Pennacchio, Anna (2015) ADVANCED BIOTECH APPLICATIONS FOR FOOD AND FEED SAFETY. [Tesi di dottorato]

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Abstract

The growing concern for human and animal health and the existence of Community directives, which set maximum permitted levels of certain mycotoxins, antibiotics and hormones in many food products, require the need for methods of analysis for the strict monitoring of the levels of mycotoxins, antibiotics and hormones in raw materials and in processed products. A mycotoxin is a toxic secondary metabolite produced by fungi. Among the micotoxins, we are interested in patulin that is associated with fruits and vegetables, in particular apples and figs. Food is also contaminated by antibiotics because of their use in veterinary medicine. The antibiotics widely used are β-lactams, which include penicillin G, present in milk. Hormones, in particular estradiol, are often used to increase milk production in cows, stimulate the growth of livestock and reduce the amount of feed they need to grow. The main aim of this work is the development of advanced diagnostic systems for a simple determination of mycotoxins, antibiotics and hormones in foods, to ensure the safety of use and the quality of raw materials, with particular reference to milk, apple juices, processed food and feed products. Optical biosensors, offer several advantages compared to conventional analysis such as HPLC and LC-MS. These include a low cost per sample, high selectivity, high sensitivity, and high throughput, no extensive extraction and sample clean-up, a fast response and the use of non-trained personnel for their utilization. This objective is pursued through the study and the development of methodologies that utilize proteins, enzymes, antibodies and/or aptamers as specific probes for the design of advanced optical biosensors (assays such as Surface plasmon resonance (SPR) and fluorescence polarization assays). I immobilized patulin, penicillin G and estradiol conjugated with a carrier protein, Glutamine binding protein, on the SPR sensing surface and I produced the antibodies anti patulin, anti penicillin G and anti estradiol. This has been successfully implemented in a new, efficient SPR-based competitive immuno-assay for the detection of the analyte of interest. Then I have combined an immuno-chemical approach with SPR spectroscopy to develop an efficient biosensor to detect an analyte of interest in foods outside the laboratory. Moreover, I present a novel and sensitive polarization-based method for the detection of patulin, penicillin G and estradiol directly in apple juice and in milk. The proposed method is based on the use of patulin, penicillin G and estradiol conjugate, labeled with a NIR probe and the use of properly produced anti-analyte antibodies. The fluorescence biosensor works in the near infrared-region of light. In fact, in this spectral region, there is almost no unspecific absorption of food matrices making possible both qualitative and quantitative assays. The obtained results confirm that this methodology can be applied outside the laboratory and directly in apple-juice and in milk without interference. In addition, these techniques are more sensitive than commonly used techniques. The results of the SPR and fluorescence polarization assay show the possibility to detect an amount of patulin, penicillin G and estradiol less than the MRL of the EU regulation limit.

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