CN103091312A - Enzyme-colorimetric method for detecting residual hydrogen peroxide in foods - Google Patents
Enzyme-colorimetric method for detecting residual hydrogen peroxide in foods Download PDFInfo
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Abstract
The invention discloses an enzyme-colorimetric method for detecting residual hydrogen peroxide in foods. The enzyme-colorimetric method comprises the following steps of: weighing a sample and adding zinc sulfate and potassium ferrocyanide to precipitate fat and protein; filtering and adjusting the pH (Potential of Hydrogen) value of filtering liquid to 7.6; filtering again and adding a hydrogen peroxide enzyme reagent solution into filtering liquid; enabling the volume to be constant; determining the absorbance at a part of 470nm; and calculating the content of the hydrogen peroxide in the sample. The method disclosed by the invention is simple, fast and accurate and has small interference.
Description
Technical field
The present invention relates to the detection of hydrogen peroxide in food safety detection, especially adopt enzyme-colourimetry to the mensuration of residual hydrogen dioxide in food.
Background technology
Hydrogen peroxide is commonly called as hydrogen peroxide, and is colourless, tasteless, and the effects such as oxidisability, deodorizing, decolouring and sterilization are arranged.Simultaneously, hydrogen peroxide is the germifuge of commonly using at sterile food packing sterilization process, must use hydrogen peroxide sterilant as Lile packaging equipment in Dairy Production.Yet due to the special nature of hydrogen peroxide, lawless people with its be added to make appearance become better in food or the shelf-life longer, the past detects in the food such as milk, dried bean curd, aquatic products, Water soaking products, meat products.There is the people to abuse the breeding that industrial hydrogen peroxide comes anti-bacteria in the milk production run, reduces the content of nitrite.But the hydrogen peroxide of high concentration not only can affect the quality of food, health that also can the grievous injury consumer.
In GB2760-2007 " food additives use hygienic standard ", hydrogen peroxide is deleted from food additives, has been put into " food industry is used list with processing aid ", can use as auxiliary agent in food production processing.According to " the law of food safety " regulation, the residual peroxide in food must not detect.Therefore study a kind of effectively, fast, high specificity, method that detectability is low be very necessary.
In food, the detection method of residual hydrogen dioxide mainly contains titrimetry (permanganimetric method or iodimetric titration), spectrophotometric method, fluorescence method, high performance liquid chromatography.The method that residual hydrogen dioxide in food detects in China is standard GB/T/T23499-2009 " assay method of residual hydrogen dioxide in food ", and what adopt in this standard is iodimetric titration and titanium salt colourimetry.And liquid phase chromatography needs more expensive instrument, and also more complicated of sample pre-treatments, and detection time is long.Test paper and the detector of fast detecting hydrogen peroxide are also arranged simultaneously, as the hydrogen peroxide test strips, based on the portable hydrogen peroxide detector of sensor and electroanalysis etc. on market.According to the characteristic of hydrogen peroxide, the common fast method of developing has test paper method, the peroxidase test-strips (CN200710047154 detects hydrogen peroxide quick test strip in food) of utilizing the light typical biosensor to make as Chai Chunyan etc.; The test strips of the Catalyzed Synthesis By Peroxidase tetramethyl benzidine colour developing of the development such as Xie Li (development of hydrogen peroxide Rapid detection test strip in milk, food science and technology, 2012,37 (3), 301-303); Wang Weijun etc. disclose a kind of enzyme test peper that utilizes peroxidase and developer to react to measure concentration of hydrogen peroxide (CN100516843C enzyme test peper that is used for measuring concentration of hydrogen peroxide).
The present invention has set up a kind of enzyme-colourimetry that detects residual hydrogen dioxide in food, and the method is simple, quick, accurate and effective, cost is low, can realize that the fast qualitative of gross sample quantitatively detects, and detection limit is low.
Summary of the invention
The purpose of this invention is to provide a kind of method of measuring residual hydrogen dioxide in food.For this reason, the technical solution used in the present invention is:
(1) solid sample is pulverized evenly, sampling 5-10g (being accurate to 0.001g) is to measuring 10-20mL in the 50mL volumetric flask after the fluid sample mixing.Add successively the 5mL solution of zinc sulfate, 5mL potassium ferrocyanide solution protein precipitation and fat, mixing adds water and is settled to scale.Place 10-20min.With filter paper or 0.45 μ m membrane filtration.
If the sample solution after protein precipitation and fat contains color, can add 1g activated charcoal jolting 1min, Filter paper filtering, or sample solution is passed through the activated charcoal solid phase extraction column, except look, collection and purification liquid, stand-by.If still have color to disturb through the charcoal absorption test liquid, should deduct the area color of test liquid during mensuration.
(2) filtrate with diluted acid and diluted alkaline regulator solution pH value 7.6, after mixing with filter paper or 0.45 μ m membrane filtration.
(3) get 10-20mL filtrate in 25 or the 50mL volumetric flask in, add peroxidase reagent solution 1-3mL, 30-40 ℃ of constant temperature water bath colour developing 10-20min is cooled to room temperature, is settled to scale.
(4) adopt spectrophotometric method to detect, the place measures absorbance at the 470nm wavelength.Absorbance is directly proportional to the content of hydrogen peroxide, thus the content of hydrogen peroxide in calculation sample.
Solution of zinc sulfate used in step (1) is prepared: take the 53.5g white vitriol soluble in water, and dilution is settled to 100mL.
Potassium ferrocyanide solution used in step (1) is prepared: take 17.2g three water potassium ferrocyanides soluble in water, and dilution is settled to 100mL.
Diluted acid used in step (2) is watery hydrochloric acid or dilution heat of sulfuric acid, mass concentration 0.01-1.0%.
Diluted alkaline used in step (2) is dilute sodium hydroxide or potassium hydroxide solution, and mass concentration is 0.01-1.0%.
Step (3) peroxidase reagent solution used is comprised of the phosphate buffer developer of horseradish peroxidase, o-methoxyphenol.The phosphate buffer (pH=7.6) that contains the 0.2mol/L of o-methoxyphenol 0.0050mol/L.During use, the horseradish peroxidase pressed powder is joined in mixed solution, mixing, be mixed with enzyme activity 5U (unit of activity)/mL gently, can preserve 1 month under 4 ℃.
Bath temperature used in step (3), preferred 37 ℃.
If contain hydrogen peroxide in sample, the pink or bronzing of solution, colour developing is obviously.The novelty of this method is the catalytic action through the superoxide enzyme, hydrogen peroxide and o-methoxyphenol reaction, reaction product is directly proportional to content of hydrogen peroxide, the absorbance of assaying reaction product, according to the typical curve of reaction product absorbance and concentration of hydrogen peroxide, calculate the content of hydrogen peroxide.
Embodiment:
The below is described in further details the present invention.
Embodiment 1:
The accurate proven superoxol of weighing, the standard solution of preparation 1000mg/L.The standard intermediate liquid of preparation 100mg/L.Respectively preparation contain 0,1,2,5,10, the hydrogen peroxide standard solution 20mL of 20mg/L, transfer in the 25mL color comparison tube, add 2mL peroxidase reagent solution, constant temperature 20min in 37 ℃ of water-baths is cooled to room temperature.Be settled to 25mL with intermediate water, and respectively at 470nm wavelength place, the absorbance of test gained solution.Take absorbance as Y-axis, concentration of hydrogen peroxide is that X-axis is made typical curve, gets the typical curve regression equation.
Embodiment 2:
analytical procedure: pipette the packed fresh milk 20mL of certain brand in the 50mL volumetric flask, add successively 5mL solution of zinc sulfate and 5mL potassium ferrocyanide solution, mixing, add water and be settled to 50mL, standing 20min, obtain filtrate with Filter paper filtering, then regulate pH value to 7.6 with 0.01% sodium hydroxide solution, Filter paper filtering, filtrate is test solution, pipette the 20mL test solution in the 25mL volumetric flask, add 2mL peroxidase reagent solution, shake up, constant temperature 20min in 37 ℃ of water-baths, solution shows light red, be cooled to room temperature, constant volume is to 25mL, measure absorbance at the 470nm place, typical curve equation according to embodiment 1 hydrogen peroxide, calculate and contain hydrogen peroxide 5.78mg/L in this sample.The method quantitatively is limited to 2.0mg/L.
Embodiment 3:
analytical procedure: get certain country fair Omasum, after being cut into small pieces, food masher is pulverized, take 10.0g (being accurate to 0.001g) in the 50mL volumetric flask, add successively 5mL solution of zinc sulfate and 5mL potassium ferrocyanide solution, mixing, add water and be settled to 50mL, standing 20min, Filter paper filtering obtains filtrate, then 0.01% sodium hydroxide solution is regulated pH value to 7.6, Filter paper filtering, filtrate is test solution, pipette the 20mL test solution in the 25mL volumetric flask, add 2mL peroxidase reagent solution, shake up, constant temperature 20min in 37 ℃ of water-baths, the aobvious pink of solution, be cooled to room temperature, constant volume is to 25mL, measure absorbance at the 470nm place, typical curve equation according to embodiment 1 hydrogen peroxide, calculate and contain hydrogen peroxide 9.54mg/kg in this sample.The method quantitatively is limited to 5.0mg/kg.
Above-described embodiment is only for explanation the present invention, and is not to be restriction to patent of the present invention; Should be pointed out that for those of ordinary skill in the art, conceive scope in the situation that do not break away from the present invention, can also make various variations and modification, these all belong to protection scope of the present invention; Therefore, all equalizations of doing with claim scope of the present invention change and modify, and all should belong to the coverage of claim of the present invention.
Claims (1)
1. enzyme-colourimetry that detects residual hydrogen dioxide in food, its feature comprises the steps:
(1) add respectively solution of zinc sulfate, potassium ferrocyanide solution protein precipitation and fat in sample, mix, constant volume, standing, filter, collect filtrate;
(2) regulate filtrate pH value to 7.6 with diluted acid or diluted alkaline, filter;
(3) get filtrate, add the peroxidase reagent solution, constant temperature water bath, colour developing, constant volume;
(4) spectrophotometry absorbance, absorbance is directly proportional to the content of hydrogen peroxide, thus the content of hydrogen peroxide in calculation sample.
Method described according to claims 1 is characterized in that, described peroxidase reagent solution is comprised of the phosphate buffer developer of horseradish peroxidase, o-methoxyphenol.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103713061A (en) * | 2013-12-30 | 2014-04-09 | 华南理工大学 | Method for determining content of hydrogen peroxide by determining concentration of oxygen |
| CN104535516A (en) * | 2015-01-09 | 2015-04-22 | 新希望六和股份有限公司 | Total choline measurement method |
| CN104596954A (en) * | 2014-12-10 | 2015-05-06 | 中国科学院上海微系统与信息技术研究所 | Method for detection of peroxide by ortho-para aldehyde group substituted aryl phenol |
| CN106644992A (en) * | 2017-03-10 | 2017-05-10 | 深圳市瑞赛生物技术有限公司 | Method for rapid detection of L-carnitine and kit thereof |
| CN108613972A (en) * | 2018-04-08 | 2018-10-02 | 浙江大学 | A kind of colorimetric sensing method based on enzymatic inductive formation inorganic nanoparticles |
| CN109946294A (en) * | 2019-05-13 | 2019-06-28 | 华北电力大学 | A rapid visual colorimetric detection method for sulfur ions of environmental pollutants based on peroxidase-like DNase |
| CN119119540A (en) * | 2024-11-15 | 2024-12-13 | 浙江锦盛装饰材料股份有限公司 | Composite reinforced PP decorative film and preparation process thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103713061A (en) * | 2013-12-30 | 2014-04-09 | 华南理工大学 | Method for determining content of hydrogen peroxide by determining concentration of oxygen |
| CN103713061B (en) * | 2013-12-30 | 2015-06-03 | 华南理工大学 | Method for determining content of hydrogen peroxide by determining concentration of oxygen |
| CN104596954A (en) * | 2014-12-10 | 2015-05-06 | 中国科学院上海微系统与信息技术研究所 | Method for detection of peroxide by ortho-para aldehyde group substituted aryl phenol |
| CN104596954B (en) * | 2014-12-10 | 2017-11-17 | 中国科学院上海微系统与信息技术研究所 | A kind of method that aryl phenol using the substitution of ortho para position aldehyde radical detects peroxide |
| CN104535516A (en) * | 2015-01-09 | 2015-04-22 | 新希望六和股份有限公司 | Total choline measurement method |
| CN104535516B (en) * | 2015-01-09 | 2018-01-02 | 新希望六和股份有限公司 | A kind of assay method of total choline |
| CN106644992A (en) * | 2017-03-10 | 2017-05-10 | 深圳市瑞赛生物技术有限公司 | Method for rapid detection of L-carnitine and kit thereof |
| CN106644992B (en) * | 2017-03-10 | 2020-04-07 | 深圳市瑞赛生物技术有限公司 | Method and kit for rapidly detecting L-carnitine |
| CN108613972A (en) * | 2018-04-08 | 2018-10-02 | 浙江大学 | A kind of colorimetric sensing method based on enzymatic inductive formation inorganic nanoparticles |
| CN109946294A (en) * | 2019-05-13 | 2019-06-28 | 华北电力大学 | A rapid visual colorimetric detection method for sulfur ions of environmental pollutants based on peroxidase-like DNase |
| CN119119540A (en) * | 2024-11-15 | 2024-12-13 | 浙江锦盛装饰材料股份有限公司 | Composite reinforced PP decorative film and preparation process thereof |
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Application publication date: 20130508 |