Determination Of Calcium Peroxide In Flour Based On Methanobactin Peroxidase-like Activity

Calcium peroxide(CaO2)is a very important inorganic peroxide and can be used as a whitening agent for flour in the food industry.However,excessive consumption of calcium peroxide will bring great harm to the human body,such as affecting metabolism and inducing cancer.Peroxidase(POD)can catalyze the oxidation of a variety of organic compounds by hydrogen peroxide,and can produce color changes,which has been widely used in the enzymatic determination of hydrogen peroxide.POD can also be used to detect the content of calcium peroxide,but natural peroxidase has disadvantages such as expensive and easy to inactivate.Methanotrophy is a kind of bacteria that use methane as the only energy and carbon source.It can release a small copper capture peptide,namely Methanobactin(Mb).In this Thesis,a rapid method for the determination of calcium peroxide in flour was established based on the specific binding of Mb with Cu2+,which had POD-like activity.Mb had a low requirement on reaction conditions,which solved the problem that natural enzymes were easy to inactivate.The main research contents are as follows:Firstly,Methylosinus trichosporium OB3b was cultured by fermentation in copper-limited medium.The fermentation broth was extracted to obtain Mb with complete structure.Then Mb was combined with copper to generate Mb-Cu.The 4-thio-5-hydroxyimidazole(THI)and 4-hydroxy-5-thioimidazole(HTI)in Mb structure could coordinate with Cu2+and have POD activity.Calcium peroxide could react with acid to generate hydrogen peroxide(H2O2)and then H2O2 could rapidly oxidize hydroquinone in the catalysis of Mb-Cu.The UV spectrophotometer was used to detect the change in the absorbance of the mixture at 245 nm,thereby establishing a rapid and accurate method for detecting the content of CaO2 in flour by using Mb-Cu peroxidase mimetic enzyme.In addition,the influence of Mb-Cu concentration,reaction temperature,pH,reaction time and other factors on the catalytic process were discussed.The results showed that when the concentration of Mb-Cu was 3.9×10-6mol/L,the reaction temperature was 60 ?,and the reaction time was 180 s,the CaO2 in the concentration range of 0-8 mg/L had a linear relationship with the absorbance at 245 nm.The linear equation obtained in this case was y=0.13214x+0.08317,R2=0.99013,detection limit=4.54 ?g/g,average recovery of standard addition was 98.2?100.6%,relative standard deviation RSD was 0.4%?1.3%.In the presence of Mb-Cu,the detection method was optimized by trying to replace the reaction substrate.The H2O2 generated by the reaction of CaO2 and acid could react with the mixed solution of phenol and 4-aminoantipyrine under the catalysis of Mb-Cu mimic enzyme to form red quinoneimine,and the change of absorbance could be detected at the wavelength of 505 nm.In addition,the reaction temperature,Mb-Cu concentration,pH,and reaction time were optimized,and standard colorimetric bands were made according to the color change relationship produced by different CaO2 concentrations,so as to realize the colorimetric method to visually determined the content of CaO2 in flour.The results showed that the best reaction conditions were the reaction temperature of 50?,the Mb-Cu concentration of 3.9×10-6 mol/L and the reaction time of 5 min.Under these conditions,the calcium peroxide concentration in the range of 0-10 mg/L had a good linear relationship with the absorbance.The linear equation obtained in this case was y=0.0673x+0.02835,R2=0.99397,detection limit LOD=3.34 ?g/g.Moreover,the average recovery of standard addition was 99.6%?100.5%,and the relative standard deviation RSD was 0.1%?0.4%.The color change of the system during the reaction process was fitted to a gradient image by Photoshop to obtain a CaO2 standard colorimetric card.The CaO2 content could be obtained by comparing the color change after the reaction with the colorimetric card.This method realized the rapid detection of CaO2 content in flour,and had good precision and accuracy.Compared with the previous method,it was more convenient,and the sensitivity and accuracy were better.It could be used for rapid visual inspection on site,and had more practical advantages.The effect of common additives in flour on the determination of calcium peroxide in flour by Mb-Cu ultraviolet spectrophotometry and color method was discussed.The results showed that the influence of lipase added in flour on the absorbance of the two methods had no obvious regularity,and the influence was small.In the Mb-Cu simulated peroxidase method,benzoyl peroxide increases the absorbance of the system.After acetone extraction,the absorbance decreases,indicating that an acetone extraction step should be added to the method.In the chromogenic method,the relative standard deviation before and after acetone extraction was 0.2%to 0.8%,respectively.This result indicated that the influence of benzoyl peroxide was small.In addition,the solution was more turbid after acetone extraction,which was not conducive to the operation of subsequent steps.In the chromogenic method,it was found that carotene and lutein interfered little with the result,which could be concluded that the chromogenic method was more specific and more accurate.