Molecular tools to exploit the biotechnological potential of brettanomyces bruxellensis : A review

The Brettanomyces bruxellensis species plays various roles in both the industrial and food sectors. At the biotechnological level, B. bruxellensis is considered to be a promising species for biofuel production. Its presence in alcoholic beverages can be detrimental or beneficial to the final product; B. bruxellensis can contribute to spoilage of wine and beer, but can also produce good aromas. However, little is known about its genetic characteristics and, despite the complete sequencing of several B. bruxellensis genomes and knowledge of its metabolic pathways, the toolkits for its efficient and easy genetic modification are still underdeveloped. Moreover, the different ploidy states and the high level of genotype diversity within this species makes the development of effective genetic manipulation tools challenging. This review summarizes the available tools for the genetic manipulation of B. bruxellensis and how they may be employed to improve the quality of wine and beer

Wine industry’s attitude towards oenological yeasts : Italy as a case study

Yeast inoculation is a widespread practice in winemaking in order to control the must fermentation. However, the use of indigenous wine yeasts can enrich wine quality and differentiate wine styles. Yeast cream preparation (CRY), recently accepted by the International Organization of Vine and Wine, could allow an easier usage of autochthonous yeasts. This work aimed at investigating the actual Italian wine industry\u2019s attitude towards the available formulations of commercial wine yeasts with attention to CRY. Moreover, this study evaluated the perception of wineries toward indigenous yeasts in both winemaking and marketing viewpoints. Data show different levels of knowledge and use about the available yeast formulations. In general, there is not a predominantly positive or negative participants\u2019 opinion regarding the use of indigenous yeasts. Wineries using CRY (4% of the sample) mainly adopt them as a part of the production in order to compare the wines with the ones traditionally obtained with commercial yeasts. CRY is perceived by some interviewees as a potential tool to increase communication and product differentiation. This survey could have anticipated future trends in the use of yeast formulations, determined by the market demands for diversified, unique, and environmentally sustainable products, that can allow an accessible application of precision enology

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier "indolome" to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us "indolome") in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100-1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition

Genotypic Characterization and Biofilm Formation of Shiga-toxin producing Escherichia coli

Shiga toxin producing Escherichia coli (STEC) are recognized as one of the most dangerous food-borne pathogens. The production of Shiga toxins together with intimin protein are among the main virulence factors. However, the ability to form biofilm can protect bacteria against environmental factors (i.e. desiccation, exposure to UV rays, predation, etc) and sanitization procedures (cleaning, rinsing, chlorination), increasing their survival on food products and in manufacturing plants. Forty-five isolates collected from food and fecal samples were genotyped by Pulsed Field Gel Electrophoresis (PFGE) analysis with XbaI restriction enzyme and investigated by searching for toxins (stx1, stx2) and intimin (eae) genes and serogroup (O157, O26, O145, O111, O103 and O104). Afterward, the ability to develop biofilm in microtiter assay and the production of adhesive curli fimbriae and cellulose on agar plates were tested. Our study demonstrated that biofilm formation has a great variability among STEC strains and cannot be related to a specific pulsotype nor even to serogroup or presence of virulence genes

CRISPR/Cas9 system as a valuable genome editing tool for wine yeasts with application to decrease urea production

An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial "starter" strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8-12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts

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

Gluten-Free Bread : Influence of Sourdough and Compressed Yeast on Proofing and Baking Properties

The use of sourdough is the oldest biotechnological process to leaven baked goods, and it represents a suitable technology to improve traditional bread texture, aroma, and shelf life. A limited number of studies concerning the use of sourdough in gluten-free (GF) breadmaking have been published in comparison to those on traditional bread. The aim of this study was to compare the properties of GF breads obtained by using a previously in-lab developed GF-sourdough (SD), compressed yeast (CY; Saccharomyces cerevisiae) or their mixture (SDCY) as leavening agents; more specifically, it aims to confirm the findings of a previous studies and to further improve (both in terms of recipe and process) the features of the resulting GF breads. Dough pH and rheological properties were measured. Fresh and stored breads were characterized for weight, height, specific volume, crust and crumb color, moisture, water activity, crumb hardness, and porosity. The combination SDCY was effective in improving bread volume and softness when compared to SD only. Furthermore, SD- and SDCY-crumbs exhibited a less crumbly behavior during storage (69 h, 25 \ub0C, 60% of relative humidity) in comparison to CY-breads. This study confirms the positive effect of SD in GF breadmaking, in particular when used in combination with CY

Airway inflammation in patients affected by obstructive sleep apnea

Obstructive sleep apnea (OSA) is characterised by repetitive episodes of upper airway occlusion during sleep. OSA has been shown to be associated with a variable degree of nasal inflammation, uvula mucosal congestion and airway hyperreactivity. The upper airway inflammation, whose clinical importance is uncertain, is characterised by leukocytes infiltration and interstitial oedema. In addition, recent data has shown the presence of neutrophilic inflammation in the lower airways. The current opinion is that airway inflammation is caused by the local, repeated mechanical trauma related to the intermittent airway occlusion typical of the disease. Another potential mechanism involves the intermittent nocturnal hypoxemia that through the phenomenon of the ischemia- reperfusion injury may induce the production of oxygen free radicals and therefore cause local and systemic inflammation. Finally, a state of low-grade systemic inflammation may be related to obesity per se with the pro-inflammatory mediators synthesised in the visceral adipose cells. Several authors stress the role of circulating and local inflammatory mediators, such as proinflammatory cytokines, exhaled nitric oxide, pentane and 8-isoprostane as the determinants of inflammation in OSA

Cold exposure affects carbohydrates and lipid metabolism, and induces Hog1p phosphorylation in Dekkera bruxellensis strain CBS 2499

Dekkera bruxellensis is a yeast known to affect the quality of wine and beer. This species, due to its high ethanol and acid tolerance, has been reported also to compete with Saccharomyces cerevisiae in distilleries producing fuel ethanol. In order to understand how this species responds when exposed to low temperatures, some mechanisms like synthesis and accumulation of intracellular metabolites, changes in lipid composition and activation of the HOG-MAPK pathway were investigated in the genome sequenced strain CBS 2499. We show that cold stress caused intracellular accumulation of glycogen, but did not induce accumulation of trehalose and glycerol. The cellular fatty acid composition changed after the temperature downshift, and a significant increase of palmitoleic acid was observed. RT-PCR analysis revealed that OLE1 encoding for \u3949-fatty acid desaturase was up-regulated, whereas TPS1 and INO1 didn't show changes in their expression. In D. bruxellensis Hog1p was activated by phosphorylation, as described in S. cerevisiae, highlighting a conserved role of the HOG-MAP kinase signaling pathway in cold stress response