Milk and dairy products: evaluation of bioactive components by analytical techniques

Milk and dairy products are important source of bioactive compounds useful to satisfy the nutritional and physiological needs of any newborns of mammalian species and useful to guarantee adequate growth and development of infants as well as provide a complete nourishment of adults. Physico-chemical, nutritional and organoleptic properties of the main constituents and the “minor” components have a crucial role in the quality of milk and milk products. Although in the past decades dietary milk fat was often regarded as harmful for the human health, recent researches suggest that milk contains specific fatty acids with nutritional and physiological health benefits. For these reasons, a major attention is given to the quantity and quality of total fat intake. In the recent years, as a result of the new concept of multifunctional agriculture and the changing behaviours about diet, consumer demands in favor of high-quality, security and safety dairy products are increased. Moreover, milk proteins and milk-derived bioactive peptides are recognized to have a high nutritive value, several health-promoting functional activities and excellent technological properties. Accordingly, growing interest in the development of functional dairy products and preparation of infant formulae for babies who cannot be breast-fed, has been give in order to meet the specific consumer’s requests. This manuscript presents the main results obtained during my PhD research aimed to evaluate the main bioactive lipids and proteins in milk and dairy products using innovative analytical techniques. The experimental section of this manuscript is divided in two sections where are reported the main results obtained during my research activities on dairy products and human milks in order to characterize their bioactive compounds for functional food applications.

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.

Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL?1 and 100 ng mL?1 of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g?1 (110e120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

The orphan nuclear receptor LXRα is positively and negatively regulated by distinct products of mevalonate metabolism

LXRα is an orphan member of the nuclear hormone receptor superfamily that displays constitutive transcriptional activity. We reasoned that this activity may result from the production of an endogenous activator that is a component of intermediary metabolism. The use of metabolic inhibitors revealed that mevalonic acid biosynthesis is required for LXRα activity. Mevalonic acid is a common metabolite used by virtually all eukaryotic cells. It serves as a precursor to a large number of important molecules including farnesyl pyrophosphate, geranylgeranyl pyrophosphate, cholesterol, and oxysterols. Inhibition of LXRα could be reversed by addition of mevalonic acid and certain oxysterols but not by other products of mevalonic acid metabolism. Surprisingly, the constitutive activity of LXRα was inhibited by geranylgeraniol, a metabolite of mevalonic acid. These findings suggest that LXRα may represent a central component of a signaling pathway that is both positively and negatively regulated by multiple products of mevalonate metabolism.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

An introduction to the chemistry of plant products by Paul Haas and T. G. Hill.

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An Introduction to the chemistry of plant products / by Paul Haas, ... and TG. Hill, ... Vol. 2 : Metabolic processes.

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