Screening of native Saccharomyces cerevisiae strains from Chile for beer production.

Introduction: Beer is one of the most consumed alcoholic drinks in the world, and this industry is a growing market that demands different properties to satisfy new consumers. The yeasts are used in different fermented beverages to contribute to new flavors. However, yeast strains used in the beer industry are limited so far, thus the diversity of flavors is very restricted. Therefore, the use of native yeast strains has been taking more importance with the purpose of conferring differentiated organoleptic properties to the product. Based on this observation the potentiality of native Saccharomyces cerevisiae strains obtained from different localities in Chile was researched. Methods: In this work was selected those strains that produced the highest ethanol concentration (nearly 6% v/v), consumed the highest amounts of sugars, and produced the lowest amounts of organic acids in the resulting beers. Finally, we did a beer tasting to select those strains that added different flavors to the final beer compared with a commercial strain used. Results and discussion: In this study, two native strains that produced fruity descriptors are described, which could be used in the future in brewing, craft or industrial production.

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.

Nonionizing Electromagnetic Field: A Promising Alternative for Growing Control Yeast.

In the food industry, some fungi are considered to be common spoilage microorganisms which reduce the shelf life of products. To avoid this outcome, different technologies are being developed to control their growth. Electromagnetic fields (EMF) have been used to combat bacterial growth, but there are few studies on yeasts and their possible action mechanisms. For this reason, we studied the effect of EMF between 1 to 5.9 GHz bands on the growth of Saccharomyces cerevisiae yeast and observed that all the frequencies of the band used cause the reduction of the viability of this yeast. In addition, we observed that the distance between the antenna and the sample is an important factor to consider to control the growing yeast. By using transmission electron microscopy, we found that the EMF caused a loss of continuity of the yeast cell membrane. Therefore, EMF may be used as a control method for yeast growth.

Purification and characterization of Saccharomyces eubayanus killer toxin: Biocontrol effectiveness against wine spoilage yeasts.

Microbiological contamination by spoilage yeasts species are frequent during winemaking, and biological control using antagonistic yeasts is considered a more beneficial alternative to conventional synthetic antimicrobials. Saccharomyces eubayanus killer toxin (SeKT) was produced and purified in a synthetic optimized medium. Purification procedure allowed the identification of SeKT as protein with an apparent molecular mass of 70 kDa and activity at physicochemical conditions suitable for winemaking process. Purified SeKT reduced the levels of volatile phenols produced by the spoilage yeasts Brettanomyces bruxellensis, Pichia membranifaciens, Meyerozyma guilliermondii and Pichia manshurica in wine-like medium. The putative mode of action of SeKT on sensitive yeast strains comprises cell wall disruption through ß-glucanase and chitinase activities as well as necrotic and apoptotic death in a toxin dose dependent manner. Thus, SeKT appears to be a promising biocontrol agent against spoilage yeasts during wine aging and storing.

Biodiversity among Brettanomyces bruxellensis Strains Isolated from Different Wine Regions of Chile: Key Factors Revealed about its Tolerance to Sulphite.

Brettanomyces bruxellensis is regarded as the main spoilage microorganism in the wine industry, owing to its production of off-flavours. It is difficult to eradicate owing to its high tolerance of adverse environmental conditions, such as low nutrient availability, low pH, and high levels of ethanol and SO2. In this study, the production of volatile phenols and the growth kinetics of isolates from various regions of Chile were evaluated under stressful conditions. Through randomly amplified polymorphic DNA (RAPD) analysis, 15 strains were identified. These were grown in the presence of p-coumaric acid, a natural antimicrobial and the main precursor of off-flavours, and molecular sulfur dioxide (mSO2), an antimicrobial synthetic used in the wine industry. When both compounds were used simultaneously, there were clear signs of an improvement in the fitness of most of the isolates, which showed an antagonistic interaction in which p-coumaric acid mitigates the effects of SO2. Fourteen strains were able to produce 4-vinylphenol, which showed signs of phenylacrylic acid decarboxylase activity, and most of them produced 4-ethylphenol as a result of active vinylphenol reductase. These results demonstrate for the first time the serious implications of using p-coumaric acid, not only for the production of off-flavours, but also for its protective action against the toxic effects of SO2.

Production of a novel killer toxin from Saccharomyces eubayanus using agro-industrial waste and its application against wine spoilage yeasts.

The juicing industry generates large amounts of waste that mostly lack commercial value and, in the absence of waste treatment policies, produces environmental pollution. Also, microbiological spoilage is a major concern in the wine industry and control tools are limited. Taking these challenges into account, agro-industrial waste coming from ultrafiltrated apple and pear juice were used to grow Saccharomyces eubayanus and to produce its killer toxin (SeKT). A Plackett-Burman screening was performed in order to optimize SeKT production in ultrafiltrated apple and pear juice. The optimized medium was characterized: 75% v/v WUJ, 0.5% m/v KH2PO4, 0.5% m/v MgSO4, 0.5% m/v (NH4)SO4, 0.5% g/L urea, 10% v/v glycerol and 0.1% v/v Triton X-100. SeKT produced in WUJ optimised medium was used to perform killer assays against wine spoilage yeasts and showed antagonistic activity against Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens. Different inhibition percentages against spoilage species in a wine environment (49-69%) were detected and preserved for at least 48 h. For the first time, this work reports the ability of S. eubayanus to produce a killer toxin with potential use as a biocontrol tool in winemaking. Producing SeKT using agro-industrial waste as an alternative medium to cultivate S. eubayanus would have industrial, economic and ecological benefits.

Novel antimicrobial peptides produced by Candida intermedia LAMAP1790 active against the wine-spoilage yeast Brettanomyces bruxellensis.

Brettanomyces bruxellensis negatively impacts on the sensorial quality of wine by producing phenolic compounds associated with unpleasant odors. Thus, the control of this spoilage yeast is a critical factor during the winemaking process. A recent approach used to biocontrol undesired microorganisms is the use of yeast released antimicrobial peptides (AMPs), but this strategy has been poorly applied to wine-related microorganisms. The aim of this study was to evaluate the antifungal capacity of Candida intermedia LAMAP1790 against wine-spoilage strains of B. bruxellensis and fermentative strains of Saccharomyces cerevisiae, and also to determine the chemical nature of the compound. The exposure of strains to the supernatant of C. intermedia saturated cultures showed antifungal activity against B. bruxellensis, without affecting the growth of S. cerevisiae. By fractionation and concentration of C. intermedia supernatants, it was determined that the antifungal activity was related to the presence of heat-labile peptides with molecular masses under 5 kDa. To our knowledge, this is the first report of AMPs secreted by C. intermedia that control B. bruxellensis. This could lead to the development of new biocontrol strategies against this wine-spoilage yeast.

Volatile phenols are produced by strains of Dekkera bruxellensis under Brazilian fuel ethanol industry-like conditions.

Dekkera bruxellensis is a spoilage yeast in wine and fuel ethanol fermentations able to produce volatile phenols from hydroxycinnamic acids by the action of the enzymes cinnamate decarboxylase (CD) and vinyphenol reductase (VR) in wine. However, there is no information about this ability in the bioethanol industry. This work evaluated CD and VR activities and 4-ethylphenol production from p-coumaric acid by three strains of D. bruxellensis and PE-2, an industrial Saccharomyces cerevisiae strain. Single and multiple-cycle batch fermentations in molasses and sugarcane juice were carried out. Dekkera bruxellensis strains showed similar CD activity but differences in VR activity. No production of 4-ethylphenol by S. cerevisiae in any fermentation system or media was observed. The concentrations of 4-ethylphenol peaked during active growth of D. bruxellensis in single-cycle fermentation but they were lower than in multiple-cycle fermentation. Higher concentrations were observed in molasses with molar conversion (p-coumaric acid to 4-ethylphenol) ranging from 45% to 85%. As the first report on 4-ethylphenol production in sugarcane musts by D. bruxellensis in industry-like conditions, it opens up a new avenue to investigate its effect on the viability and fermentative capacity of S. cerevisiae as well as to understand the interaction between the yeasts in the bioethanol industry.

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.

Development and characterization of hybrids from native wine yeasts

For commercial purposes, the winemaking industry is constantly searching for new yeast strains. Historically, this has been achieved by collecting wild strains and selecting the best for industrial use through an enological evaluation. Furthermore, the increasing consumer demands have forced the industry to incorporate new strategies such as genetic engineering to obtain improved strains. In response to the lack of public acceptance of this methodology, alternative strategies based on breeding have gained acceptance in recent years. Through the use of conjugation of individual spores without the support of genetic engineering methods we generated intraspecific hybrids from wild strains with outstanding enological characteristics and interdelta fingerprinting was used to confirm the hybrid condition. A detailed enological characterization of the hybrids in synthetic and natural must indicates that physiological parameters such as sporulation, residual sugar, ethanol yield and total nitrogen uptake are within the levels determined for the parental strains, however, other parameters such as growth rate, lag phase and ethanol production show statistical differences with some parental or commercial strains. These findings allow us to propose these hybrids as new wine-making strains.

Development and characterization of hybrids from native wine yeasts.

For commercial purposes, the winemaking industry is constantly searching for new yeast strains. Historically, this has been achieved by collecting wild strains and selecting the best for industrial use through an enological evaluation. Furthermore, the increasing consumer demands have forced the industry to incorporate new strategies such as genetic engineering to obtain improved strains. In response to the lack of public acceptance of this methodology, alternative strategies based on breeding have gained acceptance in recent years. Through the use of conjugation of individual spores without the support of genetic engineering methods we generated intraspecific hybrids from wild strains with outstanding enological characteristics and interdelta fingerprinting was used to confirm the hybrid condition. A detailed enological characterization of the hybrids in synthetic and natural must indicates that physiological parameters such as sporulation, residual sugar, ethanol yield and total nitrogen uptake are within the levels determined for the parental strains, however, other parameters such as growth rate, lag phase and ethanol production show statistical differences with some parental or commercial strains. These findings allow us to propose these hybrids as new wine-making strains.

Cinnamic acid, ethanol and temperature interaction on coumarate decarboxylase activity and the relative expression of the putative cd gene in D. bruxellensis

Dekkera bruxellensis is one of the main contaminating yeasts in wine due to its ability to metabolize cinnamic acids into volatile phenols. This yeast metabolizes p-coumaric acid into 4-vinylphenol through a coumarate decarboxylase (CD) and then transforms it into to 4-ethylphenol (EF) through a vinylphenol reductase. In this work we investigated the influence of the interaction between the concentration of p-coumaric acid, ferulic acid and ethanol as well as growth temperature on the production of CD activity and the expression of a putative gene that codes for this enzymatic activity. For this, a Box Behnken experimental design was used. The concentration of p-coumaric acid (5-26 ppm) and ferulic acid (3-9 ppm) alone did not show any significant effect on any of the studied response variables. However, the interaction between (ethanol concentration * cinnamic acid concentration) and (ethanol concentration * temperature) had a significant statistical effect on the production of CD activity. Additionally, a higher growth temperature negatively affected the expression of the putative cd gene and the production of CD activity. This is the first work that studies the effect of cinnamic acids on the production of CD activity and the relative expression of its putative gene, using natural concentrations of cinnamic acid found in wine.

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.

Production of Rhodotorula glutinis: a yeast that secretes alpha-L-arabinofuranosidase

Rhodotorula glutinis is a yeast that secretes the enzyme alpha-L-arabinofuranosidase (E.C. 3.2.1.55) into the culture medium and thus has an interesting biotechnological potential. To determine improved culture conditions of this organism, different factors of the culture media were evaluated such as the use of peptone as nitrogen source, salts composition, pH and growth temperature. Likewise, beet molasses and beet cosette were tested as industrial carbon sources to induce the production of the enzyme and how they influence the yeast growth. Based on these studies a culture medium is proposed for growth of this yeast in a continuous system. By assaying different dilution rates an average specific activity for the enzyme of 82.4 U/mg of protein was obtained.

The arginine deiminase locus of Oenococcus oeni includes a putative arginyl-tRNA synthetase ArgS2 at its 3'-end.

Oenococcus oeni is the most important lactic acid bacteria of the winemaking process involved in malolactic fermentation. Most O. oeni strains are able to catabolyze arginine via the arginine deiminase (ADI) pathway. The arcR, A, B, C, D1, and D2 cluster of O. oeni bacteria has been characterized. Here, we completed the ADI locus sequence. Downstream of arcD2 gene, we found an additional gene which encodes a putative arginyl-tRNA synthetase (argS2). It is not the same arginyl-tRNA synthetase which was sequenced in O. oeni MCW strain. Transcriptional analyses have shown that argS2 was induced by arginine. In addition, systematic polymerase chain reaction amplification of each arc gene and argS2 has provided a characteristic feature of the ADI locus within the O. oeni species: all genes of ADI locus are present or absent according to the strains.