme gel microspheres.

[0028] Figure 2 A schematic view of appearance, particle size and magnetic performance of nanoscale gel microspheres.

[0029] Figure 3 An absorption spectrum graph of different colorimetric sensing systems.

[0030] Figure 4 A condition investigation result graph of catalytic activity colorimetric detection of nanoscale enzyme gel microspheres.

[0031] Figure 5 A storage stability and reusability stability investigation result graph of nanoscale enzyme gel microspheres.

[0032] Figure 6 An absorption spectrum graph of colorimetric detection of plant edible oils with different POVs.

[0033] Figure 7 A standard curve of the enzyme-labeled instrument colorimetric method for detecting the peroxide value of edible oil.

[0034] Figure 8 Standard curve of intelligent mobile phone digital picture colorimetry for detecting the peroxide value of edible oil.

[0035] Figure 9 B value comparison results chart of color development pictures taken by different brands of smart phones.

[0036] Figure 10 Color development chart of different types and different added amounts of vegetable oil.

[0037] Figure 11 Comparison chart of detection results of enzyme-labeled instrument colorimetry and national standard method.

[0038] Figure 12 Comparison chart of detection results of intelligent mobile phone digital image colorimetry and national standard method.

[0039] Figure 13 Color development chart of different detection methods.

[0040] Figure 14 Investigation results chart of the influence of plant-based edible oil in different oxidation modes on enzyme-labeled instrument colorimetry and intelligent mobile phone digital image colorimetry. DETAILED DESCRIPTION

[0041] The application will be further described in detail below in combination with the drawings and examples.

[0042] In the application, the reagents and equipment involved are all conventional commercially available products, or can be obtained by conventional technical means in the art.

[0043] Example 1 Preparation of nano-enzyme gel microspheres

[0044] Different concentrations of sodium alginate solution (10, 14, 18, 22, 26, 30, 34 g / L) and calcium chloride solution (2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 g / L), Fe3O4@COOH nano-enzyme solution addition amount (1, 3, 7, 9, 11, 13 mL) and curing reaction time (5, 10, 15, 20, 25, 30, 35 min) were selected for the condition optimization of Fe3O4@COOH nano-enzyme gel microsphere preparation, and the results are shown in Figure 1 Among them, Figure 1 -A is the sodium alginate concentration investigation results chart; Figure 1 -B is the calcium chloride concentration investigation results chart; Figure 1 -C is the Fe3O4@COOH nano-enzyme solution addition amount investigation results chart; Figure 1 -D is the curing reaction time investigation results chart.

[0045] The Fe3O4@COOH nano-enzyme gel microspheres, the optimized preparation method thereof comprises the following steps:

[0046] 1) Take 5.02 g FeCl3·6H2O, 1.9 g FeCl2·4H2O, ultrasonic dissolve in 100 mL ultrapure water and transfer to a 250 mL four-necked flask, under the conditions of nitrogen protection, 80℃ water bath and 550 r / min stirring speed, add 40 mL 2 mol / L NaOH solution, 1.2 g citric acid monohydrate in turn, and react for 60 min;

[0047] 2) After the reaction is completed, cool, remove the supernatant by magnetic separation, and wash with ultrapure water until neutral, concentrate to obtain Fe3O4@COOH nanoscale enzyme solution;

[0048] 3) 9 mL of the Fe3O4@COOH nanoscale enzyme solution (concentration of 0.17 g / mL) is added to 50 mL of 26 g / L sodium alginate solution, and ultrasonic treatment is carried out at the same time, and stirring is carried out to obtain a mixed solution; then the mixed solution is sucked by a syringe, and is dropped into 100 mL of 7.5 g / L calcium chloride solution at a uniform speed of 0.722 mL / min by a peristaltic pump, and after solidification for 30 min, after washing with ultrapure water, drying is carried out to obtain nanoscale enzyme gel microspheres, which are stored at 4℃.

[0049] Example Two Investigation on Physical and Chemical Properties of Nanoscale Enzyme Gel Microspheres

[0050] Take the Fe3O4@COOH nanoscale enzyme gel microspheres prepared in Example One, observe the morphology characteristics, and measure the particle size and magnetic properties, and the results are shown in Figure 2 . Among them, Figure 2 -A is the morphology diagram of Fe3O4@COOH nanoscale enzyme gel microspheres; Figure 2 -B is a schematic diagram of Fe3O4@COOH nanoscale enzyme gel microspheres particle size test; Figure 2 -C is a schematic diagram of Fe3O4@COOH nanoscale enzyme gel microspheres magnetic property test.

[0051] Take one Fe3O4@COOH nanoscale enzyme gel microsphere prepared in Example One in a 1.5 mL centrifuge tube, then add 800 μL (0.2 M, pH = 3.5) sodium acetate buffer, 100 μL H2O2 (0.08 M), 50 μL TMB (0.008 M, dissolved with DMSO) in turn, mix uniformly, and stand for reaction under light shielding conditions for 5 min. After the reaction is completed, take 100 μL supernatant in a 96 enzyme label well plate, scan the absorption spectrum in the range of 550-750 nm, and the results are shown in Figure 3 . It can be known from Figure 3 that the Fe3O4@COOH nanoscale enzyme gel microspheres prepared in Example One have a catalytic activity similar to that of POD, and the maximum absorption peak wavelength is 652 nm when TMB is used as a chromogenic substrate.

[0052] Taking the Fe3O4@COOH nanoscale enzyme gel microspheres prepared in Example One, the catalytic peroxide to obtain hydroxyl radicals can oxidize the color developing substrate TMB to ox-TMB, and the absorbance value is detected at 652 nm. The effects of nanoscale enzyme gel microspheres addition amount (1, 2, 3, 4, 5, 6) and buffer pH value (3, 3.5, 4, 4.5, 5, 5.5, 6), TMB concentration (0.006, 0.0008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10 mol / L) and reaction time (1, 2, 3, 4, 5, 6, 7, 8) on the catalytic activity of Fe3O4@COOH nanoscale enzyme gel microspheres are investigated to realize the optimization of colorimetric detection conditions, and the results are shown in Figure 4 , wherein, Figure 4 -A is the addition amount of Fe3O4@COOH nanoscale enzyme gel microspheres; Figure 4 -B is the buffer pH value; Figure 4 -C is the TMB concentration; Figure 4 -D is the reaction time.

[0053] Taking the nanoscale enzyme gel microspheres prepared in Example One, the storage stability and repeated use stability are investigated, and the results are shown in Figure 5 From Figure 5 it can be seen that the storage stability and repeated use stability of the Fe3O4@COOH nanoscale enzyme gel microspheres prepared by the application are good, which is convenient for popularization and application and reduces the cost.

[0054] Example Three Colorimetric method for detecting the peroxide value of edible oil by using nanoscale enzyme gel microspheres

[0055] In order to verify the feasibility of the colorimetric detection of plant edible oil POV by the nanoscale enzyme gel microspheres prepared in Example One, colorimetric experiments were carried out on oil samples with different oxidation degrees. 2 nanoscale enzyme gel microspheres, 800 μL of HAc-NaAc buffer (0.2 M, pH=4), 200 μL of oil sample (0.0562 g / 100 g with different oxidation degrees), and 100 μL of TMB (0.06 M) were added to a centrifuge tube, mixed uniformly, reacted for 6 min, and an equal volume of supernatant was taken to a 96-well enzyme-labeled hole plate, the color change of the reaction system solution was observed, and the absorption spectrum in the range of 550-750 nm was scanned, and the results are shown in Figure 6 From Figure 6 it can be seen that without pretreatment of plant edible oil, the Fe3O4@COOH nanoscale enzyme gel microspheres provided by the application can be directly applied to the detection of plant edible oil POV.

[0056] The microsphere gel of Fe3O4@COOH nanoscale enzyme is used for detecting the peroxide value of edible oil by microplate colorimetry, including the following steps: the microsphere gel of Fe3O4@COOH nanoscale enzyme, HAc-NaAc buffer, a series of edible oil with specific peroxide value or edible oil sample, 3, 3', 5, 5'-tetramethylbenzidine are added into a centrifuge tube, mixed uniformly, after 6 min of reaction, the microsphere gel of Fe3O4@COOH nanoscale enzyme is separated by magnetic attraction, the supernatant is taken to detect the absorbance value at 652 nm in the microplate, a standard curve for detecting the peroxide value of edible oil is established according to the absorbance value of the series of edible oil, and the peroxide value of the edible oil sample is calculated. The standard curve is established as shown in Figure 7 .

[0057] 2 microspheres of Fe3O4@COOH nanoscale enzyme, 800 μL of HAc-NaAc buffer (0.2 M, pH = 4), 200 μL of oil sample (0.0889 g / 100 g with different oxidation degrees), 100 μL of TMB (0.06 M) are added into a centrifuge tube, mixed uniformly, after a certain time of reaction, a color developing picture is taken by a smart phone and the RGB value is calculated, and the results are shown in Table 1. According to the RGB value in Table 1, a quantitative detection model is tried to be established, as shown in Table 2. It can be known from Table 2 that the linear equation established by B value and POV value has a better correlation.

[0058] Table 1 RGB value of color after developing of oil sample with different POV

[0059]

[0060] Table 2 Different quantitative models and linear equations

[0061]

[0062] The microsphere gel of Fe3O4@COOH nanoscale enzyme is used for detecting the peroxide value of edible oil by microplate colorimetry, including the following steps: the microsphere gel of Fe3O4@COOH nanoscale enzyme, HAc-NaAc buffer, a series of edible oil with specific peroxide value or edible oil sample, 3, 3', 5, 5'-tetramethylbenzidine are added into a centrifuge tube, mixed uniformly, after 6 min of reaction, the microsphere gel of Fe3O4@COOH nanoscale enzyme is separated by magnetic attraction, the supernatant is taken to detect the absorbance value at 652 nm in the microplate, a standard curve for detecting the peroxide value of edible oil is established according to the absorbance value of the series of edible oil, and the peroxide value of the edible oil sample is calculated. The standard curve is established as shown in Figure 8 . The B value of the color developing picture taken by different brands of smart phones is compared as shown in Figure 9As shown in the table, the B values of the color-developed pictures taken by different brands of smartphones are very close, indicating that the method has no strict requirements for smartphones and is highly practical.

[0063] Further, the present application takes different types and different amounts of vegetable oil as the object, and simultaneously detects the RGB values of the color-developed pictures by the above-mentioned enzyme photometer colorimetric method. The color-developed pictures are as shown in the table, wherein A: peanut oil; B: sunflower oil; C: soybean oil; D: corn oil; E: rapeseed oil. The POV, RGB and absorbance values of the vegetable oils of different types and different amounts after colorimetric detection are as shown in Table 3. Figure 10

[0064] Table 3: POV, RGB and absorbance values of vegetable oils of different types and different amounts after colorimetric detection

[0065]

[0066] The present application adopts the determination coefficient (R 2 ) and the root mean square error (RMSE) as the evaluation indexes of the colorimetric detection method. The colorimetric detection methods (enzyme photometer colorimetric method and smartphone digital image colorimetric method) are used to quantitatively detect the POV (the measured results are identified as prediction values) of different types of vegetable edible oils with unknown oxidation degree, and the results of the titration method (GB5009.227-2016) in the national standard method are used as the standard (the measured results are identified as true values). The comparison of the detection results of the enzyme photometer colorimetric method and the national standard method is as shown in the table. Figure 11 The comparison of the detection results of the smartphone digital image colorimetric method and the national standard method is as shown in the table. Figure 12 From the tables, Figure 11 and Figure 12 It can be seen that the results of the enzyme photometer colorimetric method and the smartphone digital image colorimetric method provided by the present application in a large number of sample detection are highly consistent with the detection results of the national standard method, indicating that the accuracy is better.

[0067] In order to ensure the universal applicability of the test method, five representative types of vegetable edible oils (rapeseed oil, peanut oil, sunflower oil, corn oil, and soybean oil) in the market and edible oils of different oxidation degrees produced by different manufacturers (a total of more than 200 oil samples) are selected, and the peroxide value of the oil samples is detected by using the enzyme photometer colorimetric method, the smartphone digital image colorimetric method and the commercially available peroxide value rapid detection test paper. The color-developed pictures of different detection methods are as shown in the table. Figure 13 The measured results are compared with the results of the commercially available detection test paper, and the average value is as shown in Table 4. From Table 4, it can be seen that the accuracy of the measured results of the enzyme photometer colorimetric method and the smartphone digital image colorimetric method provided by the present application is better than that of the commercially available detection test paper method.

[0068] Table 4: Comparison of the measured results of different detection methods​

[0069]

[0070] In addition, the present application also investigates the influence of different oxidation methods of plant edible oil on the detection of enzyme marker colorimetric method and smart phone digital image colorimetric method, and the results are shown in Figure 14 Figure 14 It can be seen that different oxidation methods of plant edible oil samples do not have significant influence on the detection of the enzyme marker colorimetric method and the smart phone digital image colorimetric method provided by the present application, which shows that the enzyme marker colorimetric method and the smart phone digital image colorimetric method provided by the present application have wide application range and strong practicability.

[0071] The above is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application cannot be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, some simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be regarded as falling within the protection scope of the present application.​

Claims

1. A nanoscale enzyme gel microsphere, characterized in that: The preparation method comprises the following steps: 1) weigh ferric chloride or ferric chloride hydrate, ferrous chloride or ferrous chloride hydrate, and ultrasonically dissolve them in ultrapure water; under the conditions of nitrogen protection, 78-82 DEG C water bath and stirring, sodium hydroxide solution, citric acid or citric acid hydrate are sequentially added to react for 55-65 min; 2) after the reaction is completed, cooling, magnetic separation and washing with ultrapure water to neutral, and concentration to obtain Fe3O4@COOH nanoscale enzyme solution; 3) the Fe3O4@COOH nanoscale enzyme solution is added to sodium alginate solution, and ultrasonic treatment and stirring are carried out to obtain a mixed solution; then the mixed solution is taken with a syringe and dropped into a calcium chloride solution at a constant speed, and after solidification, washing with ultrapure water and drying, Fe3O4@COOH nanoscale enzyme gel microspheres are obtained.

2. The Fe304@COOH nanoszyme hydrogel microspheres according to claim 1, characterized in that: The solidification time is 28-35 min; the average particle size of the Fe3O4@COOH nanoscale enzyme gel microspheres is 2.8-3.2 mm.

3. The Fe304@COOH nanoszyme hydrogel microspheres according to claim 2, characterized in that: The solidification time is 29.5-30.5 min; the average particle size of the Fe3O4@COOH nanoscale enzyme gel microspheres is 2.9-3.1 mm.

4. The Fe304@COOH nanoszyme hydrogel microspheres according to claim 1, characterized in that: The concentration of the sodium alginate solution is 25.5-26.5 g / L; the concentration of the calcium chloride solution is 7.2-7.8 g / L.

5. The Fe304@COOH nanoszyme hydrogel microspheres according to claim 4, characterized in that: The concentration of the sodium alginate solution is 25.8-26.2 g / L; the concentration of the calcium chloride solution is 7.4-7.6 g / L.

6. A method for preparing a nano-enzyme gel microsphere, characterized in that: comprise the following steps: 1) weigh ferric chloride or ferric chloride hydrate, ferrous chloride or ferrous chloride hydrate, and ultrasonically dissolve them in ultrapure water; under the conditions of nitrogen protection, 78-82 DEG C water bath and stirring, sodium hydroxide solution, citric acid or citric acid hydrate are sequentially added to react for 55-65 min; 2) after the reaction is completed, cooling, magnetic separation and washing with ultrapure water to neutral, and concentration to obtain Fe3O4@COOH nanoscale enzyme solution; 3) the Fe3O4@COOH nanoscale enzyme solution is added to sodium alginate solution, and ultrasonic treatment and stirring are carried out to obtain a mixed solution; then the mixed solution is taken with a syringe and dropped into a calcium chloride solution at a constant speed, and after solidification, washing with ultrapure water and drying, Fe3O4@COOH nanoscale enzyme gel microspheres are obtained.

7. The use of the nanoscale enzyme gel microspheres according to any one of claims 1 to 5 in the detection of edible oils, characterized by: The detection method adopts enzyme label instrument colorimetric method or smart phone digital picture colorimetric method; the index of edible oil detection is peroxide value.

8. The application of Fe304@COOH nanoszyme gel microspheres in edible oil detection according to claim 7, characterized in that: The edible oil comprises rapeseed oil, peanut oil, corn oil, soybean oil and sunflower seed oil.

9. An enzyme label instrument colorimetric method for detecting the peroxide value of edible oil by using nanoscale enzyme gel microspheres, characterized by: The method comprises the following steps: adding Fe3O4@COOH nanoscale enzyme gel microspheres according to any one of claims 1 to 5, HAc-NaAc buffer, a series of edible oils with specific peroxide values or edible oil samples, and 3,3',5,5'-tetramethylbenzidine into a centrifuge tube, mixing uniformly, separating the Fe3O4@COOH nanoscale enzyme gel microspheres by magnetic attraction after 5.5-7.5 min of reaction, taking the supernatant to detect the absorbance value at 652 nm in an enzyme-labeled well plate, establishing a standard curve for detecting the peroxide value of edible oil according to the absorbance values of the series of edible oils, and calculating the peroxide value of the edible oil sample.

10. A smartphone digital picture colorimetric method for detecting the peroxide value of edible oil by using nanoscale enzyme gel microspheres, characterized by: The method comprises the following steps: adding Fe3O4@COOH nanoscale enzyme gel microspheres according to any one of claims 1 to 5, HAc-NaAc buffer, a series of edible oils with specific peroxide values or edible oil samples, and 3,3',5,5'-tetramethylbenzidine into a centrifuge tube, mixing uniformly, separating the Fe3O4@COOH nanoscale enzyme gel microspheres by magnetic attraction after 5.5-7.5 min of reaction, taking the supernatant to detect the absorbance value at 652 nm in an enzyme-labeled well plate, establishing a standard curve for detecting the peroxide value of edible oil according to the absorbance values of the series of edible oils, and calculating the peroxide value of the edible oil sample.

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