Studio della formazione di ammine biogene e di altri composti azotati negli alimenti

The aim of this thesis was the qualitative and quantitative determination of biogenic amines (BA) and amino acids (AA), in particular the essential AA, in different food matrices typical of Sardinia (Italy). The study was focused on wines and table olives, which are potential source of biogenic amines due to their fermentation processes involved in their production. The decision to follow this research field is based on the importance of having updated information to assess the actual risk from ingesting BA, produced by decarboxylation of AA by microorganisms. These molecules, in fact, have direct or indirect effects on the human vascular and nervous system, especially in people who are sensitive to these substances or in people with specific diseases and/or subjected to particular therapies that affect the normal metabolism. From the legal standpoint, the legislation is developing and it is necessary to collect data in order to improve the control measures for food safety. First step of the doctorate project was to set up an analytical method of high sensitivity simultaneous detection and quantification of BA and AA in food. The method was developed using selective derivatisation with dansyl chloride and HPLC with fluorescence detection and chromatographic separation of 36 compounds, 13 BA and 22 AA, ammonium ion was successfully achieved. BA and AA were dosed using calibration curves built with the internal standard method (norvaline). This method was validated according to ICH Topic Q 2 (R1) 1995 guidelines with the determination of a linearity range, r2, limit of detection (LOD), limit of quantification (LOQ), intra and inter-day repeatability, and recovery. In cooperation with several local vineries, a survey on BA and AA amount in Cannonau, Vermentino, Carignano e Vernaccia wines, the four most typical “Controlled Designation of Origin” wines from Sardinia was performed. Sixty-one samples were analyzed, and 33 compounds (between BA and amino acids AA) were identified and quantified in the wines analyzed. Carignano wine is particularly rich in BA compounds, while Vermentino wine has a concentration of BA lower than other wine analyzed. Putrescine, histamine and tyramine are the BA found in higher concentration, but usually their amount is lower than 10 mg/L. -Aminobutyric acid is very concentrate in Vermentino wine, with an average value of 130.3±44.8 mg/L. About AA compounds, the highest total concentration is in Vermentino wine, and proline is always the most representative AA. Subsequently, a trial has been set in order to investigate the production of biogenic amines during the winemaking fermentation processes. The experiment was carried out in collaboration with the Agris (Sardinian Regional Agency for Agricultural Research) on three types of characteristic wines of Sardinia: Cannonau, Vermentino and Moscato. The musts of Cannonau, Vermentino and Moscato were treated with different strains of yeast and bacteria starter, and the evolution of the concentration of BA and AA (especially the essential AA) was monitored. Comparing the data obtained, it was observed that the three types of wine at the end of fermentation have interesting differences. It can be noted that the wine Cannonau is particularly rich in nitrogen compounds compared to the two white wines, probably because of the malo-lactic fermentation that does not take place in the other two wines. The Vermentino and Moscato are more similar to each other. In general, it was observed that the choice of starter turns out to be of significant impact in the formation of BA, though not all of the starter can make a difference. Furthermore, neither worrying values of histamine nor of tyramine were observed. Finally, an experimentation regarding the table olives, a fermented product very popular in all Mediterranean area, was developed. Sampling was carried out on a typical Sardinian olive cultivar, the ‘Nera di Gonnos’, and transformation followed the traditional natural fermentation. The HPLC-FLD method used for the wines was modified and validated for the olives. The analyses were conducted during the different steps of fermentation and after one year. Fifteen compounds, between AA and AB, were identified. The obtained data revealed that during the production process there is an increase of formation of nitrogen compounds in general, but the presence in table olives of histamine and tyramine is significantly below the values considered dangerous. In conclusion, a useful, reliable and easily applied HPLC-FLD method to monitor and safeguard the quality and safety of food products was developed. The traditional products from Sardinia showed a general good quality. It would be interesting also to extend the research by selecting and testing other microbial strains in order to obtain products with a lower content of BA and higher essential AA or other useful AA, such as Arg, that have important beneficial effects, yielding products with high qualit