Determination Of Sodium Benzoate In Food Products By Immunoassay

Sodium benzoate, the sodium salt of benzoic acid, is generally used as a chemical preservative to prevent alteration or degradation caused by microorganisms during storage. Both sodium benzoate and benzoic acid exhibited inhibitory activity against a wide range of fungi, yeasts, molds and bacteria. Sodium benzoate is more widely used in a great variety of foods and beverages because of its good stability and excellent solubility in water. However, excessive intake of these preservatives might be potentially harmful to the consumers, because they have the tendency to induce allergic contact dermatitis, convulsion, hives and hepato cellular damage and others. Moreover, with the growing use of the additives, the preservative residues can be considered as environmental contaminants. For these reasons, benzoic acid and its salt are limitedly used as preservatives in some food products. In China, the maximum permitted utilization of benzoates in different types of food ranges from0.2to2.0g/kg (GB2760-2011). However, benzoates might be still excessively added in foods, because they are inexpensive and easily available. Therefore, developing a simple, rapid and economic analytical method for benzoates monitoring and controlling is important for food safety.There are various techniques studied for benzoates determination. Traditionally, benzoates are analyzed mainly by thin layer chromatography (TLC), gas chromatography (GC), capillary electrophoresis (CE), and micellar electro kinetic chromatography. So far, the dominant way to detect benzoates is high-performance liquid chromatography (HPLC). Other methods are also reported for benzoates analysis, including second-order derivative spectrophotometry, chemometrics enhanced spectrophotometry, near-infrared reflectance spectroscopy, polarography and enzymatic determination. Nevertheless, these methods do not allow an easy analysis, because the instruments are expensive, the assays are relatively time-consuming and in some cases, require steps of extraction, laborious manipulation or sample pretreatment. Immunochemical methods, especially enzyme-linked immunosorbent assay (ELISA), provide simple, sensitive, specific and inexpensive tools for analysis of various targeted analytes. However, ELISA is a heterogeneous method which involves repeated washing and a certain degree of reaction time (1-2h). Fluorescence polarization immunoassay (FPIA) is a homogeneous technique (no separation or washing), which is an excellent screening tool in food and environmental analysis because of its rapidity, reliability and ease of use. To our knowledge, development of ELISA and FPIA for sodium benzoate or benzoic acid in real samples has not been reported yet. Based on this, an ELISA and a FPIA for sodium benzoate was developed and the application of FPIA for food products was studied.Based on the structure of benzoic acid, the immunogen, coating antigen and fiuorescein-labeled analyte conjugate were successfully synthesized, and a variety of polyclonal antibody was obtained. Under the optimal assay conditions, a FPIA and an ELISA for sodium benzoate were developed respectively. The FPIA shows a detection range of0.3-20.0μg/mL for sodium benzoate with a detection limit of0.26μg/mL. In addition, the IC50value was2.48μg/mL, and the cross-reactivity of the antibodies with eleven structurally and functionally related analogs were detected respectively. The accuracy and precision were detected in borate buffer and real systems. The recoveries were91.41-106.27%and86.97-109.87%, and coefficients of variation of this method were all lower than11.25%. Compared with the detection results of HPLC, the developed FPIA has comparative performance in the real sample determination. The IC50value of ELISA was2.53μg/mL with a detection limit of0.77μg/mL and a detection range of0.5μg/mL-10μg/mL for sodium benzoate. The cross-reactivity of the antibodies with some related analogs was detected additional.In this study, the results suggest that immunoassay is a rapid, convenient and simple method, which is suitable to be used as a screening tool for detection of sodium benzoate in food products. This research opened a way for the application of immunoassay in other areas of food additives.