RESUMO
Kluyveromyces marxianus yeasts represent a valuable industry alternative due to their biotechnological potential to produce aromatic compounds. 2-phenylethanol and 2-phenylethylacetate are significant aromatic compounds widely used in food and cosmetics due to their pleasant odor. Natural obtention of these compounds increases their value, and because of this, bioprocesses such as de novo synthesis has become of great significance. However, the relationship between aromatic compound production and yeast's genetic diversity has yet to be studied. In the present study, the analysis of the genetic diversity in K. marxianus isolated from the natural fermentation of Agave duranguensis for Mezcal elaboration is presented. The results of strains in a haploid and diploid state added to the direct relationship between the mating type locus MAT with metabolic characteristics are studied. Growth rate, assimilate carbohydrates (glucose, lactose, and chicory inulin), and the production of aromatic compounds such as ethyl acetate, isoamyl acetate, isoamyl alcohol, 2-phenylethyl butyrate and phenylethyl propionate and the diversity in terms of the output of 2-phenylethanol and 2-phenylethylacetate by de novo synthesis were determinate, obtaining maximum concentrations of 51.30 and 60.39 mg/L by ITD0049 and ITD 0136 yeasts respectively.
Assuntos
Kluyveromyces , Álcool Feniletílico , Álcool Feniletílico/metabolismo , Odorantes , Kluyveromyces/genética , Leveduras/genética , Leveduras/metabolismo , Fermentação , Lactose/metabolismoRESUMO
AIMS: Yeasts produce 2-phenylethanol (2-PE) from sugars via de novo synthesis; however, its synthesis is limited due to feedback inhibition on the isofunctional 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases (Aro3p and Aro4p). This work aimed to select Kluyveromyces marxianus mutant strains with improved capacity to produce 2-PE from sugars. METHODS AND RESULTS: Kluyveromyces marxianus CCT 7735 mutant strains were selected from UV irradiation coupled with screening of p-fluoro-dl-phenylalanine (PFP) tolerant strains on culture medium without l-Phe addition. Most of them produced 2-PE titres higher than the parental strain and the Km_PFP41 mutant strain stood out for displaying the highest 2-PE specific production rate. Moreover it showed higher activity of DAHP synthase than the parental strain. We sequenced both ARO3 and ARO4 genes of Km_PFP41 mutant and identified mutations in ARO4 which caused changes in both size and conformation of the Aro4p. These changes seem to be associated with the enhanced activity of DAHP synthase and improved production of 2-PE exhibited by that mutant strain. CONCLUSIONS: The Km_PFP41 mutant strain presented improved 2-PE production via de novo synthesis and enhanced DAHP synthase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant strain obtained in this work may be exploited as a yeast cell factory for high-level synthesis of 2-PE.
Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Proteínas Fúngicas/metabolismo , Kluyveromyces/metabolismo , Álcool Feniletílico/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , Sequência de Bases , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Kluyveromyces/genética , Mutagênese , Mutação , Conformação Proteica , p-Fluorfenilalanina/metabolismoRESUMO
Endophytic fungi belonging to Muscodor genus are considered as promising alternatives to be used in biological control due to the production of volatile organic compounds (VOCs). The strains LGMF1255 and LGMF1256 were isolated from the medicinal plant Schinus terebinthifolius and, by morphological data and phylogenetic analysis, identified as belonging to Muscodor genus. Phylogenetic analysis suggests that strain LGMF1256 is a new species, which is herein introduced as Muscodor brasiliensis sp. nov. The analysis of VOCs production revealed that compounds phenylethyl alcohol, α-curcumene, and E (ß) farnesene until now has been reported only from M. brasiliensis, data that supports the classification of strain LGMF1256 as a new species. M. brasiliensis completely inhibited the phytopathogen P. digitatum in vitro. We also evaluated the ability of VOCs from LGMF1256 to inhibit the development of green mold symptoms by inoculation of P. digitatum in detached oranges. M. brasiliensis reduced the severity of diseases in 77%, and showed potential to be used for fruits storage and transportation to prevent the green mold symptoms development, eventually reducing the use of fungicides.
Assuntos
Antifúngicos/farmacologia , Agentes de Controle Biológico/farmacologia , Penicillium/efeitos dos fármacos , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/farmacologia , Xylariales/metabolismo , Anacardiaceae/microbiologia , Antifúngicos/metabolismo , Agentes de Controle Biológico/metabolismo , Fungicidas Industriais , Penicillium/crescimento & desenvolvimento , Álcool Feniletílico/metabolismo , Sesquiterpenos/metabolismo , Xylariales/isolamento & purificaçãoRESUMO
Vinasse is a waste obtained from the production of beverages, such as tequila and cachaça. The presence of acids, alcohols, sugars, minerals, amino acids, peptides, and nitrogen salts make vinasse a hazardous liquid waste to the environment, affecting the fauna, flora, and microbiota of rivers and lagoons. This study used biological treatment concomitant to volatile compound production. The yeasts used in the study were Saccharomyces cerevisiae (CCMA 0187 and CCMA 0188), Candida parapsilosis (CCMA 0544), and Pichia anomala (CCMA 0193). A higher percentage reduction in chemical and biochemical oxygen demand was observed in the tequila vinasse than in the cachaça vinasse. However, a higher production of volatile compounds was observed in the cachaça vinasse. C. parapsilosis CCMA 0544 produced the highest concentration of 2-phenylethanol (162 mg L-1). These results indicated that the environmental damage of vinasse can be reduced by treating vinasse with yeasts, and this treatment produces aroma compounds. This biological treatment has high economic potential, especially for the tequila industry.
Assuntos
Bebidas Alcoólicas , Aromatizantes/metabolismo , Resíduos Industriais , Compostos Orgânicos Voláteis/metabolismo , Gerenciamento de Resíduos/métodos , Leveduras/metabolismo , Agave/química , Agave/microbiologia , Álcoois/metabolismo , Análise da Demanda Biológica de Oxigênio , Biomassa , Candida/metabolismo , Poluição Ambiental/prevenção & controle , Fermentação , Concentração de Íons de Hidrogênio , Álcool Feniletílico/metabolismo , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharum , Temperatura , Compostos Orgânicos Voláteis/análise , Leveduras/crescimento & desenvolvimentoRESUMO
A diversity of yeast strains belonging to the cryotolerant fermentative species S. uvarum and S. eubayanus have been recovered from natural habitats and traditional fermentations in North Patagonia. The aim of this work was to evaluate the most relevant physiological features in a set of Patagonian strains belonging to S. uvarum and S. eubayanus, in order to analyze their potentiality to be used as starter cultures for cidermaking elaborated at low temperature. We evidenced that S. uvarum strains isolated from natural habitats (Araucaria araucana bark) showed similar physiological features to S. eubayanus strains obtained from the same habitat, and different from S. uvarum strains from fermentative environments (apple chichas). We also confirm the capacity of S. uvarum to produce high glycerol levels, low acetic acid and elevated production of the higher alcohol 2-phenylethanol and 2-phenylethyl acetate and demonstrated similar properties in S. eubayanus. Finally, we evidenced for the first time the antagonistic activity of S. eubayanus and selected three strains (two S. uvarum and one S. eubayanus) bearing the best combination of features to be used as a starter culture in cidermaking.
Assuntos
Fermentação/fisiologia , Malus/metabolismo , Saccharomyces/metabolismo , Vinho/microbiologia , Acetatos/metabolismo , Ácido Acético/análise , Argentina , Etanol/metabolismo , Glicerol/análise , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismoRESUMO
Phytotoxic metabolites produced in liquid culture by six species of Lasiodiplodia isolated in Brazil and causing Botryosphaeria dieback of grapevine were chemically identified. As ascertained by LC/MS, L. brasiliense, L. crassispora, L. jatrophicola, and L. pseudotheobromae produced jasmonic acid, and L. brasiliense synthesized, besides jasmonic acid, also (3R,4S)-4-hydroxymellein. L. euphorbicola and L. hormozganensis produced some low molecular weight lipophilic toxins. Specifically, L. euphorbicola produced (-)-mellein, (3R,4R)-(-)- and (3R,4S)-(-)-4-hydroxymellein, and tyrosol, and L. hormozganensis synthesized tyrosol and p-hydroxybenzoic acid. This is the first report on the production of the above cited metabolites from L. euphorbicola and L. hormozganensis. The phytotoxic activity of the metabolites produced is also discussed and related to the symptoms these pathogens cause in the grapevine host plants.
Assuntos
Ascomicetos/metabolismo , Micotoxinas/metabolismo , Vitis/microbiologia , Ascomicetos/química , Ascomicetos/patogenicidade , Brasil , Cromatografia Líquida de Alta Pressão , Ciclopentanos/metabolismo , Isocumarinas/metabolismo , Espectroscopia de Ressonância Magnética , Peso Molecular , Micotoxinas/química , Ocratoxinas/metabolismo , Oxilipinas/metabolismo , Parabenos/metabolismo , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Doenças das Plantas/microbiologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
In several grape varieties, the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoid-related compounds, such as glycosylated benzyl and 2-phenylethyl alcohol, which contribute to wine with floral and fruity aromas after being hydrolysed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora, are known to predominate during the first stages of alcoholic fermentation. Although non-Saccharomyces yeast strains have a well-recognized genetic diversity, understanding of their impact on wine flavour richness is still emerging. In this study, 11 Hansenisapora vineae strains were used to ferment a chemically defined simil-grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape-derived secondary metabolites. GC-MS analysis was performed to determine volatile compounds in the produced wines. Our results showed that benzyl alcohol, benzyl acetate and 2-phenylethyl acetate are significantly synthesized by H. vineae strains. Levels of these compounds found in fermentations with 11 H. vineae different strains were one or two orders of magnitude higher than those measured in fermentations with a known S. cerevisiae wine strain. The implications for winemaking in response to the negative correlation of benzyl alcohol, benzyl acetate and 2-phenylethyl acetate production with yeast assimilable nitrogen concentrations are discussed. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Fermentação , Hanseniaspora/metabolismo , Nitrogênio/metabolismo , Fenóis/metabolismo , Vinho , Acetatos/metabolismo , Compostos de Amônio/química , Compostos de Amônio/metabolismo , Álcool Benzílico/metabolismo , Compostos de Benzil/metabolismo , Aromatizantes/análise , Aromatizantes/química , Cromatografia Gasosa-Espectrometria de Massas , Nitrogênio/química , Fenóis/análise , Fenóis/química , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Saccharomyces cerevisiae/metabolismo , Vitis/químicaRESUMO
The prevention of adhesion of Candida cells to acrylic surfaces can be regarded as an alternative to prevent denture stomatitis. The use of quorum sensing molecules, such as tyrosol, could potentially interfere with the adhesion process. Therefore, the aim of this study was to assess the effect of tyrosol on adhesion of single and mixed cultures of Candida albicans and Candida glabrata to acrylic resin surfaces. Tyrosol was diluted in each yeast inoculum (10(7) cells/ml in artificial saliva) at 25, 50, 100, and 200 mM. Then, each dilution was added to wells of 24-well plates containing the acrylic specimens, and the plates were incubated at 37°C for 2 h. After, the effect of tyrosol was determined by total biomass quantification, metabolic activity of the cells and colony-forming unit counting. Chlorhexidine gluconate (CHG) was used as a positive control. Data were analyzed using analysis of variance (ANOVA) and the Holm-Sidak post hoc test (α = 0.05). The results of total biomass quantification and metabolic activity revealed that the tyrosol promoted significant reductions (ranging from 22.32 to 86.16%) on single C. albicans and mixed cultures. Moreover, tyrosol at 200 mM and CHG significantly reduced (p < 0.05) the number of adhered cells to the acrylic surface for single and mixed cultures of both species, with reductions ranging from 1.74 to 3.64-log10. In conclusion, tyrosol has an inhibitory effect on Candida adhesion to acrylic resin, and further investigations are warranted to clarify its potential against Candida infections.
Assuntos
Acrilatos , Candida albicans/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Microbiologia Ambiental , Álcool Feniletílico/análogos & derivados , Biomassa , Candida albicans/fisiologia , Candida glabrata/fisiologia , Contagem de Colônia Microbiana , Álcool Feniletílico/metabolismo , Temperatura , TempoRESUMO
Background Natural 2-phenylethanol (2-PE) is an important flavoring that emits the aroma of roses. During biotransformation, the aroma quality of natural 2-PE is affected by its main by-products, which include butanol, isobutyric acid, butyric acid, and isovaleric acid. Thus, controlling undesirable by-product formation can reduce the effect of odor on 2-PE aroma quality. Results 2-PE was produced through biotransformation using l-phenylalanine as a substrate and glucose as a carbon source. Ascorbic acid was added to the system to improve the redox reaction and suppress the generation of by-products. Principal component analysis of the aroma quality of 2-PE was performed using an electronic nose. Similarity analysis revealed that the effects of four by-products on 2-PE aroma quality may be ranked in the following order: isovaleric acid > butyric acid > isobutyric acid > butanol. The sample that exhibited the best similarity to the standard 2-PE sample (99.19%) was the sample to which ascorbic acid had been added during glucose metabolism. Conclusions 2-PE produced through the addition of ascorbic acid exhibited the closest aroma similarity to the standard 2-PE sample.
Assuntos
Álcool Feniletílico/metabolismo , Ácido Ascórbico/metabolismo , Biotransformação , Odorantes , Análise de Componente Principal , Nariz EletrônicoRESUMO
Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth.