Impact of a Shorter Brine Soaking Time on Nutrient Bioaccessibility and Peptide Formation in 30-Months-Ripened Parmigiano Reggiano Cheese

Reducing the salt content in food is an important nutritional strategy for decreasing the risk of diet-related diseases. This strategy is particularly effective when applied to highly appreciated food having good nutritional characteristics, if it does not impact either upon sensory or nutritional properties of the final product. This work aimed at evaluating if the reduction of salt content by decreasing the brine soaking time modifies fatty acid and protein bioaccessibility and bioactive peptide formation in a 30-month-ripened Parmigiano Reggiano cheese (PRC). Hence, conventional and hyposodic PRC underwent in vitro static gastrointestinal digestion, and fatty acid and protein bioaccessibility were assessed. The release of peptide sequences during digestion was followed by LC–HRMS, and bioactive peptides were identified using a bioinformatic approach. At the end of digestion, fatty acid and protein bioaccessibility were similar in conventional and hyposodic PRC, but most of the bioactive peptides, mainly the ACE-inhibitors, were present in higher concentrations in the low-salt cheese. Considering that the sensory profiles were already evaluated as remarkably similar in conventional and hyposodic PRC, our results confirmed that shortening brine soaking time represents a promising strategy to reduce salt content in PRC

Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing "anti-nitrite feeling" has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative "clean label" salamis without negatively affecting their nutritional value

Synthesis of novel hybrid nanosystems composed by core-shell SiC/SiOx nanowires conjugated with porphyrins for X-ray-excited PDT

My PhD thesis dealt with the preparation and characterization of hybrid nanosystems for possible application in nanomedicine, in particular for the treatment of deep solid tumours by X-Ray activated PhotoDynamic Therapy, according to the “self lighting photodynamic therapy” recently proposed in the literature. The activity has been performed in the framework of the BioNiMED Project funded by the CARIPARMA foundation and coordinated by IMEM-CNR (Parma). The project aims to develop a nanosystem capable of causing oxidative stress through the production of singlet oxygen, a cytotoxic species, when exposed to a source of X-Ray. During my PhD work, I functionalized cubic SiC/SiOx nanowires, grown at IMEM (CNR), and prepared nanosystems consisting of “core shell” cubic SiC@SiOx nanowires conjugated with porphyrins. SiC@SiOx nanowires work as scintillators: indeed, when they are irradiated with X-Ray, they emit light in the wavelength range corresponding to the porphyrin absorption. This energy transfer excites the porphyrin, which in turns produces singlet oxygen. Indeed, porphyrins are a wide class of photosensitizers, largely used in conventional PDT. Cubic SiC is known to be a biocompatible material, employed in biomedical field, and the biocompatibility of the SiC/SiOx nanowires was also assessed and previously published. In a previous PhD work developed in our laboratory the tetra-(4- carboxyphenyl)porphyrin (H2TCPP ) was covalently linked to the SiC/SiOx NWs by a ‘click’ reaction, giving a novel nanosystem able to promote X-ray- excited PDT, as evidenced by in vitro studies. During my thesis work, I conjugated the selected porphyrin to the nanowires by the formation of the covalent amide bond and introduced different ending chains in the porphyrin moiety in order to increase the dispersion of the nanosystem in aqueous medium. The formation of the amide bonds required the previously functionalization of the nanowire surface with amino groups reacting the silica hydroxyl groups with APTES (aminopropyltriethoxysilane). To bind the porphyrin to the nanowire surface, the carboxylic groups of H2TCPP porphyrin were previously activated with typical condensation agents (EDC, HOBt, and DMAP) and then reacted with the amino groups to give the amide bond formation. This conjugation approach resulted to give a higher degree of porphyrin loading, as evidenced by fluorescence spectra, and occurred under very mild conditions (r.t. vs. high temperature used in the thermal click reaction). In addition, it was possible to bind polar chains to the conjugated porphyrin. It was planned to introduce short PEG chains to modulate the polarity of the nanosystem. In particular, two different ending NH2-PEG chains were introduced, PEG6-CH2COOH and –PEG8-OH. In the first case, I prepared NH2CH2CH2(OCH2CH2)5OCH2CO2H by a multistep synthesis starting from hexaethylene glycol. Then this chain was bound to the residual activated carboxylic groups of the porphyrin conjugated to the nanowires. Finally, deprotection with trifluoroacetic acid gave free acid carboxylic functions at the end of the PEG- chains. The nanosystem was characterized by fluorescence spectroscopy that confirmed that functionalization occurred successfully. Last, the nanowires were detached from the support using an ultrasound microtip. In vitro experiments (clonogenic tests) performed on the adenocarcinoma human alveolar basal epithelial (A549) cell line evidenced the ability to significantly reduce the survival fraction with respect to simple radiotherapy. To prepare the second chain, NH2-PEG8-OH, without the ionizable acid group, I started from tetraethylene glycol by a multistep synthesis. The residual activated carboxylic groups of the H2TCPP porphyrin conjugated to the nanowires were reacted with this chain giving a less polar nanosystem. The activity of the nanowires, after detachment from the support, was tested by in vitro experiment on A549 tumoural cell line. The lower activity observed could be attributed to lower internalization due to the formation of boundles in the biological medium. To increase the cytotoxic activity, a further aim was to obtain a thicker porphyrin coating. Thus, it was planned to link a second different porphyrin on the conjugated H2TCPP porphyrin. In particular, tetra(4- aminophenyl)porphyrin could be successfully reacted and in vitro experiments are in progress. To face the open problem of the number of porphyrin arms involved in the conjugation, we planned to apply XPS spectroscopy to study the nanosystem conveniently modified by the presence of bromine atom. To check the possibility of evaluating the C=O/Br ratio in XPS spectra, I synthesized the bromophenyl tetra-derivative of H2TCPP porphyrin as reference compound. Then H2TCPP and the bromine derivative porphyrins were deposited on Pt-metalized wafer by drop casting. XPS spectra will give us information on the utility of this approach. Last, to evaluate the porphyrin loading, I synthesized the Cu-TCPP porphyrin, a metal derivative stable enough to be conjugated to the nanowires. Indeed, the complete removal of copper from the porphyrin requires the treatment with conc. sulfuric acid. The determination of Cu amount by atomic absorption was performed. To explore a different type of linker to anchor a porphyrin to a solid support, I successfully synthesized a metal tetra-phosphonated porphyrin. In particular, starting from the tetra(4-hydroxyphenyl)porphyrin (H2THPP) it was possible to obtain the Zn-THPP functionalized with four phosphonic acid chain ( –CH2)6PO3H), compound that is not reported in the literature. This type of porphyrins is of great interest for both energy transfer process and electron transfer process. The anchorage of this porphyrin on a flat silicon support will be useful to study the porphyrin position on the surface, parallel or not, by X-ray-excited optical luminescence (XEOL)

Elucidation of the Structure and Fluxionality of a Dinuclear Organometallic Complex Reluctant to Crystallize: An Experimental and Theoretical Integrated Approach

The organometallic dication 12+ has been constructed by combining the angular py-functionalized ligand N,N’-bis-(3- aminopyridine)-1,4,5,8-naphthalenetetracarboxydiimide (L) with [(p-cymene)RuCl] moieties. In 12+ two ligands L bridge two organometallic nodes. Repeated crystallization attempts of 12+ failed and then a combined DFT/VT-1HNMR/UV-Vis spectroscopy study has been performed to define the most plausible structure of the dication. These considers the two naphthalenediimide platforms unstacked, thus giving rise to a very open conformation where the two chloride and the two p-cymene ligands lye on the same side of the cationic cleft. The high fluxionality of 12+ evidenced by 1HNMR spectroscopy has been elucidated by a combined PES (Potential Energy Surface)/MD (Molecular Dynamic)/DFT-GIAO (Gauge Including Atomic Orbitals) study, thus furnishing a detailed description of the dynamic character of 12+

Solid-state and solution characterization of half-sandwich Ru(II) complexes with carboxylic ligands containing N-based coordinating functions

The aminobenozic ligands 3-(4-aminophenyl)propionic acid (4APPA), 4-(4-aminophenyl)butyric acid (4APBA) and 5-aminoisophthalic acid (5AIPA) have been reacted with [(h6-p-cymene)RuCl2]2 giving rise to the corresponding half-sandwich Ru(II) complexes of formula [(h6-p-cymene)Ru(ligand)Cl2] (1, 2 and 3, respectively). The solid state structures of all three organometallic compounds have been elucidated by single crystal X-ray analysis conducted on crystals grown in MeOH or THF. The aminobenzoic ligands interact with the metal through the amine function, the pseudo-octahedral geometry being satisfied by a h6-coordinated p-cymene ring and by two chloride ligands. Dissolution of the complexes in coordinating solvents, such as dmso or acetonitrile led to ligand solvolysis, as evidenced by 1H NMR analysis. The lability of monodentate N-based ligands toward the organometallic fragment [(h6-p-cymene)RuCl2] was evidenced by the structural outcomes deriving from crystals grown in acetonitrile of the related compounds [(h6-p-cymene)Ru(INA)Cl2] (4) (INA ¼ isonicotinic acid) and [(h6-p-cymene)Ru(4ABA)Cl2] (5) (4ABA ¼ 4-aminobenzoic acid). These revealed, although in low yields, the occurred ligand solvolysis with formation of a cocrystal of formula {[(h6-p-cymene)Ru(INA)Cl2](H2O) (INA)} in the case of complex 4 and formation of the tetrakiseacetonitrile complex trans-[Ru(CH3CN)4Cl2] in the case of complex 5

Processing of raw donkey milk by pasteurisation and UV-C to produce freeze-dried milk powders: The effect on protein quality, digestibility and bioactive properties

Non-thermal processing of milk has been considered over the past decade as an alternative or adjunct to thermal processes. UV-C radiation in combination with turbulent flow of opaque liquids seems to be a promising non-thermal method for the reduction of bacterial populations in milk. Apart from confirming the efficacy of UV-C in destroying pathogens and spoilage bacteria, there is a need for assessment of the quality characteristics of the end-product and especially in added-value dairy products where bioactivity of constituents should be preserved during processing. Under this context, freeze-dried donkey milk powder processing by UV-C was studied and the effect on protein quality, digestibility and bioactive properties were assessed. Results show that UV-C treatment retains the protein's quality characteristics highly comparable to the not-treated milk (i.e. raw) rather than the pasteurized milk where some deterioration (i.e. lower bioactivities) was detected

Reduction in the Brining Time in Parmigiano Reggiano Cheese Production Minimally Affects Proteolysis, with No Effect on Sensory Properties

Brine soaking is one of the most important steps in the production of Parmigiano Reggiano cheese, since it determines the amount of salt in the final product. Reduction in salt in Parmigiano Reggiano cheese might be important for improving its nutritional profile, but it could affect the manufacturing processes by altering proteolysis and consequently the product quality. In this study, for the first time, salt reduction was explored at the industrial level on real cheese samples manufactured in a local dairy. In particular, 20 wheels were produced with conventional (18 days, 10 wheels) and shorter (12 days, 10 wheels) brining steps. In every group, wheels were studied at two different ripening times, 15 and 30 months. A shorter brining time resulted in an average 12% decrease in salt content. A full characterization of free amino acids and peptides was performed by LC-MS on all samples. Free amino acids and peptides, as expected, increased with ripening, due to proteolysis, with samples having low salt content showing a slightly faster increase when compared to standard ones, hinting to a slightly accelerated proteolytic process. Nonetheless, low-salt and conventional cheeses shared similar sensory profiles at both ripening times

A novel approach based on enzymatic hydrolysis for the valorisation of edible Parmigiano Reggiano cheese rinds

The growing market for shredded cheeses (such as flakes, internal cylinders, snacks and portions without rind) generates a large amount of Parmigiano Reggiano (PRRE) rind by-products. With the aim to provide a valorisation route for edible PRRE rinds, enzymatic hydrolysis methods were performed to produce a protein hydrolysate starting from the rinds at two ripening stages (12 and 24 months), which were previously characterised for their proximate composition. The results showed that, compared with the inner, the rind section has significantly higher protein (42.4% versus 29.7%) and lipid percentages (36.6% versus 29.7%), due to the lower moisture content (15.7% versus 32.4%). Then, after a single step of enzymatic hydrolysis, high-quality high-protein ingredients with low lipid amount (10–20%) and high digestibility (especially for the high amount of free amino acids at about 50 g 100 g−1 cheese) were obtained, to be potentially used in different food applications