Caracterización química y bioactividad de compuestos producidos por levaduras y derivados de la vid.

Abstract

In recent years, melatonin has been identified in different foods, mainly from plant origin such us fruits (grapes, strawberries) and vegetables (tomatoes and peppers). However, the identification of melatonin in fermented foods as beer and wine has attracted an increasing interest in research. Literature data indicate that the presence of this molecule in fermented food is due to the yeast metabolism, especially from Saccharomyces cerevisiae, during fermentative process. Melatonin is a well-known neurohormone synthesized by the pineal gland of mammals. Its main function is related with circadian rhythms depending of light/darkness conditions. Besides, it has reported additional bioactive properties such as antioxidant, being an effective oxygen scavenger. On the other hand, it has also been described in plants and microorganism, although its role in both organism in not well elucidated. Melatonin is formed from the amino acid tryptophan that belongs to the aromatic amino acids groups. Mammal’s metabolism of tryptophan encompasses different secondary metabolites such as 5-hydroxytryptophan, serotonin and N-acetylserotonin. In plants, tryptamine can be included on the melatonin pathway instead of 5-hydroxytryptophan. However, the metabolic pathway that produce melatonin in microorganisms is not completely defined. Nevertheless, amino acid catabolism by yeast known as the Ehrlich pathway mainly produces indole acetic acid and tryptophol. Analytical techniques to quantify melatonin in biological fluids and foodstuff is quite diverse, covering from radioimmunoassay, being ELISA the most representative, to high resolution liquid chromatographic techniques coupled to a high resolution mass spectrometer. The main challenge of these techniques is to improve the detection and quantification limits of these compounds. ELISA technique is well performed when biological fluids are analysed. Nevertheless, its application to foodstuffs has been less repeatable, maybe due to the presence of interference in the food matrix that can react with the ELISA antibodies. For this reason, High Resolution Liquid Chromatography has proved to be the best technique for the melatonin and derived tryptophan compounds analysis. At first, chromatographic methods with a fluorescence detector were used to determine melatonin in food. However, the low concentrations of melatonin presented in foods, and the limits of detection and quantitation of this detector make it ineffective to describe the presence of melatonin unequivocally. Nowadays, liquid chromatography coupled to mass spectrometers results the best option to study melatonin. However, most of the reported methods only analysed melatonin. Thus, they cannot bring to light to a fully view of the likely formation pathways in fermented foods. Therefore, one of the aims of the present doctoral thesis is to develop a chromatographic method couple to mass spectrometry that simultaneously identify different tryptophan derived compounds related with melatonin formation pathway. Additionally, the study of the compounds stability in different storage conditions was also included. Furthermore, fermentation experiments were performed in synthetic and natural must in order to find yeast with the best ability to synthesize indolic compounds derived from tryptophan. Eventually, commercial beers were analysed to dilucidate whether in other matrixes apart from wine indolic compounds are also produced. Moreover, the bioactivity of stilbenoids compounds derived from the amino acid phenylalanine and present in grapevine stalks of Vitis vinífera, was also study. These experiments have the aim of evaluate the potential ability of these compounds to inhibit angiogenesis. This physiological process is involved in the development of cancer and cardiovascular diseases in adult population. Both diseases are consider the world’s leading cause of death, according to the last data reported by the World Health Organisation. Since vascular endothelial growth factor (VEGF) is the main proangiogenic factor, its inhibition is a plausible molecular mechanism that shows a direct cause-effect for decreasing cardiovascular and cancer risk. One of the most remarkable proofs of VEGF role in angiogenesis is that some cancer treatments are based in an anti- VEGF drugs such as Avastin®, which is the most currently used. However, their lengthy treatment causes serious side effects, increasing the risk of hypertension. Certain polyphenols that are present in foods such as epillogatechin gallate and quercetin, among others, have proved a potential anti-VEGF activity, allowing the activation at the same time of the endothelial nitric oxide synthase, responsible of vasodilation. Thus, anti-VEGF potential of twelve stilbenes was evaluated in human umbilical vein endothelial cells. In addition, the possible effect on the signalling intracellular downstream, particularly on phospholipase γ1 (PLC γ1, main responsible of cell proliferation), protein kinase B and eNOS was also studied. Most remarkable results lead from the development and validation of an analytical method that identified simultaneously nine compounds derived from the amino acid tryptophan for the first time by UHPLC/HRMS technique in fermented synthetic must. The present method has been useful to evaluate the possible matrix effect of fermented samples, resulting beer as the matrix that caused less decrease of indolic compound composition and establishing a storage temperature of 4ºC as the best alternative of conservation. On the other hand, melatonin and related compounds derived from tryptophan metabolism such as serotonin, indole acetic acid, tryptophol and ethyl ester of tryptophan was confirm during the alcoholic fermentation of synthetic must, being the S.cerevisiae Aroma White the most melatonin producer. Additionally, it was proved the reproducibility of results in natural must of different grape varieties (Corredera, Chardonnay, Moscatel, Palomino Fino, Sauvignon Blanc, Tempranillo, Vijiriega), where Aroma White proved again to be the highest melatonin producer. Moreover, for the first time N-acetylserotonin was quantified during the alcoholic fermentation of Tempranillo must. The analysis of indolic compounds in beers pinpoints the identification of 5-hydroxytryptophan, Nacetylserotonin and indole acetic acid in beers as a novelty. Regarding stilbenes bioactivity, astringin, piceatannol, pallidol, ε- and ω-viniferina inhibited VEGF activity (IC50 = 2.9-39.4 μM). In addition, both ε- and ω-viniferin were also able to inhibited PLCγ1, while activated eNOS, likely avoiding the side effects of hypertension, currently related with the anti-VEGF drugs treatment. With no doubt, present results are remarkable to improve wine-making process by enhancing the presence of stilbenes from grapevine and the indolic compounds derived from the amino acid tryptophan by yeast metabolism in wines during the alcoholic fermentation, giving an added value to the final product and trying to emulate this practices to the production of other fermented beverages such as beer.