Propolis efficacy on SARS-COV viruses: a review on antimicrobial activities and molecular simulations.

This current study review provides a brief review of a natural bee product known as propolis and its relevance toward combating SARS-CoV viruses. Propolis has been utilized in medicinal products for centuries due to its excellent biological properties. These include anti-oxidant, immunomodulatory, anti-inflammatory, anti-viral, anti-fungal, and bactericidal activities. Furthermore, studies on molecular simulations show that flavonoids in propolis may reduce viral replication. While further research is needed to validate this theory, it has been observed that COVID-19 patients receiving propolis show earlier viral clearance, enhanced symptom recovery, quicker discharge from hospitals, and a reduced mortality rate relative to other patients. As a result, it appears that propolis could probably be useful in the treatment of SARS-CoV-2-infected patients. Therefore, this review sought to explore the natural properties of propolis and further evaluated past studies that investigated propolis as an alternative product for the treatment of COVID-19 symptoms. In addition, the review also highlights the possible mode of propolis action as well as molecular simulations of propolis compounds that may interact with the SARS-CoV-2 virus. The activity of propolis compounds in decreasing the impact of COVID-19-related comorbidities, the possible roles of such compounds as COVID-19 vaccine adjuvants, and the use of nutraceuticals in COVID-19 treatment, instead of pharmaceuticals, has also been discussed.

Effect of Light and p-Coumaric Acid on the Growth and Expression of Genes Related to Oxidative Stress in Brettanomyces bruxellensis LAMAP2480.

Brettanomyces bruxellensis is considered the most significant contaminant yeast in the wine industry since it causes a deterioration in the organoleptic properties of the wine and significant economic losses. This deterioration is due to the production of volatile phenols from hydroxycinnamic acids. These compounds possess antimicrobial properties; however, B. bruxellensis can resist this effect because it metabolizes them into less toxic ones. Recent studies have reported that B. bruxellensis grows under different stress conditions, including p-coumaric acid (pCA) but effective methods for its control have not been found yet. Since that in other yeasts, such as Saccharomyces cerevisiae, it has been described that light affects its growth, and we evaluated whether the light would have a similar effect on B. bruxellensis. The results show that at light intensities of 2,500 and 4,000 lux in the absence of pCA, B. bruxellensis LAMAP2480 does not grow in the culture medium; however, when the medium contains this acid, the yeast adapts to both factors of stress managing to grow. The expression of genes related to oxidative stress in B. bruxellensis LAMAP2480, such as SOD1, GCN4, and ESBP6, showed a higher relative expression when the yeast was exposed to 2,500 lux compared to 4,000 lux, agreeing with the growth curves. This suggests that a higher expression of the genes studied would be related to stress-protective effects by pCA.

Adaptive Laboratory Evolution of Native Torulaspora delbrueckii YCPUC10 With Enhanced Ethanol Resistance and Evaluation in Co-inoculated Fermentation.

Torulaspora delbrueckii is a yeast species typically present in the early stages of the fermentation process. T. delbrueckii positively modifies the aromatic properties of wines. However, its contribution to the final quality of the wine is restricted by its low tolerance to ethanol. T. delbrueckii is capable of fermenting and tolerating an ethanol concentration ranging from 7.4% (v/v) to slightly higher than 9% (v/v). For this reason, it cannot complete fermentation, when alcohol reach levels higher than 12% (v/v), limiting their use in the industry. The objective of this work was to obtain new variants of T. delbrueckii with improved resistance to ethanol through adaptive laboratory evolution. Variants capable of tolerating ethanol levels of 11.5% (v/v) were obtained. These presented improved kinetic parameters, and additionally showed an increase in resistance to SO2 in ethanol compared to the original strain. Co-inoculated fermentations were performed with the original strain (FTd/Sc) and with the evolved strain (FTdF/Sc), in addition to a control fermentation using only Saccharomyces cerevisiae EC1118 (FSc). The results obtained show that FTdF/Sc present higher levels of 2-Ethylhexanol, compared to FTd/Sc and FSc. Furthermore, FTdF/Sc presents higher levels of total alcohols, total aldehydes, total phenolic derivatives, and total sulfur compounds with significant differences with FSc. These results provide a T. delbrueckii YCPUC10-F yeast with higher resistance to ethanol, which can be present throughout the fermentation process and be used in co-inoculated fermentations. This would positively impact the performance of T. delbrueckii by allowing it to be present not only in the early stages of fermentation but to remain until the end of fermentation.

Genomics Perspectives on Metabolism, Survival Strategies, and Biotechnological Applications of Brettanomyces bruxellensis LAMAP2480.

Wine production is an important commercial issue for the liquor industry. The global production was estimated at 275.7 million hectoliters in 2015. The loss of wine production due to Brettanomyces bruxellensis contamination is currently a problem. This yeast causes a "horse sweat" flavor in wine, which is an undesired organoleptic attribute. To date, 6 B. bruxellensis annotated genome sequences are available (LAMAP2480, AWRI1499, AWRI1608, AWRI1613, ST05.12/22, and CBS2499), and whole genome comparisons between strains are limited. In this article, we reassembled and reannotated the genome of B. bruxellensis LAMAP2480, obtaining a 27-Mb assembly with 5.5 kb of N50. In addition, the genome of B. bruxellensis LAMAP2480 was analyzed in the context of spoilage yeast and potential as a biotechnological tool. In addition, we carried out an exploratory transcriptomic analysis of this strain grown in synthetic wine. Several genes related to stress tolerance, micronutrient acquisition, ethanol production, and lignocellulose assimilation were found. In conclusion, the analysis of the genome of B. bruxellensis LAMAP2480 reaffirms the biotechnological potential of this strain. This research represents an interesting platform for the study of the spoilage yeast B. bruxellensis.

Identification of a second PAD1 in Brettanomyces bruxellensis LAMAP2480.

Volatile phenols are aromatic compounds produced by some yeasts of the genus Brettanomyces as defense against the toxicity of hydroxycinnamic acids (p-coumaric acid, ferulic acid and caffeic acid). The origin of these compounds in winemaking involves the sequential action of two enzymes: coumarate decarboxylase and vinylphenol reductase. The first one converts hydroxycinnamic acids into hydroxystyrenes, which are then reduced to ethyl derivatives by vinylphenol reductase. Volatile phenols derived from p-coumaric acid (4-vinylphenol and 4-ethylphenol) have been described as the major contributors to self-defeating aromas associated with stable, gouache, wet mouse, etc., which generates large economic losses in the wine industry. The gene responsible for the production of 4-vinylphenol from p-coumaric acid has been identified as PAD1, which encodes a phenylacrylic acid decarboxylase. PAD1 has been described for many species, among them Candida albicans, Candida dubliniensis, Debaryomyces hansenii and Pichia anomala. In Brettanomyces bruxellensis LAMAP2480, a 666 bp reading frame (DbPAD) encodes a coumarate decarboxylase. Recent studies have reported the existence of a new reading frame belonging to DbPAD called DbPAD2 of 531 bp, which could encode a protein with similar enzymatic activity to PAD1. The present study confirmed that the transformation of Saccharomyces cerevisiae strain BY4722 with reading frame DbPAD2 under the control of the B. bruxellensis ACT1 promoter, encodes an enzyme with coumarate decarboxylase activity. This work has provided deeper insight into the origin of aroma defects in wine due to contamination by Brettanomyces spp.

Comparative transcriptome assembly and genome-guided profiling for Brettanomyces bruxellensis LAMAP2480 during p-coumaric acid stress.

Brettanomyces bruxellensis has been described as the main contaminant yeast in wine production, due to its ability to convert the hydroxycinnamic acids naturally present in the grape phenolic derivatives, into volatile phenols. Currently, there are no studies in B. bruxellensis which explains the resistance mechanisms to hydroxycinnamic acids, and in particular to p-coumaric acid which is directly involved in alterations to wine. In this work, we performed a transcriptome analysis of B. bruxellensis LAMAP248rown in the presence and absence of p-coumaric acid during lag phase. Because of reported genetic variability among B. bruxellensis strains, to complement de novo assembly of the transcripts, we used the high-quality genome of B. bruxellensis AWRI1499, as well as the draft genomes of strains CBS2499 and0 g LAMAP2480. The results from the transcriptome analysis allowed us to propose a model in which the entrance of p-coumaric acid to the cell generates a generalized stress condition, in which the expression of proton pump and efflux of toxic compounds are induced. In addition, these mechanisms could be involved in the outflux of nitrogen compounds, such as amino acids, decreasing the overall concentration and triggering the expression of nitrogen metabolism genes.

Comparative proteome analysis of Brettanomyces bruxellensisunder hydroxycinnamic acid growth

Background: Brettanomyces bruxellensis is an important spoilage yeast in the winemaking process. The capacity of this yeast to generate an undesired off-flavor constitutes a significant loss in the Chilean wine industry. Results: The proteomic profile of B. bruxellensis in the presence of p-coumaric acid was determined by 2D gel electrophoresis, gel image analysis and differential spot selection. A set of 41 proteins showed a differential accumulation of ±2 and a p-value <0.0001. The homology sequence analysis was performed using the databases available. Differential proteins belonged to the categories of 'energy production and conversion' and 'amino acid transport and metabolism'. Conclusions: The proteomic profile of B. bruxellensis cultivated in the presence of p-coumaric acid in synthetic wine, agrees with the hypothesis of metabolic flux regulation, allowing a better conditioning to an adverse environment. This study involved the translational level of B. bruxellensis in the production of ethylphenols and corroborated that this yeast presented an advantage in these stress conditions. Thus, this work will allow an understanding of the regulation and processes involved in the production of ethyl-derivate compounds by B. bruxellensis. Furthermore, it allows the development of newer and better techniques for spoilage yeast control.

Draft genome sequence and transcriptome analysis of the wine spoilage yeast Dekkera bruxellensis LAMAP2480 provides insights into genetic diversity, metabolism and survival.

Dekkera bruxellensis is the major contaminant yeast in the wine industry worldwide. Here, we present the draft genome sequence of D. bruxellensis LAMAP2480 isolated from a Chilean wine. Genomic evidence reveals shared and exclusive genes potentially involved in colonization and survival during alcoholic fermentation.

Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis.

The presence of Brettanomyces bruxellensis has been correlated with an increase of phenolic aromas in wine. The production of these aromas results from the metabolization of cinnamic acids, present in the wine, to their ethyl derivatives. Hence, the participation of two enzymes has been proposed: a p-coumarate decarboxylase (CD) and a vinylphenol reductase (VR). Both enzymes were purified and characterized from B. bruxellensis. In denaturing conditions, the CD enzyme had a molecular mass of 21 kDa, while in native conditions its mass was 41 kDa. The optimal activity was obtained at a temperature of 40 degrees C and a pH of 6.0. For p-coumaric acid, the Km value and Vmax were 1.22+/-0.08 mM and 98+/-0.15 micromol/min mg, respectively. The VR enzyme had a molecular mass of 37 kDa in SDS-PAGE, while in natural conditions its mass was 118 kDa. The Km value was > 3.37+/-2.05 mM and its Vmax was 107.62+/-50.38 micromol/min mg for NADPH used as a cofactor. Both enzymatic activities were stable at pH 3.4, but in the presence of ethanol the CD activity decreased drastically while the VR activity was more stable. This is the first report that shows the presence of a CD and a VR enzyme in B. bruxellensis.