BIOCHEMICAL METHODOLOGIES APPLIED TO FOODS
Learning outcomes of the course unit
The course is designed to provide students with concepts regarding the principal biochemical techniques applied to the food sector.
Course contents summary
Introduction to the use of antibodies in immunochemical techniques. Preparation of polyclonal and monoclonal antibodies. Antigen-antibody interaction and immunoprecipitation. Labeling of antibodies using fluorescent probes, radioisotopes and enzymes. Use of antibodies conjugated with alkaline phosphatase and peroxidase. Western blotting, immunoenzymatic measuring techniques (ELISA) and radioimmunological measuring (RIA).
Prion diseases. Biochemical bases of bovine spongiform encephalopathy (BSE). Official immunochemical diagnostic tests for diagnosis of BSE: Prionics test, Platelia-BSE test and Enfer test. Innovative techniques for diagnosis of BSE: Protein misfolding cyclic amplification (PMCA). Theoretical assumptions, application, sensitivity and future perspectives of the PMCA method. BSE and food safety; methods for identifying bovine nervous tissue in foods destined for human and animal consumption. Production of a bovine knockout for the gene coding for prion protein.
Use of antibodies in identifying adulteration of proteic foods : (soft wheat in semolinas and pasta, analysis of milk and milk products, analysis of meat products). Use of antibodies in apten detection in food products: preparation of strips for immunochromatographic tests and their use in identification of mycotoxins and antibiotics.
Adverse reactions to foods. Food allergies. Allergenic molecules of food origin and identification of linear and conformational epitopes. Immunochemical techniques used in the study of allergies: skin prick test, radio-allergosorbent test (RAST), enzyme-allergosorbent test (EAST), component resolved diagnosis and use of microarray. Cross-reactivity between food allergens: case study of allergens identified in mango. Influences of technological processes on allergenicity of foods: effect of various heat treatments on the allergenicity of the peanut and apple. European standards regarding the presence of potential allergens in food products. Methods for detection of allergens in foods: direct methods (identification of the allergenic protein) and maximum security methods (identification of the ingredient that is the source of the allergy). Preparation of immunochemical methods for the identification of sesame in food and the primary peanut allergen in food
Allergenicity of new foods: bioinformatic approach (homology sequence) and experimental approach (cross-reactivity, stability in the gastric environment, cross-linking). Allergenicity in GM-derived foods.
Xenoestrogens with estrogenic-like or anti-estrogenic activity as contaminants of food products. Estrogen mechanism of action. In vitro and in vivo methods for testing substances with estrogenic activity. Use of reporter genes for analysis purposes. Preparation of a method developed in yeast cells to detect the presence of compounds with estrogenic activity.
Production of food-related protein in recombinant form. Expression of heterologous protein in E. coli: transcription and translation efficiency, the pET system, production of fusion protein, inclusion bodies. Expression of heterologous protein in yeast. Use of the P. pastoris yeast. Production of recombinant bovine chymosin used in the dairy industry.
Use of RFLP for the analysis of the heterozygote or homozygote genotype in swine affected by malignant hyperthermia, cause of PSE (pale, soft, exudative) meat not suitable for the cured meat industry.
Ninfa & Ballou, Metodologie di base per la biochimica e la biotecnologia, Zanichelli Ed.
Wilson & Walker, Metodologia Biochimica, Cortina Ed.