Biogenic amine detection kit based on double-enzyme nanoparticles as well as preparation method and application of biogenic amine detection kit

Abstract

The invention discloses a biogenic amine detection kit based on double-enzyme nanoparticles as well as a preparation method and application of the biogenic amine detection kit. The biogenic amine detection kit based on the double-enzyme nanoparticles comprises a biogenic amine recognition agent, a color developing agent and a solvent, the biogenic amine recognition agent is horse radish peroxidase / diamine oxidase-zinc phosphate nanoparticles, the color developing agent is dopamine, and the kit can specifically recognize histamine and diamine biogenic amine and can be used for detecting the content of histamine and diamine biogenic amine in a sample; when the biogenic amine detection kit is used for biogenic amine content detection, the cost is low, the operation is simple and rapid, the detection kit is environment-friendly, expensive detection equipment and professional technicians are not needed, the result can be obtained in the whole detection process within a short time, and the biogenic amine detection kit is suitable for rapid detection of biogenic amine in food.

Description

Technical Field

[0001] The present invention relates to the field of food safety detection, and in particular to a biogenic amine detection kit based on dual-enzyme nanoparticles, and a preparation method and application thereof. Background Art

[0002] Biogenic amines are alkaline nitrogen compounds produced by amino acid decarboxylation during food spoilage due to microbial activity or endogenous tissue metabolism. Aquatic foods are particularly prone to biogenic amine accumulation due to their own biochemical characteristics. Histamine (HA) is the most toxic substance among biogenic amines. It is produced by histidine under the action of decarboxylase. Studies have shown that histamine intake of 80 to 400 mg kg -1 Fish meat may have a mild poisoning reaction, and when the intake concentration exceeds 1000 mg kg -1 There will be serious poisoning reactions, including vomiting, dizziness, diarrhea, allergies and other symptoms. Severe cases can also lead to shock and even endanger life.

[0003] At present, the detection methods of biogenic amines mainly include high performance liquid chromatography, fluorescence quantitative method, ion exchange chromatography, capillary electrophoresis and enzyme-linked immunosorbent assay. For example, the invention patent with application publication number CN 111175403 A discloses a method for detecting histamine in products by liquid chromatography. Although these methods have high sensitivity, they cannot meet the market demand for rapid detection due to high requirements for sample pretreatment, time-consuming and laborious operation, the need for professional personnel, high detection costs, and expensive instruments. Therefore, there is a need for a rapid and sensitive method for detecting biogenic amines. Summary of the invention

[0004] To solve the above problems, in a first aspect, the present invention provides a biogenic amine detection kit based on dual-enzyme nanoparticles, which can quickly and cost-effectively detect the content of biogenic amines in food.

[0005] The technical solution of the present invention to solve the above problems is as follows:

[0006] A biogenic amine detection kit based on dual-enzyme nanoparticles comprises: a biogenic amine recognition agent, a color developer and a solvent; the biogenic amine recognition agent is horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles; the color developer is a color developer that can react in the presence of a reaction product of a biogenic amine and the biogenic amine recognition agent.

[0007] Preferably, the biogenic amines include histamine and diamine biogenic amines.

[0008] Preferably, the concentration of the biogenic amine recognition agent is 10-40 mg / mL.

[0009] Preferably, the developer is dopamine.

[0010] Preferably, the developer is dopamine hydrochloride, and the concentration of the developer is 0.0625-1.250 g / mL.

[0011] Preferably, the solvent is selected from at least one of sodium acetate buffer, Tris-HCl buffer, and phosphate buffered saline.

[0012] Preferably, the preparation method of the horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles is: adding zinc salt solution to a phosphate buffered saline solution containing horseradish peroxidase and diamine oxidase, mixing evenly, and centrifuging to obtain a precipitate to obtain the horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles.

[0013] Preferably, the mass concentration ratio of the horseradish peroxidase to the diamine oxidase is (1-2):(1-2).

[0014] Preferably, the concentration of the horseradish peroxidase is 0.5-10 mg / mL; the concentration of the diamine oxidase is 0.5-10 mg / mL.

[0015] Enzymes are the most commonly used biorecognition elements in biosensors, but natural enzymes have problems such as easy inactivation and poor chemical stability. Therefore, how to ensure that the enzyme has high activity and stability, improve the convenience and effect of detection, and improve the detection efficiency has become a problem worth exploring. The use of biomineralization to prepare inorganic hybrid nanoparticles can effectively solve the above problems. The method is simple, and the prepared enzyme has high activity and stability and can be used in detection experiments.

[0016] Diamine oxidase (DAO) is widely present in animal tissues (intestinal mucosa, lungs, liver, kidneys, etc.), plant tissues and microorganisms. It is an enzyme that catalyzes the oxidation of histamine and diamine biogenic amines to produce substances such as aldehydes, hydrogen peroxide and ammonia.

[0017] Horseradish peroxidase (HRP) is widely distributed in the plant kingdom and is found in high concentrations in horseradish. It is a glycoprotein composed of a colorless enzyme protein and a brown iron porphyrin. It is composed of multiple isozymes and its main function is to catalyze the decomposition of hydrogen peroxide into oxygen and water.

[0018] The present invention adopts the biomineralization method to simultaneously fix horseradish peroxidase and diamine oxidase, thereby ensuring that the two enzymes can have high activity and stability, and uses the prepared horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticle solution as a biogenic amine recognition agent.

[0019] Dopamine (DA) is an endogenous nitrogen-containing organic compound. It is an intermediate product produced by tyrosine metabolism via dihydroxyphenylalanine. It is also the most abundant catecholamine neurotransmitter in the brain. Dopamine also has a special property. Under the combined action of alkaline solution (pH=8.5) and oxygen, it will self-polymerize to produce black polydopamine.

[0020] Diamine oxidase catalyzes histamine and diamine biogenic amines to produce aldehydes and hydrogen peroxide, and then the hydrogen peroxide can react with horseradish peroxidase to generate oxygen, and the oxygen reacts with a color developer to generate a colored substance, and the content of histamine and diamine biogenic amines in the sample can be obtained by measuring the absorbance. Therefore, the biogenic amine detection kit of the present invention can accurately detect the content of histamine and diamine biogenic amines in the sample to be tested, and the detection reagent can also be well preserved, and it is not easy to cause the detection reagent to become ineffective due to the inactivation of the detection enzyme.

[0021] In a second aspect, the present invention provides an application of the above-mentioned dual-enzyme nanoparticle-based biogenic amine detection kit, which can be used to detect the content of histamine and diamine biogenic amines in food.

[0022] The present invention also provides a method for detecting biogenic amines in food, which requires the use of the above-mentioned biogenic amine detection kit based on dual-enzyme nanoparticles. The detection method comprises the following steps:

[0023] S1. Using a biogenic amine standard solution, a standard curve is obtained between the absorbance value of the biogenic amine solution at 465 nm and the biogenic amine concentration;

[0024] S2. Add the biogenic amine recognition agent and the color developer to the solvent, mix well and then add the sample solution to be tested. After the reaction is completed, detect the absorbance value of the reaction solution at 465nm and substitute it into the standard curve of S1 to obtain the biogenic amine concentration in the sample to be tested.

[0025] Preferably, in step S2, the reaction temperature is 25-37° C., the reaction time is 2-3 h, and the reaction pH is 5.5-7.5.

[0026] In addition, the concentration of biogenic amines in the sample to be tested can be obtained by observing the color change of the solution after the reaction in S2 is completed and comparing the standard correspondence between the solution color and the histamine concentration, or the concentration of biogenic amines in the sample to be tested can be roughly determined by the color change of the solution.

[0027] The present invention has the following beneficial effects:

[0028] (1) The biogenic amine detection kit of the present invention uses horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles as biogenic amine recognition agents and dopamine hydrochloride as a color developer, which can specifically recognize histamine and diamine biogenic amines and can be used to detect the content of histamine and diamine biogenic amines in samples;

[0029] (2) The cost of using the biogenic amine detection kit of the present invention to detect the biogenic amine content is low, the operation is simple, fast, and environmentally friendly. It does not require expensive detection equipment and professional technicians. The entire detection process can obtain results in a short time. It is suitable for the rapid detection of histamine and diamine biogenic amines in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a standard curve diagram of the detection system in Example 1;

[0031] Figure 2 This is a graph showing the specific detection results of the detection system in Example 2. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Preparation of histamine recognition agent

[0035] 50 mg of diamine oxidase and 50 mg of horseradish peroxidase were dissolved in 50 mL of phosphate buffered saline (pH = 7.4) to prepare an enzyme solution. Zinc sulfate solution was slowly added to the enzyme solution, and the mixed solution was centrifuged to obtain a precipitate, rinsed 3 times, and ultrasonically placed to obtain a supernatant to obtain a histamine recognition agent, i.e., horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles.

[0036] 2. Establishment of standard curve

[0037] Prepare 0.025, 0.25, 2.5, 5 mg / mL histamine solutions respectively, the solvent is phosphate buffered saline (PB), its pH = 6.5, take 20uL to make the final concentration of 0.5, 5, 50, 100ug / mL; prepare 125mg / mL dopamine solution as color developer, the solvent is PB, its pH = 6.5, take 40uL to make the final concentration of 5mg / mL; the concentration of histamine recognition agent is 20mg / mL, take 50uL to make the final concentration of 1mg / mL; the solvent is PB, its pH = 6.5, the system is 1mL; incubate the solution for 2h, and measure the absorbance at 465nm. Draw a standard curve with histamine concentration as the horizontal axis and absorbance value as the vertical axis. The results are as follows Figure 1 As shown, from Figure 1 It can be concluded that the detection limit is 20.40ug / mL.

[0038] Example 2

[0039] Prepare 5 mg / mL solutions of histamine, spermine, spermidine, tyramine and tryptamine respectively, with the solvent being PB and its pH being 6.5, and take 20uL to make its final concentration be 100ug / mL; prepare 125 mg / mL dopamine solution as the color developer, with the solvent being PB and its pH being 6.5, and take 40uL to make its final concentration be 5mg / mL; the concentration of the histamine recognition agent is 20mg / mL, and take 50uL to make its final concentration be 1mg / mL; the solvent is PB and its pH being 6.5, and the system is 1mL.

[0040] Control group setting: prepare 125 mg / mL dopamine solution as the color developer, the solvent is PB, its pH = 6.5, take 40uL to make its final concentration 5mg / mL; the concentration of histamine recognition agent is 20mg / mL, take 50uL to make its final concentration 1mg / mL; the solvent is PB, its pH = 6.5, and the system is 1mL.

[0041] The above solution was incubated for 2 hours and the absorbance at 465 nm was measured. Figure 2 As shown, it shows that the detection system has certain specificity for histamine and diamine biogenic amines.

[0042] Example 3

[0043] Take 5g of salmon and add it to 5mL of PB solution with a pH of 6.5. Homogenize and centrifuge to obtain the supernatant. Take 100uL of salmon as the sample solution. Prepare a 125mg / mL dopamine solution as a color developer, the solvent is PB, its pH is 6.5, take 40uL to make its final concentration 5mg / mL; take the histamine recognition agent in Example 1, prepare a solution with a concentration of 20mg / mL, take 50uL to make its final concentration 1mg / mL; the solvent is PB, its pH is 6.5, and the system is 1mL; incubate the solution for 2h, measure the absorbance at 465nm, and substitute Figure 1 The histamine content in salmon was calculated using the formula in the experiment and was found to be 0.364 mg / mL.

[0044] Example 4

[0045] Take 5g of shrimp and add it to 5mL of PB solution with a pH of 6.5. Homogenize and centrifuge to obtain the supernatant. Take 50uL of the shrimp as the sample solution. Prepare a 125mg / mL dopamine solution as a color developer, the solvent is PB, its pH is 6.5, take 40uL to make its final concentration 5mg / mL; take the histamine recognition agent in Example 1, prepare a solution with a concentration of 20mg / mL, take 50uL to make its final concentration 1mg / mL; the solvent is PB, its pH is 6.5, and the system is 1mL; incubate the solution for 2h, measure the absorbance at 465nm, and substitute Figure 1 The histamine content in shrimp was calculated using the formula in , and the content was 0.021 mg / mL.

Claims

1. A biogenic amine detection kit based on dual-enzyme nanoparticles, characterized in that: include: A biogenic amine recognition agent, a color developer and a solvent; the biogenic amine recognition agent is horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles; the color developer is a color developer that can react in the presence of a reaction product of a biogenic amine and the biogenic amine recognition agent.

2. A biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 1, characterized in that: The biogenic amines include histamine and diamine biogenic amines.

3. A biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 1, characterized in that: The concentration of the biogenic amine recognition agent is 10-40 mg / mL.

4. A biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 1, characterized in that: The color developing agent is dopamine.

5. A biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 4, characterized in that: The color developer is dopamine hydrochloride, and the concentration of the color developer is 0.0625-1.250 g / mL.

6. The biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 1, characterized in that: The solvent is selected from at least one of sodium acetate buffer, Tris-HCl buffer, and phosphate buffered saline.

7. The biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 1, characterized in that: The preparation method of the horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles comprises the following steps: adding a zinc salt solution to a phosphate buffered saline solution containing horseradish peroxidase and diamine oxidase, mixing the mixture evenly, and centrifuging to obtain a precipitate to obtain the horseradish peroxidase / diamine oxidase-zinc phosphate nanoparticles.

8. The biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 7, characterized in that: The mass concentration ratio of the horseradish peroxidase to the diamine oxidase is (1-2):(1-2).

9. The biogenic amine detection kit based on dual-enzyme nanoparticles according to claim 7, characterized in that: The concentration of the horseradish peroxidase is 0.5-10 mg / mL; the concentration of the diamine oxidase is 0.5-10 mg / mL.

10. A method for detecting biogenic amines in food, characterized in that: Using the biogenic amine detection kit based on dual-enzyme nanoparticles according to any one of claims 1 to 9, the detection method comprises the following steps: S1. Using a biogenic amine standard solution, a standard curve is obtained between the absorbance value of the biogenic amine solution at 465 nm and the biogenic amine concentration; S2. Add the biogenic amine recognition agent and the color developer to the solvent, mix well and then add the sample solution to be tested. After the reaction is completed, detect the absorbance value of the reaction solution at 465nm and substitute it into the standard curve of S1 to obtain the biogenic amine concentration in the sample to be tested.

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