Healthy and adverse effects of plant-derived functional metabolites: the need of revealing their content and bioactivity in a complex food matrix.

In recent years, both food quality and its effect on human health have become a fundamental issue all over the world. As a consequence of this new and increased awareness, American, European, and Asian policymakers have strongly encouraged the research programs on food quality and safety thematic. Attempts to improve human health and to satisfy people's desire for healthcare without intake of pharmaceuticals, has led the food industry to focus attention on functional or nutraceutical food. For a long time, compounds with nutraceutical activity have been produced chemically, but the new demands for a sustainable life have gradually led the food industry to move towards natural compounds, mainly those derived from plants. Many phytochemicals are known to promote good health, but, sometimes, undesirable effects are also reported. Furthermore, several products present on the market show few benefits and sometimes even the reverse - unhealthy effects; the evidence of efficacy is often unconvincing and epidemiological studies are necessary to prove the truth of their claims. Therefore, there is a need for reliable analytical control systems to measure the bioactivity, content, and quality of these additives in the complex food matrix. This review describes the most widespread nutraceutics and an analytical control of the same using recently developed biosensors which are promising candidates for routine control of functional foods.

The NUTRA-SNACKS project: basic research and biotechnological programs on nutraceutics.

The Nutra-Snacks project aims at creating novel high quality ready-to-eat foods with functional activity, useful for promoting public health. The team is composed of seven research institutes and three SMEs from different countries whose activities span from basic to applied research providing the right technological transfer to small and medium industries involved in the novel food production chain. Strategic objectives include the application of plant cell and in vitro culture systems to create very large amounts of high-value plant secondary metabolites with recognized anticancer, antilipidemic, anticholesterol, antimicrobial, antiviral, antihypertensive and anti-inflammatory properties and to include them in specific food products. To this end, the screening of a vast number of working organisms capable of accumulating the desired compounds and the characterization of their expression profiles represent fundamental steps in the research program. The information allows the identification of plant species hyper-producing metabolites and selection of those metabolites capable of specifically counteracting the oxidative stress that underlies the development of important pathologies and diseases. In addition, devising safe metabolite extraction procedures is also crucial in order to provide nutraceutical-enriched extracts compatible with human health. New biotechnological approaches are also undertaken including the exploitation of photosynthetic algal strains in bio-farms to enhance the synthesis ofantioxidant compounds and the design of novel bioreactors for small and large scale biomass production. Further outstanding objectives include the development of (i) safety and quality control protocols (ii) biosensor techniques for the analysis of the emerging ready-to-eat food and (iii) a contribution to define a standard for new regulations on nutraceutics.

Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli.

The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein. To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies. The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent. Enhanced protein refolding was observed by reducing the protein concentration at 0.6-0.8 mg/ml. SDS-PAGE and gel filtration chromatography indicated an apparent molecular mass of approximately 40 kDa and the occurrence of the protein as monomers. The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity. Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding. The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of beta structures (higher than 60%) and a small contribution from alpha helices (less than 12%). The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites.

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants.

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.