Method for detecting content of heavy metals in food additive

Abstract

The invention belongs to the technical field of detection, and particularly relates to a food additive heavy metal content detection method. The detection method comprises the following steps: (1) pre-treating a sample; (2) preparing heavy metal standard solutions with a series of concentrations, and testing the net intensity of the heavy metal standard solutions by adopting an ICP-MS (Inductively Coupled Plasma Mass Spectrometry) instrument to obtain a standard working curve; and (3) testing the net strength of the sample solution and the blank solution by using an ICP-MS (Inductively Coupled Plasma Mass Spectrometry) instrument, and combining with the standard working curve to obtain the concentration of the heavy metal in the food additive so as to further obtain the content of the heavy metal in the food additive. The detection method disclosed by the invention can be used for detecting the heavy metal arsenic in the compound meat product thickening agent, the sample structure is further destroyed by virtue of enzymolysis-auxiliary extract ultrasonic synergistic extraction and gradient ultrasonic combined with temperature control, and the dissolution efficiency of the arsenic is improved; eDTA and citric acid are used for complexing metal ions, multiple components cooperate to reduce the fixation effect of other ions on arsenic, and the accuracy of a detection result is improved through the synergistic effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a method for detecting heavy metal content of food additives. BACKGROUND

[0002] With the rapid development of the food industry, compound food additives are widely used in food processing due to their versatility and process convenience. However, the residual heavy metal arsenic (As) in compound additives is an important risk factor for food safety due to its high toxicity and cumulative harm.

[0003] Currently, the detection of arsenic in compound additives mainly relies on wet digestion-atomic fluorescence spectrometry and dry ashing method, but the above methods have the following problems: wet digestion requires a large amount of strong acid (nitric acid, sulfuric acid, etc.), and the digestion process needs to be supplemented with acid several times and strictly controlled temperature to avoid charring, which is complicated to operate and easy to produce harmful gas; dry ashing method requires long time high temperature ashing, which may cause arsenic volatilization loss, and the recovery rate fluctuates greatly. At the same time, the composition of compound additives is complex (such as containing phosphate, organic acid, etc.), which may inhibit the reduction of arsenic or complex with detection reagent, resulting in false negative or signal inhibition. The existing technology solves the problem by multiple dilution or separation and purification, but prolongs the detection cycle.

[0004] Chinese patent CN104198417B discloses a method for determining heavy metal arsenic in compound food additives. The sample of compound food additives is digested by wet method to prepare a sample solution. Using graphite furnace atomic absorption spectrometry (GF-AAS), under the irradiation of light source, the sample is introduced into the atomizer through dry, ashing, atomization and other three steps, enters the optical system, monochromator, detector, and electronic circuit, and its absorbance is detected, which is proportional to the intensity. The standard curve method is used for quantitative measurement, and the content of total arsenic in compound food additives is calculated.

[0005] Compound meat product thickener is a food additive specially used to improve the texture, taste and stability of meat products. It is usually compounded by multiple single thickeners, stabilizers and other functional ingredients, which can enhance the water holding capacity, elasticity and shape retention of meat products, and prevent delamination or water separation. Compound meat product thickener of Qingdao Dehui Marine Biological Technology Co., Ltd. Figure 1 ) is a product with a large market share, which is deeply recognized by industry customers, especially in the production of quick-frozen surimi (shrimp paste) products such as fish balls, crab sticks, shrimp paste, fish paste, fish tofu and cuttle balls. However, due to the differences in the requirements of heavy metal content in food additives in different countries, in order to cope with these differences, food production enterprises need to carry out quality management according to the specific requirements of the target market. Due to the complexity of the matrix of compound meat product thickener, the existence form of arsenic and the potential interference in the pretreatment process, the accuracy of the determination of arsenic content in the existing technology is not ideal.

[0006] The article "Simultaneous Detection of Common Food Additives and Heavy Metals in Food by Reversed-Phase HPLC-ICP-MS" (Yan Tingzhao, et al.) discloses the use of zinc sulfate and potassium ferrous hydride for pretreatment of food additives. However, the accuracy of this method for detecting arsenic in compound meat thickeners is not good.

[0007] Therefore, there is an urgent need for a method specifically for detecting the heavy metal content of food additives in compound meat thickeners. Summary of the Invention

[0008] The purpose of this invention is to provide a method for detecting the heavy metal content in food additives.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A method for detecting heavy metal content in food additives includes the following steps: (1) Sample pretreatment: A: Weigh the food additives and add water, add papain and neutral protease, hydrolyze, inactivate the enzymes, continue to add auxiliary extraction solution, mix evenly, perform gradient sonication, centrifuge after completion, take the supernatant, filter with a filter membrane, wash the filtrate with water into a volumetric flask, add rhodium solution as an internal standard element, dilute with water to the mark of the volumetric flask, shake well, and use as the sample solution. B: Add papain and neutral protease to water, heat to inactivate enzymes, continue to add auxiliary extraction solution, then add rhodium solution as internal standard element, dilute to the mark of volumetric flask with water, shake well, and use as blank solution; (2) Prepare a series of heavy metal standard solutions of different concentrations, and test their net strength using an ICP-MS instrument to obtain a standard working curve; (3) The net strength of the sample solution and blank solution was tested using an ICP-MS instrument. Combined with the standard working curve, the concentration of heavy metals in the food additive was obtained, and the content of heavy metals in the food additive was further obtained.

[0010] Furthermore, the method includes the following steps: (1) Sample pretreatment: A: Weigh 5g of food additive, accurate to 0.0001g, add 100mL of water, add papain and neutral protease, enzymatically hydrolyze at 35-37℃ for 4-5h, inactivate the enzyme in a boiling water bath for 10 minutes, continue to add 250mL of auxiliary extraction solution, mix well, and then perform gradient sonication. After the process is complete, centrifuge for 10-15min, take the supernatant, filter it using a filter membrane, wash the filtrate with water into a 500mL volumetric flask, add 10mL of 50μg / L rhodium solution as an internal standard element, dilute to the mark of the volumetric flask with water, shake well, and use as the sample solution; B: 100 mL water is added with papain and neutral protease, heated at 35-37 DEG C for 4-5 h, and the enzyme is inactivated by boiling in water bath for 10 min, 250 mL auxiliary extraction solution is continuously added, 10 mL rhodium solution with a concentration of 50 ug / L is added as an internal standard element, and water is added to a 500 mL volumetric flask to the calibration mark, and then shaken uniformly to serve as a blank solution; (2) A series of heavy metal standard solutions are prepared, and the net intensity is tested by using an ICP-MS instrument to obtain a standard working curve; (3) The net intensity of the sample solution and the blank solution is tested by using an ICP-MS instrument, and the concentration of the heavy metal in the food additive is obtained in combination with the standard working curve, and the content of the heavy metal in the food additive is further obtained.

[0011] Further, the food additive is a compound meat product thickening agent.

[0012] Further, the heavy metal is arsenic.

[0013] Further, the amount of papain and neutral protease in step (1) is 0.5-1 wt% of the food additive.

[0014] The present application is extracted by enzyme hydrolysis-assisted extraction and ultrasonic synergistic extraction, the sample is decomposed by papain and neutral protease to release the combined arsenic, and the sample structure is further damaged by using gradient ultrasonic combined with temperature control to improve the dissolution efficiency of arsenic.

[0015] Further, the auxiliary extraction solution in step (1) comprises the following components: 0.05-0.1 wt% EDTA, 0.1-0.3 wt% citric acid, 0.03-0.05 wt% zinc sulfate, and the balance is water.

[0016] Further, the gradient ultrasonic conditions in step (1) are as follows: the first stage is 100 W ultrasonic for 5 min; the second stage is 120 W ultrasonic for 5 min, the water bath temperature is controlled at 25-30 DEG C during ultrasonic process, and the sample is placed for 10-20 min after ultrasonic.

[0017] The present application adopts EDTA and citric acid to complex metal ions to avoid the combination of arsenic and the sample matrix; citric acid can promote the desorption of arsenic from the adsorption sites in the sample matrix by reducing the pH value of the solution, and zinc sulfate adjusts the ionic strength and reduces the fixation of arsenic by other ions, so that the method accuracy is improved through synergistic effect.

[0018] Further, 0.22 mu m filter membrane is used for filtration in step (1).

[0019] Further, the step (2) is freshly prepared with 0, 0.2, 1, 2, 5, 10, 20, 50 µg / L of metal standard solution using metal standard solution, 10 mL of rhodium solution with a concentration of 50 µg / L is added as an internal standard element before constant volume, and a standard working curve is drawn; the above series of concentration standard solutions are injected into the ICP-MS instrument, the content of the metal is determined by using the online internal standard method, and a standard working curve is obtained.

[0020] Further, the conditions of the ICP-MS instrument test are as follows: High-frequency power 1.40 kW; Plasma gas flow rate 13.0 L.min -1 ; Carrier gas flow rate 0.8 L.min -1 ; He flow rate 4.0 mL.min -1 ; Sample lifting amount 0.1 mL.min -1 ; Sampling depth 10.0 mm; Sampling nickel cone: hole plate diameter 1.0 mm; Interception nickel cone: hole plate diameter 0.4 mm.

[0021] Compared with the prior art, the advantages and beneficial effects of the present application are as follows: 1. The present application uses ICP-MS technology to detect trace arsenic in food additives, which can meet the stringent standards of food safety and ensure the accurate quantification of arsenic in complex samples. The present application can reduce the detection period and labor cost by using ICP-MS; the accurate detection data of the present application support product quality declaration, which can enhance consumer trust, help brand differentiation competition, help enterprises realize arsenic pollution risk control, detection cost optimization and market competitiveness improvement, and provide technical support for food safety management and brand construction.

[0022] 2. The present application uses enzyme hydrolysis-assisted extract ultrasonic synergistic extraction, which decomposes the sample by using papain and neutral protease, releases the combined arsenic, and further destroys the sample structure by using gradient ultrasonic combined with temperature control to improve the dissolution efficiency of arsenic. The present application uses EDTA and citric acid to complex metal ions to avoid the combination of arsenic and sample matrix; citric acid can promote the desorption of arsenic from the adsorption sites in the sample matrix by reducing the pH value of the solution, and zinc sulfate adjusts the ionic strength and reduces the fixation of other ions on arsenic. Through synergistic effect, the accuracy of the method is improved.

[0023] 3, Compound meat product thickening agent is widely used in food processing, which is directly related to the health and safety of consumers. Compound meat product thickening agent is usually composed of a plurality of natural polysaccharides and other auxiliary ingredients, which may combine or adsorb arsenic, increasing the difficulty of extraction. The present application can ensure the safety of food additives and protect public health by detecting arsenic content. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Schematic diagram of commercially available compound meat product thickening agent. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0026] The raw materials used in the following embodiments of the present application are all commercially available: Neutral protease, enzyme activity 100u / mg; Shanghai Yuan Ye Biotechnology Co., Ltd. Papain, enzyme activity 800u / mg; Shanghai Yuan Ye Biotechnology Co., Ltd. Bromelain, enzyme activity 1200u / mg; Shanghai Yuan Ye Biotechnology Co., Ltd. Ficus protease, enzyme activity 100u / mg; Shanghai Yuan Ye Biotechnology Co., Ltd. Water: ultrapure water. EMBODIMENT

[0027] The present embodiment provides a food additive heavy metal content detection method, comprising the following steps: (1) Sample pretreatment: A: Take 5g of food additive, accurate to 0.0001g, add 100mL of water, add papain and neutral protease, 36℃ enzyme for 4.5h, boiling water bath for 10min to inactivate the enzyme, continue to add 250mL of auxiliary extraction solution, mix uniformly, then gradient ultrasonic treatment, after completion, centrifuge for 10min, take the supernatant, filter with filter membrane, and then wash the filtrate into a 500mL volumetric flask with water, add 10mL of rhodium solution with a concentration of 50μg / L as an internal standard element, and then dilute to the mark of the volumetric flask with water, shake well, and use as the sample solution; B: Add papain and neutral protease to 100mL of water, heat at 36℃ for 4.5h, boiling water bath for 10min to inactivate the enzyme, continue to add 250mL of auxiliary extraction solution, add 10mL of rhodium solution with a concentration of 50μg / L as an internal standard element, dilute to the mark of a 500mL volumetric flask with water, shake well, and use as the blank solution; The amount of papain and neutral protease used in step (1) is 0.5wt% of the food additive.

[0028] The auxiliary extraction solution in step (1) comprises the following components: 0.08 wt% EDTA, 0.2 wt% citric acid, 0.04 wt% zinc sulfate, and the balance being water.

[0029] The gradient ultrasonic conditions in step (1) are as follows: first stage: 100 W ultrasonic for 5 minutes; second stage: 120 W ultrasonic for 5 minutes; the water bath is controlled at 27℃ during ultrasonic, and the sample is placed for 15 minutes after ultrasonic.

[0030] In step (1), a 0.22 μm filter membrane is used for filtration.

[0031] (2) Freshly prepare 0, 0.2, 1, 2, 5, 10, 20, and 50 μg / L arsenic standard solutions using arsenic standard solution, add 10 mL of rhodium solution with a concentration of 50 μg / L as an internal standard element before constant volume, and draw a standard working curve; inject the above series of standard solutions into the ICP-MS instrument, and use the online internal standard method to determine the content of arsenic to obtain the standard working curve. (3) Use the ICP-MS instrument to test the net intensity of the sample solution and the blank solution, combine the standard working curve, obtain the concentration of heavy metals in the food additive, and further obtain the content of heavy metals in the food additive.

[0032] The conditions for testing by the ICP-MS instrument are as follows: High frequency power 1.40 kW; Plasma gas flow rate 13.0 L·min -1 ; Carrier gas flow rate 0.8 L·min -1 ; He flow rate 4.0 mL·min -1 ; Sample lifting amount 0.1 mL·min -1 ; Sampling depth 10.0 mm; Sampling nickel cone: orifice plate diameter 1.0 mm; Interception nickel cone: orifice plate diameter 0.4 mm.

[0033] Comparative Example 1 The difference between this comparative example and Example 1 is that the method in the article “Synchronous detection of common food additives and heavy metals in food by reversed-phase HPLC-ICP-MS” (Yan Tingzhao, et al.) is referred to: The food additive 5g is added into 2mL zinc sulfate and 2mL potassium ferrous hydride, 50mL water is uniformly mixed, then ultrasonic treatment is carried out for 20min, centrifugal treatment is carried out for 10min, the supernatant is taken, 0.22μm filter membrane is used, then the filtrate is washed into a 500mL volumetric flask with water, 10mL rhodium solution with a concentration of 50μg / L is added as an internal standard element, water is used to constant volume to the volumetric flask scale, and then shaking is uniformly carried out, which is used as a sample solution.

[0034] 100mL water is added into 2mL zinc sulfate and 2mL potassium ferrous hydride, 10mL rhodium solution with a concentration of 50μg / L is added as an internal standard element, water is used to constant volume to the 500mL volumetric flask scale, and then shaking is uniformly carried out, which is used as a blank solution. Comparative Example 2 The difference between the present comparative example and Example 1 is that no enzymatic hydrolysis is carried out.

[0035] (1) Sample pretreatment: A: 5g of food additive is weighed to 0.0001g, 250mL auxiliary extraction solution is added, and then uniformly mixed, gradient ultrasonic treatment is carried out, after the end, centrifugal treatment is carried out for 10min, the supernatant is taken, filter membrane is used for filtration, the filtrate is washed into a 500mL volumetric flask with water, 10mL rhodium solution with a concentration of 50μg / L is added as an internal standard element, water is used to constant volume to the volumetric flask scale, and then shaking is uniformly carried out, which is used as a sample solution; B: 100mL water is added into 250mL auxiliary extraction solution, 10mL rhodium solution with a concentration of 50μg / L is added as an internal standard element, water is used to constant volume to the 500mL volumetric flask scale, and then shaking is uniformly carried out, which is used as a blank solution.

[0036] Comparative Example 3 The difference between the present comparative example and Example 1 is that gradient ultrasonic is not used, and is replaced by 120W ultrasonic for 10min.

[0037] Comparative Example 4 The difference between the present comparative example and Example 1 is that the ultrasonic time in the second stage is different.

[0038] The gradient ultrasonic condition in the step (1) is that the first stage is 100W ultrasonic for 10min, the second stage is 120W ultrasonic for 10min, the water bath temperature is controlled to 27℃ during ultrasonic, and then standing for 15min after the end of ultrasonic.

[0039] Comparative Example 5 The present comparative example uses the method disclosed in Example of CN104198417B.

[0040] Comparative Example 6 The present comparative example adopts the first method of hydride atomic fluorescence spectrometry in GB / T5009.11-2024.

[0041] Comparative Example 7 The difference between this comparative example and Example 1 is that the neutral protease is replaced by bromelain; the papain is replaced by ficin.

[0042] Performance test The arsenic in commercially available compound meat product thickening agent (Example 1 and Comparative Examples 1-6) was detected by the detection method. Figure 1

[0043] Table 1 arsenic content detection results (n = 3) Test Mean value pg / L Standard deviation Example 1 0.028 0.0021 Example 2 0.027 0.0020 Comparative Example 1 0.019 0.0023 Comparative Example 2 0.021 0.0053 Comparative Example 3 0.023 0.0037 Comparative Example 4 0.024 0.0032 Comparative Example 5 0.027 0.0024 Comparative Example 6 0.028 0.0028 Comparative Example 7 0.022 0.0042 As can be seen from Table 1, the experimental data of Example 1 and Comparative Examples 5 and 6 are compared, and the results of the three methods are similar, so the experimental results of this method are accurate and the data are reliable.

[0044] The detection result is small by using the pretreatment method in Comparative Example 1, which shows that the above pretreatment method cannot extract the arsenic in the food additive sufficiently, and the relative standard deviation is large.

[0045] Comparative Example 2 does not perform enzymatic hydrolysis, and the detection result is small, which shows that the above pretreatment method cannot extract the arsenic in the food additive sufficiently. At the same time, the standard deviation is large, which shows that the stability of the pretreatment is poor without enzymatic hydrolysis.

[0046] Comparative Examples 3 and 4 have different ultrasonic treatment conditions, which shows that only under the ultrasonic conditions of the present application, the arsenic in the food additive can be dissolved sufficiently.

[0047] Comparative Example 7 uses different types of enzymes for pretreatment, and the detection result is small, which shows that only the use of specific types of enzymes can obtain accurate results. At the same time, the standard deviation is large, which shows that only the use of specific enzymes for enzymatic hydrolysis can make the target element be released sufficiently.

[0048] The above is the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.​

Claims

1. A method for detecting the heavy metal content of a food additive, characterized by, The method comprises the following steps: (1) sample pretreatment: A: weigh the food additive, add water, add papain and neutral protease, enzymolysis, after enzyme inactivation, continue to add auxiliary extraction solution, mix uniformly, then gradient ultrasonic treatment, after the end, centrifugal, take the supernatant, filter with filter membrane, wash the filtrate into a volumetric flask with water, then add rhodium solution as internal standard element, dilute to the mark of the volumetric flask with water, shake well, as sample solution; B: add papain and neutral protease in water, heat, inactivate the enzyme, continue to add auxiliary extraction solution, then add rhodium solution as internal standard element, dilute to the mark of the volumetric flask with water, shake well, as blank solution; (2) prepare a series of concentration of heavy metal standard solution, test the net intensity by ICP-MS instrument, obtain the standard working curve; (3) test the net intensity of sample solution and blank solution by ICP-MS instrument, combine the standard working curve, obtain the concentration of heavy metal in food additive, further obtain the content of heavy metal in food additive.

2. The method of claim 1, wherein the food additive is selected from the group consisting of a food additive listed in Table 1, a food additive listed in Table 2, and a food additive listed in Table 3. The food additive is a compound thickening agent for meat products.

3. The method of claim 1, wherein the food additive is selected from the group consisting of a food additive listed in Table 1, and a food additive listed in Table 2. The heavy metal is arsenic.

4. The method of claim 1, wherein the food additive is selected from the group consisting of a food additive having a high content of heavy metals, a food additive having a low content of heavy metals, and a food additive having no heavy metals. The amount of papain and neutral protease in step (1) is 0.5-1wt% of the food additive.

5. The method for detecting heavy metal content in food additives according to claim 1, characterized in that, The auxiliary extraction solution in step (1) comprises the following components: 0.05-0.1wt% EDTA, 0.1-0.3wt% citric acid, 0.03-0.05wt% zinc sulfate, and the rest is water.

6. The method for detecting heavy metal content in food additives according to claim 1, characterized in that, The gradient ultrasonic conditions in step (1) are as follows: the first stage: 100W ultrasonic for 5 minutes; the second stage: 120W ultrasonic for 5 minutes, the water bath temperature is controlled at 25-30℃ during ultrasonic process, and the sample is placed for 10-20 minutes after ultrasonic.

7. The method for detecting heavy metal content in food additives according to claim 1, characterized in that, 0.22μm filter membrane is used in step (1).

8. The method for detecting heavy metal content in food additives according to claim 1, characterized in that, In step (2), 0, 0.2, 1, 2, 5, 10, 20, 50µg / L of metal standard solution are freshly prepared by using metal standard solution, 10mL of rhodium solution with a concentration of 50μg / L is added as internal standard element before dilution, and the standard working curve is drawn; the above series of concentration of metal standard solution is injected into ICP-MS instrument, the content of metal is determined by using online internal standard method, and the standard working curve is obtained.

9. The method for detecting heavy metal content in food additives according to claim 1, characterized in that, The conditions of ICP-MS instrument test are as follows: high frequency power 1.40kW; Plasma gas flow 13.0 L.min -1 ; Carrier gas flow rate 0.8 L.min -1 ; He flow 4.0 mL. min -1 ; Sample uplift 0.1 mL. min -1 ; sampling depth 10.0mm; sampling nickel cone: hole plate diameter 1.0mm; cutting nickel cone: hole plate diameter 0.4mm.

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