Preparation Of Collagen Immobilized Catalase Sensor And Detection Of Hydrogen Peroxide Residues In Milk

Hydrogen peroxide?H₂O₂?is a carcinogen commonly added in milk,vegetables and water products.It is important for supervision of food safety and protection of human health to rapidly and sensitively detecting H₂O₂.However,the traditional H₂O₂ detection method has the problems of long detection time,complex sample pretreatment and low sensitivity,which is difficult to meet the requirement of rapid detection of H₂O₂ in food.Biosensor is a kind of new food safety detection technology,which has the advantages of fast detection speed and high sensitivity.Combined with enzyme-catalyzed reaction and immobilized enzyme technology,the sensitivity and specificity of the sensor can be further improved.An immobilized catalase?Col-Cat?was prepared using pig skin collagen as material,and the different enzyme immobilization methods and conditions were optimized.The content of H₂O₂ in milk was determined by measuring the content of H₂O₂ in combination with the sensor.The specific contents of the study are as follows:1.The preparation of Col-Cat and the study of its catalytic properties:Firstly,the collagen powder extracted and dried was dissolved in 0.5 mol/L acetic acid solution,and then the film was dried to form a film on a PTFE plate to prepare the collagen film carrier material.Then,it was fixed on collagen film carrier by adsorption method,embedding method and glutaraldehyde crosslinking method.The factors affecting the activity of immobilized enzyme?adsorption time,concentration of enzyme liquid,temperature of film forming,concentration of glutaraldehyde,ultrasonic time and power?were studied by single factor test.The results showed that the optimum conditions for preparing Col-Cat by adsorption method were as follows:the concentration of the enzyme solution was 0.15 mg/mL,the adsorption time was 16h,the ultrasonic power was 50W,the ultrasonic time was 30 min,the prepared Col-Cat had good catalytic activity,the initial enzyme activity was 378.96 U/g,compared with that of Col-Cat which was not treated with sonication.The activity of immobilized enzyme was increased by 16.8%.After 8times of repeated determination,the activity of immobilized enzyme remained more than50%.The optimum conditions of Col-Cat prepared by embedding and glutaraldehyde crosslinking were as follows:the concentration of enzyme solution was 0.2 mg/mL,the concentration of initial collagen was 1%,the temperature of film was 20?,the concentration of glutaraldehyde was 5%,the highest activity of Col-Cat prepared was623.9 U/g.After 14 times of reuse,the activity of Col-Cat remained more than 50%.At the same time,in order to better understand the catalytic properties of Col-Cat,we have systematically studied the optimum pH and temperature of Col-Cat.The results showed that the prepared Col-Cat had good thermal stability,the relative enzyme activity was stable over 70%in the temperature range of 25⁴5?,and the best catalytic pH value was 7.5.2.By using Col-Cat as the sensitive response element and dissolved oxygen sensor,the detection system of H₂O₂ content in the sample was established.In the study,the temperature,pH and other conditions of the detection system were optimized,and the optimal detection range and the times of reuse of Col-Cat under the detection method were investigated.The results showed that under the conditions of 30?and pH 7.5,the immobilized enzyme sensor could detect the H₂O₂ content in the system with sensitivity.The optimal H₂O₂ concentration range was 0.1¹0 mg/L,and the linear correlation coefficient?R?was 0.9982.It can maintain high accuracy after 8 times of repeated use;the enzyme sensor was used for the determination of H₂O₂ content in milk.The recovery rate was 89%⁹6%,which was convenient and rapid for the determination of H₂O₂content in samples.