Food safety in wine: optimization of analytical controls and evaluation of production technologies

This PhD thesis has been proposed to validate and then apply innovative analytical methodologies for the determination of compounds with harmful impact on human health, such as biogenic amines and ochratoxin A in wines. Therefore, the influence of production technology (pH, amino acids precursor and use of different malolactic starters) on biogenic amines content in wines was evaluated. An HPLC method for simultaneous determination of amino acids and amines with precolumnderivatization with 9-Fluorenyl-methoxycarbonyl chloride (FMOC-Cl) and UV detection was developed. Initially, the influence of pH, time of derivatization, gradient profile were studied. In order to improve the separation of amino acids and amines and reduce the time of analysis, it was decided to study the influence of different flows and the use of different columns in the chromatographic method. Firstly, a C18 Luna column was used and later two monolithic columns Chromolith in series. It appeared to be suitable for an easy, precise and accurate determination of a relatively large number of amino acids and amines in wines. This method was then applied on different wines produced in the Emilia Romagna region. The investigation permitted to discriminate between red and white wines. Amino acids content is related to the winemaking process. Biogenic amines content in these wines does not represent a possible toxicological problem for human health. The results of the study of influence of technologies and wine composition demonstrated that pH of wines and amino acids content are the most important factors. Particularly wines with pH > 3,5 show higher concentration of biogenic amines than wines with lower pH. The enrichment of wines by nutrients also influences the content of some biogenic amines that are higher in wines added with amino acids precursors. In this study, amino acids and biogenic amines are not statistically affected by strain of lactic acid bacteria inoculated as a starter for malolactic fermentation. An evaluation of different clean-up (SPE-MycoSep; IACs and LLE) and determination methods (HPLC and ELISA) of ochratoxin A was carried out. The results obtained proved that the SPE clean-up are reliable at the same level while the LLE procedures shows lowest recovery. The ELISA method gave a lower determination and a low reproducibility than HPLC method.

Pet food: quality and quality improvement

Today’s pet food industry is growing rapidly, with pet owners demanding high-quality diets for their pets. The primary role of diet is to provide enough nutrients to meet metabolic requirements, while giving the consumer a feeling of well-being. Diet nutrient composition and digestibility are of crucial importance for health and well being of animals. A recent strategy to improve the quality of food is the use of “nutraceuticals” or “Functional foods”. At the moment, probiotics and prebiotics are among the most studied and frequently used functional food compounds in pet foods. The present thesis reported results from three different studies. The first study aimed to develop a simple laboratory method to predict pet foods digestibility. The developed method was based on the two-step multi-enzymatic incubation assay described by Vervaeke et al. (1989), with some modification in order to better represent the digestive physiology of dogs. A trial was then conducted to compare in vivo digestibility of pet-foods and in vitro digestibility using the newly developed method. Correlation coefficients showed a close correlation between digestibility data of total dry matter and crude protein obtained with in vivo and in vitro methods (0.9976 and 0.9957, respectively). Ether extract presented a lower correlation coefficient, although close to 1 (0.9098). Based on the present results, the new method could be considered as an alternative system of evaluation of dog foods digestibility, reducing the need for using experimental animals in digestibility trials. The second parte of the study aimed to isolate from dog faeces a Lactobacillus strain capable of exert a probiotic effect on dog intestinal microflora. A L. animalis strain was isolated from the faeces of 17 adult healthy dogs..The isolated strain was first studied in vitro when it was added to a canine faecal inoculum (at a final concentration of 6 Log CFU/mL) that was incubated in anaerobic serum bottles and syringes which simulated the large intestine of dogs. Samples of fermentation fluid were collected at 0, 4, 8, and 24 hours for analysis (ammonia, SCFA, pH, lactobacilli, enterococci, coliforms, clostridia). Consequently, the L. animalis strain was fed to nine dogs having lactobacilli counts lower than 4.5 Log CFU per g of faeces. The study indicated that the L animalis strain was able to survive gastrointestinal passage and transitorily colonize the dog intestine. Both in vitro and in vivo results showed that the L. animalis strain positively influenced composition and metabolism of the intestinal microflora of dogs. The third trail investigated in vitro the effects of several non-digestible oligosaccharides (NDO) on dog intestinal microflora composition and metabolism. Substrates were fermented using a canine faecal inoculum that was incubated in anaerobic serum bottles and syringes. Substrates were added at the final concentration of 1g/L (inulin, FOS, pectin, lactitol, gluconic acid) or 4g/L (chicory). Samples of fermentation fluid were collected at 0, 6, and 24 hours for analysis (ammonia, SCFA, pH, lactobacilli, enterococci, coliforms). Gas production was measured throughout the 24 h of the study. Among the tested NDO lactitol showed the best prebiotic properties. In fact, it reduced coliforms and increased lactobacilli counts, enhanced microbial fermentation and promoted the production of SCFA while decreasing BCFA. All the substrates that were investigated showed one or more positive effects on dog faecal microflora metabolism or composition. Further studies (in particular in vivo studies with dogs) will be needed to confirm the prebiotic properties of lactitol and evaluate its optimal level of inclusion in the diet.

Mass Loss in Population II Red Giants: an IRAC@Spitzer Survey of Galactic Globular Clusters

With the goal of studying ML along the RGB, mid-IR observations of a carefully selected sample of 17 Galactic globular clusters (GGCs) with different metallicity and horizontal branch (HB) morphology have been secured with IRAC on board Spitzer: a global sample counting about 8000 giant has been obtained. Suitable complementary photometry in the optical and near-IR has been also secured in order to properly characterize the stellar counterparts to the Spitzer sources and their photospheric parameters. Stars with color (i.e. dust) excess have been identified, their likely circumstellar emission quantified and modelled, and empirical estimates of mass loss rates and timescales obtained. We find that mass loss rates increases with increasing stellar luminosity and decreasing metallicity. For a given luminosity, we find that ML rates are systematically higher than the prediction by extrapolating the Reimers law. CMDs constructed from ground based near-IR and IRAC bands show that at a given luminosity some stars have dusty envelopes and others do not. From this, we deduce that the mass loss is episodic and is ``on'' for some fraction of the time. The total mass lost on the RGB can be easily computed by multiplying ML rates by the ML timescales and integrating over the evolutionary timescale. The average total mass lost moderately increases with increasing metallicity, and for a given metallicity is systematically higher in clusters with extended blue HB.