Quality and safety of oils and fats obtained as co-products or by-products of the food chain and destined to animal production

The aim of the first part of this thesis was to evaluate the effect of trans fatty acid- (TFA), contaminant, polycyclic aromatic hydrocarbon (PAH)- and oxidation productenriched diets on the content of TFA and conjugated linoleic acid (CLA) isomers in meat and liver of both poultry and rabbit. The enriched feedings were prepared with preselected fatty co-and by-products that contained low and high levels of TFA (low, palm fatty acid distillate; high, hydrogenated palm fatty acid distillate), environmental contaminants (dioxins and PCBs) (two different fish oils), PAH (olive oil acid oils and pomace olive oil from chemical refining, for low and high levels) and oxidation products (sunflower-olive oil blend before and after frying), so as to obtain single feedings with three enrichment degrees (high, medium and low) of the compound of interest. This experimental set-up is a part of a large, collaborative European project (http://www.ub.edu/feedfat/), where other chemical and health parameters are assessed. Lipids were extracted, methylated with diazomethane, then transmethylated with 2N KOH/methanol and analyzed by GC and silver-ion TLC-GC. TFA and CLA were determined in the fats, the feedings, meat and liver of both poultry and rabbit. In general, the level of TFA and CLA in meat and liver mainly varied according to those originally found in the feeding fats. It must be pointed out, though, that TFA and CLA accumulation was different for the two animal species, as well as for the two types of tissues. The TFA composition of meat and liver changes according to the composition of the oils added to the feeds with some differences between species. Chicken meat with skin shows higher TFA content (2.6–5.4 fold) than rabbit meat, except for the “PAH” trial. Chicken liver shows higher TFA content (1.2–2.1 fold) than rabbit liver, except for the “TRANS” and “PAH” trials. In both chicken and rabbit meats, the TFA content was higher for the “TRANS” trial, followed by the “DIOXIN” trial. Slight differences were found on the “OXIDATION” and “PAH” trends in both types of meats. In both chicken and rabbit livers, the TFA content was higher for the “TRANS” trial, followed by those of the “PAH”, “DIOXIN” and “OXIDATION” trials. This trend, however, was not identical to that of feeds, where the TFA content varied as follows: “TRANS” > “DIOXIN” >“PAH” > “OXIDATION”. In chicken and rabbit meat samples, C18:1 TFA were the most abundant, followed by C18:2 TFA and C18:3 TFA, except for the “DIOXIN” trial where C18:3 TFA > C18:2 TFA. In chicken and rabbit liver samples of the “TRANS” and “OXIDATION” trials, C18:1 TFA were the most abundant, followed by C18:2 TFA and C18:3 TFA, whereas C18:3 TFA > C18:2 in the “DIOXIN” trial. Slight differences were found on the “PAH” trend in livers from both species. The second part of the thesis dealt with the study of lipid oxidation in washed turkey muscle added with different antioxidants. The evaluation on the oxidative stability of muscle foods found that oxidation could be measured by headspace solid phase microestraction (SPME) of hexanal and propanal. To make this method effective, an antioxidant system was added to stored muscle to stop the oxidative processes. An increase in ionic strength of the sample was also implemented to increase the concentration of aldehydes in the headspace. This method was found to be more sensitive than the commonly used thiobarbituric acid reactive substances (TBARs) method. However, after antioxidants were added and oxidation was stopped, the concentration of aldehydes decreased. It was found that the decrease in aldehyde concentration was due to the binding of the aldehydes to muscle proteins, thus decreasing the volatility and making them less detectable.

Lipid quality and oxidative stability in food products from animal origin, as affected by breeding factor

Nowadays it is requested more investigations on alternative rearing systems that are able to improve poultry welfare and to warrant high-quality and safe meat products. This thesis work was focused on the evaluation of the oxidative stability of poultry meats, obtained with different rearing systems, diets (supplemented with bioactive compounds), and packaging conditions. The thesis work was divided into the following parts: - Evaluation of the effects of different rearing systems on the quality, fatty acid composition and oxidative stability of poultry thigh and breast meat belonging to different product categories (“rotisserie” and “cut-up” carcasses); - Evaluation of the effects of different rearing systems and packaging conditions on the shelf-life of poultry thigh meat stored at 4°C for 14 days, and the effects of feed supplementation with thymol (control diet and diet with 2 different concentration of thymol) and packaging conditions on lipid oxidation of poultry thigh meat shelf-life (stored at 4°C for 14 days). The oxidative stability of poultry meat was studied by means of the spectrophotometric determinations of peroxide value and thiobarbituric acid reactive substances. - Evaluation of anti-inflammatory effects of different flavonoids (thymol, luteolin, tangeretin, sulforaphane, polymethoxyflavones, curcumin derivates) to detect their biological activity in LPS-stimulated RAW 264.7 macrophage cells in vitro, in order to study more in depth their action mechanisms. It was evaluated the cell vitality (MTT assay), nitrite concentration and protein profile. The study was focused on the identification of potential dietary bioactive compounds in order to investigate their biological activity and possible synergic effects, and to develop new suitable strategies for long-term promotion of human health, in particular against cancer.

Farming and processing stategies for improving poultry meat quality

The purpose of the PhD research was the identification of new strategies of farming and processing, with the aim to improve the nutritional and technological characteristics of poultry meat. Part of the PhD research was focused on evaluation of alternative farming systems, with the aim to increase animal welfare and to improve the meat quality and sensorial characteristics in broiler chickens. It was also assessed the use of innovative ingredients for marination of poultry meat (sodium bicarbonate and natural antioxidants) The research was developed by studying the following aspects: - Meat quality characteristics, oxidative stability and sensorial traits of chicken meat obtained from two different farming systems: free range vs conventional; - Meat quality traits of frozen chicken breast pre-salted using increasing concentrations of sodium chloride; - Use of sodium bicarbonate in comparison with sodium trypolyphosphate for marination of broiler breast meat and phase; - Marination with thyme and orange essential oils mixture to improve chicken meat quality traits, susceptibility to lipid oxidation and sensory traits. The following meat quality traits analyseswere performed: Colour, pH, water holding capacity by conventional (gravimetric methods, pressure application, centrifugation and cooking) and innovative methods (low-field NMR and DSC analysis) ability to absorb marinade soloutions, texture (shear force using different probes and texture profile analysis), proximate analysis (moisture, proteins, lipids, ash content, collagen, fatty acid), susceptibility to lipid oxidation (determinations of reactive substances with thiobarbituric acid and peroxide value), sensorial analysis (triangle test and consumer test).

New routes to enantioenriched substances through small organic molecules

In this thesis we will disclose the results obtained from the diastereoisomeric salt formation (n salt, p salt and p1,n1 salt) between non-racemic trans-chrysanthemic acid (trans-ChA) and pure enantiomers of threo-2-dimethylamino-1-phenyl-1,3-propanediol (DMPP). The occurrence of p1,n1 salt formation can have profound effects on enantiomer separation of scalemic (non-racemic) mixtures. This phenomenon when accompanied by substrate self-association impedes the complete recovery of the major enantiomer through formation of an inescapable racemate cage. A synthetic sequence for the asymmetric synthesis of bicyclo[3.2.0]heptanones and bicyclo[3.2.0]hept-3-en-6-ones through a cycloaddition strategy is reported. The fundamental step is a [2+2]-cycloaddition of an enantiopure amide derived from the reaction between a set of acids and an oxazolidinone as the chiral auxiliary. The inter- and intramolecular cycloaddition of in situ-generated keteniminium salts gives bicycles with a good enantioselection. A key intermediate of Iloprost, a chemically stable and biologically active mimic of prostacyclin PGI2 is synthesized following a ‘green approach’. An example of simple optical resolution of this racemic intermediate involving the diastereoisomeric salt formation is described.

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

New analytical LC-mass spectrometry methodologies for the quali-quantitative determination of natural substances and drugs in complex matrices

This thesis reports an integrated analytical and physicochemical approach for the study of natural substances and new drugs based on mass spectrometry techniques combined with liquid chromatography. In particular, Chapter 1 concerns the study of Berberine a natural substance with pharmacological activity for the treatment of hepatobiliary and intestinal diseases. The first part focused on the relationships between physicochemical properties, pharmacokinetics and metabolism of Berberine and its metabolites. For this purpose a sensitive HPLC-ES-MS/MS method have been developed, validated and used to determine these compounds during their physicochemical properties studies and plasma levels of berberine and its metabolites including berberrubine(M1), demethylenberberine(M3), and jatrorrhizine(M4) in humans. Data show that M1, could have an efficient intestinal absorption by passive diffusion due to a keto-enol tautomerism confirmed by NMR studies and its higher plasma concentration. In the second part of Chapter 1, a comparison between M1 and BBR in vivo biodistribution in rat has been studied. In Chapter 2 a new HPLC-ES-MS/MS method for the simultaneous determination and quantification of glucosinolates, as glucoraphanin, glucoerucin and sinigrin, and isothiocyanates, as sulforaphane and erucin, has developed and validated. This method has been used for the analysis of functional foods enriched with vegetable extracts. Chapter 3 focused on a physicochemical study of the interaction between the bile acid sequestrants used in the treatment of hypercholesterolemia including colesevelam and cholestyramine with obeticolic acid (OCA), potent agonist of nuclear receptor farnesoid X (FXR). In particular, a new experimental model for the determination of equilibrium binding isotherm was developed. Chapter 4 focused on methodological aspects of new hard ionization coupled with liquid chromatography (Direct-EI-UHPLC-MS) not yet commercially available and potentially useful for qualitative analysis and for “transparent” molecules to soft ionization techniques. This method was applied to the analysis of several steroid derivatives.