Using ethanol as postharvest treatment to increase polyphenols and anthocyanins in wine grape.

Red wine grapes are qualitatively evaluated for their content in polyphenols and anthocyanins. Due to certain conditions (weather, latitude, temperature), the concentration of these compounds may be not at the right level for reaching a high-quality wine, thus postharvest technologies can be operated as a remediation strategy. Ethanol is a secondary volatile metabolite and its application has been demonstrated to delay fruit ripening, to reduce decay, and to increase secondary metabolites. The present study investigates the effects of ethanol post-harvest application on wine grapes' metabolism and composition. Red wine grapes (Vitis Vinifera L. cv Aglianico) were exposed to different ethanol doses (0.25, 0.5, or 1 mL L-1) for 12, 24, or 36 h. Ethanol increased sugar concentration, malic acid, free amino nitrogen, polyphenols, and anthocyanins. Particularly, anthocyanins reached an average value of 1820 mg/L in treated samples versus the 1200 mg/L of control grapes already after 12 h whatever the concentration was. Moreover, the highest concentration of ethanol modified berry metabolism shifting from aerobic to anaerobic one. Obtained results suggest that 12 h of ethanol postharvest treatment could be an interesting solution to improve anthocyanins in wine grapes, especially when the quality is not as good as expected.

Microencapsulation and sonication: A multiple physical approach to attenuate the probiotic Lacticaseibacillus casei ATCC 393.

Modulation of probiotic performances represents a tool to avoid the probiotic off-flavor in probiotic food. Microencapsulation and sonication were evaluated in slowing down the Lacticaseibacillus casei ATCC 393 induced acidification. Firstly, the influence of alginate concentration and chitosan coating on acidification rate were tested. Microcapsule morphology and the entrapment efficacy were also evaluated. Then, two time of exposure to ultrasound, 6 and 8 min, were applied for L. casei attenuation. Finally, sonicated cells were encapsulated. ΔpH after 6 and 24 h of incubation at 37 °C revealed that chitosan-alginate microcapsules and the 8-min sonicated probiotic presented a significant delayed acidification. When all the systems were compared, the encapsulation of 8-min sonicated L. casei in chitosan-alginate microcapsules significantly improved the results obtained with the single technologies. These results suggest that by modulating the operating parameters and combining these two technologies an increasingly efficient attenuation system can be developed.

Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Whole-Genome Duplication and Yeast's Fruitful Way of Life.

Studies on the fate of Saccharomyces cerevisiae paralogous gene pairs that arose through a whole-genome duplication event have shown diversification of retained duplicated genes. Paralogous functional specialization often results in improved function and/or novel function that could contribute to the adaptation of the organism to a new lifestyle. Here, we analyze and discuss particular case studies of paralogous functional diversification that could have played a role in the acquisition of yeast fermentative metabolism.

Regulation of glycolytic flux and overflow metabolism depending on the source of energy generation for energy demand.

Overflow metabolism is a common phenomenon observed at higher glycolytic flux in many bacteria, yeast (known as Crabtree effect), and mammalian cells including cancer cells (known as Warburg effect). This phenomenon has recently been characterized as the trade-offs between protein costs and enzyme efficiencies based on coarse-graining approaches. Moreover, it has been recognized that the glycolytic flux increases as the source of energy generation changes from energetically efficient respiration to inefficient respiro-fermentative or fermentative metabolism causing overflow metabolism. It is highly desired to clarify the metabolic regulation mechanisms behind such phenomena. Metabolic fluxes are located on top of the hierarchical regulation systems, and represent the outcome of the integrated response of all levels of cellular regulation systems. In the present article, we discuss about the different levels of regulation systems for the modulation of fluxes depending on the growth rate, growth condition such as oxygen limitation that alters the metabolism towards fermentation, and genetic perturbation affecting the source of energy generation from respiration to respiro-fermentative metabolism in relation to overflow metabolism. The intracellular metabolite of the upper glycolysis such as fructose 1,6-bisphosphate (FBP) plays an important role not only for flux sensing, but also for the regulation of the respiratory activity either directly or indirectly (via transcription factors) at higher growth rate. The glycolytic flux regulation is backed up (enhanced) by unphosphorylated EIIA and HPr of the phosphotransferase system (PTS) components, together with the sugar-phosphate stress regulation, where the transcriptional regulation is further modulated by post-transcriptional regulation via the degradation of mRNA (stability of mRNA) in Escherichia coli. Moreover, the channeling may also play some role in modulating the glycolytic cascade reactions.

Interactions between carbon and nitrogen sources depend on RIM15 and determine fermentative or respiratory growth in Saccharomyces cerevisiae.

Nutritional homeostasis is fundamental for alcoholic fermentation in Saccharomyces cerevisiae. Carbon and nitrogen have been related to this metabolic process; nevertheless, little is known about their interactions with the media and the energetic metabolism. Rim15p kinase is a point of convergence among different nutrient-activated signaling pathways; this makes it a target to investigate the relationship between nutritional status and energetic metabolism. To improve the current knowledge of nutrient interactions and their association with RIM15, we validated the doubling time as an indicator of growth phenotype, confirming that this kinetic parameter can be related to the cellular bioenergetic status. This endorses the usefulness of a threshold in doubling time values as an indicator of fermentative (≤ 6.5 h) and respiratory growth (≥ 13.2 h). Using the doubling time as response variable, we find that (i) two second-order interactions between type and concentration of carbon and nitrogen sources significantly affected the growth phenotype of S. cerevisiae; (ii) these metabolic interactions changed when RIM15 was deleted, suggesting a dependence on this gene; (iii) high concentration of ammonium (5% w/v) is toxic for S. cerevisiae cells; (iv) proline prompted fermentative growth phenotype regardless presence or absence of RIM15; (v) RIM15 deletion reverted ammonium toxicity when cells were grown in glucose (10% w/v); and (vi) RIM15 deletion improves fermentative metabolism probably by a partial inhibition of the respiration capacity. This study reveals the existence of synergic and diverse roles of carbon and nitrogen sources that are affected by RIM15, influencing the fermentative and respiratory growth of S. cerevisiae.

Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: a strategy to obtain wines with reduced ethanol content.

Ethanol content of wine has increased over the last decades as consequence of searching phenolic maturity, requiring increased grape maturity. This may result in the production of wines with excessive alcohol levels (sometimes more than 15% (v/v)), sluggish and stuck fermentations and excessive volatile acidity. Many strategies to reduce ethanol in wines are being studied, and microbial methods have some additional advantages. However, because of the broad intra- and interspecies variability, new selection criteria should be included. Therefore, the goal of the present work was to design and evaluate a simple and integral procedure for non-Saccharomyces yeast selection. This strategy allowed selection of yeasts that presented successful implantation in grape must with high alcohol potential and their use in co-cultures could reduce the ethanol in wines. A total of 114 native non-Saccharomyces yeasts were assayed to determine their respiratory, fermentative and physiological characteristics of enological interest. Hanseniaspora uvarum BHu9 and BHu11, H. osmophila BHo51, Starmerella bacillaris BSb55 and Candida membranaefaciens BCm71 were selected as candidates to design co-culture starters.

Biochemical Basis of CO2-Related Internal Browning Disorders in Pears (Pyrus communis L. cv. Rocha) during Long-Term Storage.

This study aimed at understanding the biochemical basis of internal browning disorders (IBDs) in 'Rocha' pear. For this purpose, the effects of storage under normal controlled atmosphere (CA) (3 kPa of O2 + 0.5 kPa of CO2) and IBD-inducing CA (1 kPa of O2 + 10 kPa of CO2) on the antioxidant and fermentative metabolisms and polyphenol oxidase (PPO) activity and phenolics concentration were studied. The higher IBD incidence in high CO2-stored fruits was positively correlated with fermentative metabolites and negatively with ascorbate and H2O2 concentrations, and it was linked to PPO activation. These results indicate that both the antioxidant and fermentative metabolisms are involved in the occurrence of IBD in 'Rocha' pear. From the integration of the biochemical and enzymatic data, a schematic model illustrating the effects of high CO2 and low O2 in 'Rocha' pears during long-term storage was constructed.

Complete genome sequence and description of Salinispira pacifica gen. nov., sp. nov., a novel spirochaete isolated form a hypersaline microbial mat.

During a study of the anaerobic microbial community of a lithifying hypersaline microbial mat of Lake 21 on the Kiritimati atoll (Kiribati Republic, Central Pacific) strain L21-RPul-D2(T) was isolated. The closest phylogenetic neighbor was Spirochaeta africana Z-7692(T) that shared a 16S rRNA gene sequence identity value of 90% with the novel strain and thus was only distantly related. A comprehensive polyphasic study including determination of the complete genome sequence was initiated to characterize the novel isolate. Cells of strain L21-RPul-D2(T) had a size of 0.2 - 0.25 × 8-9 µm, were helical, motile, stained Gram-negative and produced an orange carotenoid-like pigment. Optimal conditions for growth were 35°C, a salinity of 50 g/l NaCl and a pH around 7.0. Preferred substrates for growth were carbohydrates and a few carboxylic acids. The novel strain had an obligate fermentative metabolism and produced ethanol, acetate, lactate, hydrogen and carbon dioxide during growth on glucose. Strain L21-RPul-D2(T) was aerotolerant, but oxygen did not stimulate growth. Major cellular fatty acids were C14:0, iso-C15:0, C16:0 and C18:0. The major polar lipids were an unidentified aminolipid, phosphatidylglycerol, an unidentified phospholipid and two unidentified glycolipids. Whole-cell hydrolysates contained L-ornithine as diagnostic diamino acid of the cell wall peptidoglycan. The complete genome sequence was determined and annotated. The genome comprised one circular chromosome with a size of 3.78 Mbp that contained 3450 protein-coding genes and 50 RNA genes, including 2 operons of ribosomal RNA genes. The DNA G + C content was determined from the genome sequence as 51.9 mol%. There were no predicted genes encoding cytochromes or enzymes responsible for the biosynthesis of respiratory lipoquinones. Based on significant differences to the uncultured type species of the genus Spirochaeta, S. plicatilis, as well as to any other phylogenetically related cultured species it is suggested to place strain L21-RPul-D2(T) (=DSM 27196(T) = JCM 18663(T)) in a novel species and genus, for which the name Salinispira pacifica gen. nov., sp. nov. is proposed.

Zinc cluster protein Znf1, a novel transcription factor of non-fermentative metabolism in Saccharomyces cerevisiae.

The ability to rapidly respond to nutrient changes is a fundamental requirement for cell survival. Here, we show that the zinc cluster regulator Znf1 responds to altered nutrient signals following glucose starvation through the direct control of genes involved in non-fermentative metabolism, including those belonged to the central pathways of gluconeogenesis (PCK1, FBP1 and MDH2), glyoxylate shunt (MLS1 and ICL1) and the tricarboxylic acid cycle (ACO1), which is demonstrated by Znf1-binding enrichment at these promoters during the glucose-ethanol shift. Additionally, reduced Pck1 and Fbp1 enzymatic activities correlate well with the data obtained from gene transcription analysis. Cells deleted for ZNF1 also display defective mitochondrial morphology with unclear structures of the inner membrane cristae when grown in ethanol, in agreement with the substantial reduction in the ATP content, suggesting for roles of Znf1 in maintaining mitochondrial morphology and function. Furthermore, Znf1 also plays a role in tolerance to pH and osmotic stress, especially during the oxidative metabolism. Taken together, our results clearly suggest that Znf1 is a critical transcriptional regulator for stress adaptation during non-fermentative growth with some partial overlapping targets with previously reported regulators in Saccharomyces cerevisiae.

Differences in responses to flooding by germinating seeds of two contrasting rice cultivars and two species of economically important grass weeds.

Crop productivity is largely affected by abiotic factors such as flooding and by biotic factors such as weeds. Although flooding after direct seeding of rice helps suppress weeds, it also can adversely affects germination and growth of rice, resulting in poor crop establishment. Barnyard grasses (Echinochloa spp.) are among the most widespread weeds affecting rice, especially under direct seeding. The present work aimed to establish effective management options to control these weeds. We assessed the effects of variable depths and time of submergence on germination, seedling growth and carbohydrate metabolism of (i) two cultivars of rice known to differ in their tolerance to flooding during germination and (ii) two barnyard grasses (Echinochloa colona and E. crus-galli) that commonly infest rice fields. Flooding barnyard grasses with 100-mm-deep water immediately after seeding was effective in suppressing germination and growth. Echinochloa colona showed greater reductions in emergence, shoot and root growth than E. crus-galli. Delaying flooding for 2 or 4 days was less injurious to both species. Echinochloa colona was also more susceptible to flooding than the flood-sensitive rice cultivar 'IR42'. The activity of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) in rice seedlings was increased by flooding after sowing but with greater increases in 'Khao Hlan On' compared with 'IR42'. The activity of ADH and PDC was enhanced to a similar extent in both barnyard grasses. Under aerobic conditions, the activity of ADH and PDC in the two barnyard grasses was downregulated, which might contribute to their inherently faster growth compared with rice. Aldehyde dehydrogenase activity was significantly enhanced in flood-tolerant 'Khao Hlan On' and E. crus-galli, but did not increase in flood-sensitive E. colona and 'IR42', implying a greater ability of the flood-tolerant types to detoxify acetaldehyde generated during anaerobic fermentation. Confirmation of this hypothesis is now being sought.

Complete genome sequence of Thermosphaera aggregans type strain (M11TL).

Thermosphaera aggregans Huber et al. 1998 is the type species of the genus Thermosphaera, which comprises at the time of writing only one species. This species represents archaea with a hyperthermophilic, heterotrophic, strictly anaerobic and fermentative phenotype. The type strain M11TL(T) was isolated from a water-sediment sample of a hot terrestrial spring (Obsidian Pool, Yellowstone National Park, Wyoming). Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,316,595 bp long single replicon genome with its 1,410 protein-coding and 47 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.