阅读说明：本技术 一种数字图像比色法检测食品中亚硝酸盐的方法 (method for detecting nitrite in food by digital image colorimetric method ) 是由 王晓闻 王慧慧 荆旭 武文英 贾丽艳 王腾飞 杨佳丽 于 2019-08-22 设计创作，主要内容包括：本发明涉及一种数字图像比色法检测食品中亚硝酸盐的方法。本发明利用NO<Sup>-</Sup>在酸性条件下具有氧化性,可以氧化3,3’,5,5’-四甲基联苯胺(TMB)生成黄色的TMB二亚胺,采用智能手机对实验结果进行拍照,然后用图像处理软件采用RGB模型对采集的图像进行处理,将可视化的颜色转化为可处理的数据,实现食品样品中亚硝酸盐的定量检测。本发明方法操作简单方便、使用仪器易得,实验结果准确可靠,所需成本较低,可以实现现场快速检测。(The invention relates to a method for detecting nitrite in food by a digital image colorimetric method. According to the method, NO-has oxidability under an acidic condition, and can oxidize 3,3',5,5' -Tetramethylbenzidine (TMB) to generate yellow TMB diimine, an intelligent mobile phone is used for photographing an experimental result, then an image processing software is used for processing an acquired image by using an RGB model, and visual colors are converted into processable data, so that the quantitative detection of nitrite in a food sample is realized. The method has the advantages of simple and convenient operation, easy acquisition of used instruments, accurate and reliable experimental results, lower required cost and capability of realizing on-site rapid detection.)

1. A method for detecting nitrite in food by digital image colorimetry is characterized by comprising the following steps:

S1, sample treatment: accurately weighing a sample, adding water, homogenizing, fixing the volume, cooling in a boiling water bath, filtering to obtain a supernatant, adding a potassium ferrocyanide solution and a zinc acetate solution, shaking up, centrifuging to remove fat, filtering again, and taking a filtrate as a sample treatment solution to be detected;

s2, preparing nitrite standard substances with different concentrations for preparing a standard curve;

S3, mixing and reacting the TMB color developing agent, the disodium hydrogen phosphate-citric acid buffer solution and the sample treatment solution, and photographing to obtain a sample photo; reacting nitrite standard substances with different concentrations with a TMB color developing agent and a disodium hydrogen phosphate-citric acid buffer solution, and then photographing to obtain a standard substance photo;

s4, selecting an RGB mode by using image processing software to perform data processing on data of image reading G and B channels and drawing a standard curve;

And substituting the data of the RGB mode of the sample photo into the standard curve to obtain the concentration of the nitrite in the sample.

2. The method of claim 1, wherein in step S1, the boiling water bath is used for 10-20 min; the concentration of the potassium ferrocyanide solution is 0.25mol/L, and the concentration of the zinc acetate solution is 1 mol/L; the centrifugation is 5000r/min, 5 min.

3. The method of claim 1, wherein in step S1, the sample is 5g, and water is added at 30-50 mL, 70-80 ℃; the volume is 50 mL-100 mL, the supernatant is 9 mL-15 mL, and the volume of the potassium ferrocyanide solution and the volume of the zinc acetate solution are 150 muL-300 muL respectively.

4. The method of claim 1, wherein in step S2, the linear range of the concentration of the NaNO2 standard in the nitrite standard is 10 μmol/L to 440 μmol/L.

5. The method of claim 1, wherein in step S2, the concentrations of the NaNO2 standard are 10 μmol/L, 15 μmol/L, 20 μmol/L, 30 μmol/L, 50 μmol/L, 80 μmol/L, 100 μmol/L, 200 μmol/L, 300 μmol/L, 400 μmol/L, respectively.

6. The method as claimed in claim 1, wherein in step S3, the TMB color developer contains 25 to 50% by weight of ethanol, the TMB concentration is 450 to 700 μmol/L, and the pH of the disodium hydrogen phosphate-citric acid buffer is 2.2 to 4.

7. The method of claim 1, wherein the TMB developer comprises 25% ethanol by mass and a TMB concentration of 500 μmol/L; the pH value of the disodium hydrogen phosphate-citric acid buffer solution is 2.2.

8. The method of claim 1, wherein in step S3, the TMB color reagent and the disodium hydrogen phosphate-citric acid buffer solution are used in equal amounts with the sample treatment solution or the nitrite standard for 20min or more.

9. The method as claimed in claim 1, wherein in step S4, the image processing software is Adobe Photoshop, the data processing is to calculate intensity I-1-B/R according to an intensity formula, a standard curve is drawn with nitrite concentration as abscissa, intensity Δ I-I0 as ordinate, I0 is the intensity at the concentration of NaNO2 as 0, and the intensity Δ I-I0 of the sample to be tested is I-I0.

10. The method of claim 1, wherein the device for taking the picture is a light reflector on the periphery and top, a light diffuser on the bottom, and a light source on the top to ensure consistent light intensity during each picture taking.

Technical Field

The invention relates to a method for detecting nitrite in food by a digital image colorimetric method, belonging to the technical field of food detection.

background

Nitrite is one of the most widely used industrial salts, is commonly used as a food additive and a preservative, and is commonly used as a preservative and a color former in meat processing (such as ham and sausage), so that the meat product has good color, proper flavor and antibacterial property. However, the potential toxicity poses a great threat to human health, and inhalation of small amounts of nitrite can lead to acute toxicity and long-term intake can lead to cancer. Under the action of gastric acid, nitrite can react with secondary amine, amide and the like to generate N-nitrosamine carcinogens, so that kidneys, spleens and nervous systems are easily damaged, and various cancers are caused. In addition, nitrite can generate methemoglobin through irreversible reaction with hemoglobin in human blood, and the compound can reduce the oxygen transport capacity of blood, thereby causing methemoglobin or blue infant syndrome and having great harm to pregnant women and infants. Nitrite in humans is mainly taken by diet, so monitoring the nitrite content in food is of great importance.

at present, common nitrite detection methods in the world mainly comprise a fluorescence method, a chemiluminescence method, an electrochemical method, a fluorescence capillary quenching method, an ion chromatography method, a spectrophotometry method and the like. Although these detection methods are sensitive and accurate, they require expensive instruments, professional operators, time-consuming procedures, complex sample processing procedures and a large number of samples, and the disadvantages of unstable synthesized materials, high cost and the like, which greatly limit their application in field food inspection, so that establishing a method capable of rapidly detecting nitrite on the field has great practical significance.

Disclosure of Invention

Technical problem to be solved

In order to solve the problems in the prior art, the invention provides a nitrite detection method, and particularly relates to a digital image colorimetric detection method for rapidly, accurately and conveniently detecting nitrite in a food product on site.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

A method for detecting nitrite in food by digital image colorimetry comprises the following steps:

S1, sample treatment: accurately weighing a sample, adding water, homogenizing, fixing the volume, cooling in a boiling water bath, filtering to obtain a supernatant, adding a potassium ferrocyanide solution and a zinc acetate solution, shaking up, centrifuging to remove fat, filtering again, and taking a filtrate as a sample treatment solution to be detected;

S2, preparing nitrite standard substances with different concentrations for preparing a standard curve;

S3, mixing and reacting the TMB color developing agent, the disodium hydrogen phosphate-citric acid buffer solution and the sample treatment solution, and photographing to obtain a sample photo; reacting nitrite standard substances with different concentrations with a TMB color developing agent and a disodium hydrogen phosphate-citric acid buffer solution, and then photographing to obtain a standard substance photo;

S4, selecting an RGB mode by using image processing software to perform data processing on data of image reading G and B channels and drawing a standard curve;

And substituting the data of the RGB mode of the sample photo into the standard curve to obtain the concentration of the nitrite in the sample.

In a preferred embodiment, in the step S1, the boiling water bath time is 10-20 min; the concentration of the potassium ferrocyanide solution is 0.25mol/L, and the concentration of the zinc acetate solution is 1 mol/L; the centrifugation is 5000r/min, 5 min.

Further, the sample is 5g, the water is added into the sample for 30mL to 50mL and the temperature is 70 ℃ to 80 ℃; the volume is 50 mL-100 mL, the supernatant is 9 mL-15 mL, and the volume of the potassium ferrocyanide solution and the volume of the zinc acetate solution are 150 muL-300 muL respectively.

In a preferred embodiment, in step S2, the linear range of the concentration of NaNO2 standard in the nitrite standard is 10 μmol/L to 440 μmol/L.

Further, the concentrations of the NaNO2 standard samples are 10 mu mol/L, 15 mu mol/L, 20 mu mol/L, 30 mu mol/L, 50 mu mol/L, 80 mu mol/L, 100 mu mol/L, 200 mu mol/L, 300 mu mol/L and 400 mu mol/L respectively.

in a preferred embodiment, in step S3, the TMB color developing agent contains 25 to 50% by mass of ethanol, the TMB concentration is 450 to 700 μmol/L, and the pH of the disodium hydrogen phosphate-citric acid buffer is 2.2 to 4.

further, the mass fraction of ethanol in the TMB color developer is most preferably 25%, the TMB concentration is most preferably 500 μmol/L, and the pH of the disodium hydrogen phosphate-citric acid buffer is most preferably 2.2.

in a preferred embodiment, in step S3, the TMB color reagent and the disodium hydrogen phosphate-citric acid buffer solution are used in equal amounts with the sample treatment solution or the nitrite standard, and the reaction time is 20min or more than 20 min.

In a preferred embodiment, in step S4, the image processing software is Adobe Photoshop, and the data processing is to calculate the intensity I-1-B/R according to the intensity formula, draw a standard curve with the nitrite concentration as abscissa and the intensity Δ I-I0 as ordinate, and the intensity I0 is the intensity at the concentration of NaNO2 of 0, and the intensity Δ I-I0 of the sample to be tested.

In a preferred embodiment, the periphery and the top of the photographing device are provided with a reflector, the bottom of the photographing device is provided with a light softening plate, and a light source adopts a light emitting diode and is arranged on the top; so as to ensure the light source intensity to be consistent when taking pictures.

(III) advantageous effects

The invention has the beneficial effects that:

According to the detection method of the nitrite, the newly developed TMB color developing agent is adopted, yellow TMB diimine is generated when meeting the nitrite under an acidic condition, macroscopic color change is generated, and finally generated components are stable; the intelligent mobile phone can be used for photographing for analyzing and processing subsequent results, the detection cost is low, and the detection method disclosed by the invention has the advantages of accurate result and small error in actual sample detection.

The method of the invention does not need expensive precise instruments (such as an ultraviolet spectrophotometer or a fluorescence analyzer), uses a common electronic device of a smart phone, and can accurately analyze the experimental result by using an image processing system; the color developing agent reagent used in the detection method is safe and nontoxic, no substance needs to be synthesized in the experimental process, the reaction condition is mild, the operation is simple and rapid, the experimental result is accurate and reliable, and the remote and on-site rapid detection can be realized.

Drawings

FIG. 1 is an image and UV-visible absorption spectra of different reaction systems.

fig. 2 shows the result of the mobile phone photographing.

Fig. 3 is a cut-out of the active area.

FIG. 4 is a graph of the effect of different TMB concentrations on intensity (I).

FIG. 5 is a graph showing the effect of pH on the intensity (I) of a disodium hydrogen phosphate-citric acid buffer.

FIG. 6 is a graph showing the effect of percent ethanol on strength (I).

FIG. 7 shows the effect of reaction time on intensity (I).

Fig. 8 is a linear plot of sodium nitrite.

Detailed Description

3,3',5,5' -tetramethyl benzidine (TMB) is a safe, efficient and stable color developing agent. When the oxidation of the substance to be detected is weak, TMB loses 1 electron, a blue-green mixture is generated, and a characteristic absorption peak appears at 625 nm. And when the oxidability of the substance to be detected is strong, TMB loses 2 electrons to generate a yellow mixture, and a characteristic absorption peak appears at 450 nm. The research of the invention finds that NaNO2 has strong oxidizing property under the acidic condition, TMB can be subjected to oxidation reaction without a catalyst, and the nitrite can be quantitatively detected by adopting a digital image colorimetric method. Digital Image Colorimetry (DIC) involves two processes: and acquiring an image and reading out colors, wherein the method acquires a sample image by using an image acquisition tool and then performs color analysis on the acquired image by using image processing software. Because DIC data is provided by image software, the influence of naked eyes is reduced, the accuracy of a detection result is greatly improved, and a camera, a scanner, a computer camera and a smart phone can be used for acquiring images. The smart phone becomes a ubiquitous electronic device, has the advantages of small size, convenience in use, high camera resolution, rapidness and convenience in obtaining image original data, digital platform, huge storage capacity and the like, and becomes an image acquisition tool with the most extensive application.

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.