ENHANCEMENT OF SACCHAROMYCES CEREVISIAE GLUTATHIONE AND MICRONUTRIENTS CONTENT FOR NUTRACEUTICAL APPLICATIONS.

This Ph.D. research concerns nutritional and microbiological aspects, and the final aim of this project is the development of innovative food supplements formulations containing Saccharomyces cerevisiae, enriched, by biotechnological processes, with micronutrients and antioxidant molecules. This idea was supported by the fact that even if in the developed world a clinical deficiency of micronutrients is uncommon, a suboptimal intake of certain micronutrients has been linked with an increased risk of chronic diseases such as CVD (cardiovascular disease) and cancer; moreover external factors, such as smoke, UV radiations and pollution contribute to oxidative stress and to the formation of free radicals that are considered to contribute to the risk of cancer. In this context and to contain cellular damage, an important role has recently been attributed to the possible use of molecules with antioxidant activity, such as glutathione. The yeast S. cerevisiae is one of the most studied microorganisms and is considered a model for eukaryotes. It is used both in industrial productions and in human diet. As well as leavening agent for baking products and fermenting agent for alcoholic beverages such as wine and beer, S. cerevisiae is used in the industrial production of ethanol, enzymes and dried yeast both for animal-feed and food supplement (Directive 2002/46/EC). Subsequently to the identification of some potential molecules, with which the yeast might be enriched, the research focused mainly on cell enrichment with reduced glutathione (GSH) and metal-conjugated glutathione, with particular regard to the set-up of biotechnological processes in order to increase product yields. The research also investigated the biological activity of the obtained enriched biomass, in particular the fate of GSH when the biomass is swallowed, by investigating GSH stability during gastric digestion and any possible protective role of the yeast cell, and by analysing GSH transport/absorbtion by intestinal cell lines and any possible toxicity. In the first year of the research numerous trials were carried out in order to increase intracellular GSH levels in S. cerevisiae both during cell growth and applying a post-growth procedure, identified as “activation”. The best results were obtained applying this second approach in which glucose, GSH precursors (cysteine CYS, glycine GLY and glutamic acid GLU), together with cofactors, were dissolved in a solution in which yeast cells were suspended. In this experimental phase the activation attitude of two different commercial forms of the yeast, compressed or dried, were evaluated. Results highlighted that, even though they generally performed similarly in bread-making process and possessed similar initial intracellular GSH levels (about 0.6% dcw) and for both of them higher yields were obtained during the first 24 h of activation, they evidenced different attitudes towards the tested activation mix. In particular, dried yeast samples reached high GSH levels (1.24 ±0.08% dcw) with the mix CYS-GLY-GLU, while the compressed yeast also with the CYS-GLY-SER (with serine) (1.44 ±0.12% dcw). Concerning the influence of the energy source, the dried form did not appear sensitive to the over-presence of glucose, while the compressed yeast form showed different sensitivity to glucose addition, depending on the type of the mixture employed. Glucose addition in CYS-GLY and CYS-GLY-GLU mixtures (in particular at 4 h) increased GSH yields of about 30-40% with respect to the control sample. No increase was evidenced employing the CYS-GLY-SER mixture. The possibility of obtaining GSH directly in extracellular form was also investigated; this approach can represent an interesting opportunity of reducing GSH production cost and furthering the range of application of this molecule. The second year of the research proceeded mainly with the compressed yeast form as it was found to accumulate higher GSH level than the dried one. As GSH biosynthesis is ATP-dependent, in order to increase intracellular ATP levels molecules directly or indirectly involved in its synthesis (i.e. adenosine and adenine) and an ATP-ase inhibitory agent (dithiothreitol) were added to activation solution CYS-GLY-GLU. All the tested mixtures furnished significantly higher GSH yields than the control ones; above all, adenine addition allowed to obtained 1.68±0.04 GSH % dcw. Influence of yeast shelf-life on GSH accumulation was also investigated and results showed that when yeast was at the beginning of its shelf-life (1 day) it was able to gain high intracellular GSH levels, with a three-fold increase with respect to t0; when prolonging the age, a significant change in the yeast attitude towards the activation procedure was evidenced, and a general decrease of GSH intracellular levels was found. As well as yeast shelf-life, also intracellular trehalose content was found a very important factor for determining GSH accumulation ability of yeast. Important results were then obtained applying a Design of Experiments (DoE) on four GSH precursor amino acids (A-CYS, B-GLY, C-SER, D-GLU). The Design Expert® (Statease, Minneapolis) software, used for analysing results, individuated significant models both for the compressed and dried yeast samples. All four factors were found significant together with AC and BC interactions and the quadratic terms A2 and D2 for compressed yeast, while only A2 for the dried sample. During the prosecution of the research, biological activity of GSH and of enriched biomass was investigated. This part of the research was performed at Instituto de Agrochimica y Tecnologia de los Alimentos (IATA-CSIC) in Valencia (Spain) and the evaluation of an in vitro gastrointestinal digestion followed by cellular transport throught intestinal epithelium was performed. Both standard GSH and yeast cells GSH-enriched were tested. The in vitro gastrointestinal digestion did not affect GSH levels, neither when present as a standard solution nor when it accumulated inside a yeast cell; anyway, a very limited amount of GSH (up to 25%) was found to oxidize to GSSG. In vitro GSH transport trials through the intestinal epithelium were carried out with the Caco-2 cell line model and a co-culture Caco-2:HT-29-MTX (ratios 50:50 and 70:30), comparatively. GSH standard solution was employed at three different levels (3, 10 and 30 mM). Results showed that GSH transport from the apical to the basal chamber is very low, either using Caco-2 cell line and the co-culture. Important results were obtained in experiments regarding the protective effect of GSH in intestinal cells exposed to an inducer of oxidative stress (H2O2). Cell viability reduction was lower in samples added with GSH when employing H2O2 at high concentration (10-20 mM); moreover the use of yeast-enriched was found to prevent cells viability reduction much more than the equivalent standard GSH (3 mM). In the third year of the research experiments were carried out in order to obtain S. cerevisiae copper-enriched cells. Thinking that copper can conjugate with GSH and/or GSSG, the strategy was to enrich cells with GSH and then to furnish them copper acetate with different treatments. Results highlighted that before copper addition GSH levels were in the range 1.2-1.5% dcw, while after 4 and 24 h these levels drastically decreased to 0.5-1% dcw; on the contrary, GSSG was found to increase. All biomass obtained were analysed by ICP-AES: results showed that the biomass centrifuged and resuspended in the copper solution accumulated the highest copper levels; copper direct addition to the activation mixture led to the lowest results. Summarizing, the research was aimed at obtaining GSH and copper-enriched cells of S. cerevisiae, developing an efficient delivery system for nutraceutical compounds and metals, suitable for human nutrition and therapeutic treatments. GSH enriched yeast cells can be obtained applying different post-fermentation strategies, taking in consideration the type of the yeast employed, its shelf-life and the formulation of the activation mixture (energy request and aminoacids precursors). The application of this procedure resulted in an increase of intracellular GSH levels, from initial 0.5-0.7% dcw to a final 1.7-1.9% dcw. Even if the obtained results highlighted that GSH is transported from intestinal cells in limited amount, it can cooperate to maintain important functions of the intestinal lumen. In conclusion obtained GSH and copper-enriched yeast biomass can be considered an interesting opportunity to further the range of application of yeast cell cultures for nutraceutical application.