Evaluación de los efectos inmunológicos de nuevos componentes de alto valor biológico elaborados por la industria alimentaria

Resumen

Human and animal feeding is continuously needed of new alimentary sources both healthy and highly productive, in order to accommodate increasing demographic pressure and strong demand of specialized foods. Apart from the regular primary agro-food sources, the design of alternative ways for raw foods processing, re-usage of secondary products or by-products, or the isolation of essential components from foods and their addition to functional or dedicated design-foods represent strategies of wide evolution. The present work focuses into the alternative usage of two basic food resources. On one side, olive fractionation prior to olive processing in the oil factories is a source or new and valuable products of high added value. One of these products is the flour arising from the olive stone, which is becoming highly valuated because of its high content in nutritional components and its emerging nutraceutical capacities. On the other side, the different species of sweet lupins, particularly narrowleafed lupin, represent crops in continuous growth, as its consumption in several preparations (i.e. whole seeds or flours) have shown to display anti-diabetic properties, as well as a satiating property contributing to fight obesity. We lack, however of scientific evidence supporting such properties, which were observed empirically only. Therefore, the main objective of the present Ph.D. work is to obtain such evidence, through the biological assay of components derived from these alimentary sources. In the case of the olive, the NUTRAOLEUM study was used as the basis. This study has shown to be a highly informative scenario to analyze in vivo the benefits of triterpenes of the olive oil (oleanolic and maslinic acids) on the health of healthy humans. The assay encompasses the raw intake by participants of a supplementary dose (30 mL/day) of optimized olive oil, and a functional olive oil, both created by means of olive-derived-technical procedures, in comparison with intake of an extravirgin olive oil (all of them displaying differential concentrations of these compounds). Fifty-one adults participated in this random, crossed, controlled assay. Differently from other studies emerging from this clinical assay already performed in vivo, in this Ph. D. work a new method for ex vivo culture of the patients’ blood including the induction of an artificial inflammatory state was designed, which allowed the amplification of the signaling network involved. The plasma samples, or the peripheral mononuclear blood cells isolated from the cultures were subjected to a comprehensive analysis of inflammation and oxidative stress by means of ELISA, Q-PCR, quantitative Western blotting and BioPlex [i.e. IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL12, IL-13, IL-17, granulocyte macrophage colonystimulating factor (GM-CSF), interferon (IFN)-γ, TNF-α, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1], NO concentration, presence of iNOS and changes in the protein nitration profiles), which allowed demonstrating the high anti-inflammatory and anti-oxidant capacity of these compounds (triterpenes) from the olive oil. It was also shown (by using analogous procedures) the antiinflammatory character of flours derived from olive seeds, which were added to the ex vivo blood cultures of the patients. This was particularly evident in the type-2 diabetic patients, who showed a diminution of the sub-clinic inflammatory state, subjacent to this pathology. Such assays with seed flours were also confirmed in a different experimental system, which consist in cell cultures of pancreatic cells, induced by using three different inflammation inducers. In the case of lupin proteins, recombinant forms of the narrowleafed (rβ1, rβ2, rβ3, rβ4 y rβ6), purified by affinity chromatography were used. These proteins were assayed in several biological systems including their ex vivo co-culture with the blood of healthy patients and diabetic/metabolic syndrome patients, in vitro culture of pancreatic cells, and in vitro cultures of pancreatic cells after the induction of insulin tolerance. In this case, on top of the most relevant markers of inflammation and oxidative state mentioned above, additional markers were used, as those corresponding to protein carbonylation, markers involved in the insulin signaling pathways and others like IRS-1, GLUT4, CCL5, CCR2, ICAM-1 and NF-κB1 (p50), protein kinase B (AKT). Also, analyses regarding the straightforward capacity of the recombinant conglutins to bind insulin and an analysis of IRS-1 phosphorylation were performed. Such studies concluded that rβ1, rβ3, y rβ6 conglutins are able to reduce the expression of pro-inflammatory molecules as well as the whole oxidative state, increasing on the other side the expression of insulin receptors and transporters, possibly favoring recognition and control of the insulin levels. rβ2 y rβ4 isoforms of conglutins are, contrarily, favoring the inflammatory state and had no effect insulin signaling. In the case of insulin-resistant cells, the recombinant conglutins rβ1, rβ3, y rβ6 were able to noticeably improve the levels of the most relevant markers involved in maintaining optimal inflammatory and oxidative state in the resistant cultures. The present Ph. D. allowed to develop tools and procedures able to dissect the potential nutraceutical effects of different food components, by exemplifying them in two foods of broad use like the olive (and its derivatives) and lupin. This knowledge will be of utility for the agro-food industry and develops new aspects of health, which are likely applicable to the treatment of several pathologies like type-2 diabetes.