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 \u201cactivation\u201d. 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 \ub10.08% dcw) with the mix CYS-GLY-GLU, while the compressed yeast also with the CYS-GLY-SER (with serine) (1.44 \ub10.12% 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\ub10.04 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\uae (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

Zymomonas mobilis in Bread Dough: Characterization of Dough Leavening Performance in Presence of Sucrose

Zymomonas mobilis, because of its fermentative metabolism, has potential food applications in the development of leavened baked goods consumable by people with adverse responses to Saccharomyces cerevisiae. Since Z. mobilis is not able to utilize maltose present in flour, the effect of sucrose addition (2.5 g/100 g flour) on bread dough leavening properties was studied. For comparison purposes, leavening performances of S. cerevisiae with and without sucrose were also investigated. Doughs leavened by Z. mobilis without sucrose addition showed the lowest height development (14.95 +/- 0.21 mm) and CO2 production (855 +/- 136 mL). When sucrose was added, fermentative performances of Z. mobilis significantly (p < 0.05) improved (+80% and +85% of gas production and retention, respectively), with a dough maximum height 2.6 times higher, results indicating that Z. mobilis with sucrose can be leavened in shorter time with respect to the sample without addition. S. cerevisiae did not benefit the sucrose addition in terms of CO2 production and retention, even if lag leavening time was significantly (p < 0.05) shorter (about the half) and time of porosity appearance significantly (p < 0.05) longer (about 26%) with respect to S. cerevisiae alone. Results demonstrate that in the presence of sucrose, Z. mobilis can efficiently leaven a bread dough, thus providing innovation possibilities in the area of yeast-free leavened products

Evaluation of the antioxidant/antimicrobial performance of Posidonia oceanica in comparison with three commercial natural extracts and as a treatment on fresh-cut peaches (Prunus persica Batsch)

This research aimed at extending the choice of natural antimicrobials/antioxidants for food applications. Four plant extracts, Posidonia oceanica (PO), Green Tea (GT), Grape seeds (GS) and Grape skin (GK), were analyzed to determine their total phenolic content, antioxidant activity and in vitro antimicrobial performance. PO extract showed the highest total phenolic content (711 mg gallic acid/g extract) and antifungal activity against Aspergillus niger and Penicillium chrysogenum. The highest antioxidant (3.81 mg/L EC50) and antibacterial activities (bactericidal against Gram positives and bacteriostatic against Gram negatives) were found for GT extract. The best performing extracts (PO and GT) were applied by dipping on peach slices in storage trials. Microbiological and pomological parameters were evaluated during 7 d storage. Total aerobic count, Pseudomonas as well as yeasts and moulds populations, were reduced by about 0.5 log cfu/g, mainly up to 5 d in all treated samples compared to the control. Total soluble solids, titratable acidity and colour (L*a*b*) changes were also delayed in treated fruit

An alternative encapsulation approach for production of active chitosan-propolis beads

Encapsulation is a promising technology to carry natural active substances, preventing their loss and maintaining their stability until use. Beads of chitosan-containing propolis have been prepared using a mono-pore filter device, which permits the encapsulation of natural polyphenols avoiding heat treatments, high shear rates and the use of toxic solvents. Beads proved to be active against Bacillis cereus, Escherichia coli, Listeria innocua, Pseudomonas fluorescens, Yarrovia lipolytica and three moulds strains; the highest effect was found against Staphylococcus aureus (MIC 0.8 mg beads mL-1). Results in liquid cultures of S. aureus evidenced that beads were able to release the flavonoids from propolis: the diffusion of the active compounds is a key factor in the exploitation of the microbial activity. The obtained chitosan-propolis beads represent an example of natural antimicrobial delivery system that could be used to prevent the growth of pathogenic/spoilage bacteria in food applications

Influence of substrate on beta-galactosidase production by Kluyveromyces strains

The aim of the present research was to investigate the influence of culture conditions on the levels of β-galactosidase (EC 3.2.1.23) activity produced by Kluyveromyces strains. Interest was focused on evaluating enzyme activity levels when lactose or cheese whey was employed as substrate in culture medium formulation. From an overall look at the obtained results, the tested strains were found to be able to produce β-galactosidase at promising levels. The use of cheese whey, either for strain maintenance and production trials, allowed to obtain a high cell yield associated with β-galactosidase production. The maximum β-galactosidase volumetric activity, EA max 66.5 IU/ml, corresponding to 3184 EAspec IU/g cell dw, was obtained with K. marxianus MIM 782 at 37 °C and 72 h incubation

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thincut meat: preliminary assessment

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm 122) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 \ub0C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g 121). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases

Can Zymomonas mobilis Substitute Saccharomyces cerevisiae in Cereal Dough Leavening?

Baker\u2019s yeast intolerance is rising among Western populations, where Saccharomyces cerevisiae is spread in fermented food and food components. Zymomonas mobilis is a bacterium commonly used in tropical areas to produce alcoholic beverages, and it has only rarely been considered for dough leavening probably because it only ferments glucose, fructose and sucrose, which are scarcely present in flour. However, through alcoholic fermentation, similarly to S. cerevisiae, it provides an equimolar mixture of ethanol and CO\u2082 that can rise a dough. Here, we propose Z. mobilis as a new leavening agent, as an alternative to S. cerevisiae, overcoming its technological limit with different strategies: (1) adding glucose to the dough formulation; and (2) exploiting the maltose hydrolytic activity of Lactobacillus sanfranciscensis associated with Z. mobilis. CO\u2082 production, dough volume increase, pH value, microbial counts, sugars consumption and ethanol production were monitored. Results suggest that glucose addition to the dough lets Z. mobilis efficiently leaven a dough, while glucose released by L. sanfranciscensis is not so well fermented by Z. mobilis, probably due to the strong acidification. Nevertheless, the use of Z. mobilis as a leavening agent could contribute to increasing the variety of baked goods alternative to those leavened by S. cerevisiae

Glutathione–enriched baker’s yeast: production, bioaccessibility and intestinal transport assays

AIMS: a glutathione (GSH) yeast-based biomass (S. cerevisiae) was used to investigate GSH stability, solubilization during gastrointestinal digestion and GSH intestinal transport. METHODS AND RESULTS: a post-growing procedure was applied to improve intracellular GSH yeast content. The presence of adenine (ADE) in the biotransformation solution (CYS-GLY-GLU mixture) and alternatively, a glucose shot after 4 h incubation, allowed to obtain cells containing about GSH 1.6-1.7% dcw (dry cell weight) (control 0.5%). Yeast samples were subjected to in vitro gastro-intestinal digestion and absorption assays employing Caco-2 and HT29-MTX cell lines in different proportions (100/0, 70/30 and 50/50). Trials were also performed to verify intestinal cell viability. CONCLUSIONS: at least 87% of ingested GSH is available in reduced form for intestinal absorption. In vitro GSH transport assays indicated that GSH is poorly absorbed (<20%). Nevertheless, studies in response to oxidative stress induced by H2 O2 demonstrated a protective role of the GSH-enriched biomass towards intestinal cell viability. SIGNIFICANCE AND IMPACT OF STUDY: an enriched GSH yeast-based biomass has been obtained using a post-growing procedure. Although GSH present in enriched yeasts is poorly absorbed by intestinal cells, this biomass showed an intestinal local protective effect, improving cells viability when a simulated oxidative stress was applied

Cellulose nano-fibers (CNF) - Sakacin-A active material: production, characterization and application in storage trials of smoked salmon

BACKGROUND: Sakacin-A due to its specific antimicrobial activity may represent a good candidate to develop active packagingsolutions for food items supportingListeriagrowth. In the present study a protein extract containing the bacteriocin sakacin-A,produced byLactobacillus sakeiLb 706 in a low-cost culture medium containing deproteinized cheese whey, was adsorbed ontocellulose nanofibers (CNFs) to obtain an active material to be used as a mat (or a separator) in direct contact with foods.RESULTS: The applied fermentation conditions allowed 4.51g L 121of freeze-dried protein extract to be obtained, characterizedby an antimicrobial activity of near 16 700 AU g 121, that was used for the preparation of the active material by casting. The activematerial was then characterized by infrared spectra and thermogravimetric analyses. Antimicrobial trials were carried outinvitrousingListeriainnocuaasindicatorstrain;resultswerealsoconfirmedinvivo,employingsmokedsalmonfilletsintentionallyinoculated withListeria innocua: its final population was reduced to about 2.5 \u2013 3 Log cycles after 28 days of storage at 6 18Cinpresence of sakacin-A, compared with negative control mats produced without the bacteriocin extract.CONCLUSION: This study demonstrates the possibility of producing an antimicrobial active material containing sakacin-Aabsorbed onto CNFs to decreaseListeriapopulation in smoked salmon, a ready-to eat-food product

Prezioso come uno scarto: dal permeato di siero un imballaggio sostenibile ad attivit&#224; antimicrobica

L\u2019interesse verso gli imballaggi alimentari attivi sta rapidamente crescendo. In quest\u2019ottica, il progetto NANOSAK ha consentito di sviluppare a partire da permeato da ultrafiltrazione di siero materiali attivi in grado di inibire lo sviluppo di Listeria monocytogenes