Gildo Almeida da Silva

Production of yellow-green fluorescent pigment by Pseudomonas fluorescens

A medium was prepared from brewery waste yeast with and without mineral salts to study growth and yellow-green fluorescent pigment production (YGFP) by Pseudomonas fluorescens. The Kings medium used for detection of siderophore production were expressively weaker inductors of YGFP formation when compared to FYE medium. Although FYE and CYE could be used for growth of P. fluorescens, only FYE was an attractive medium for detection of YGFP strain producers.

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.

Influence of Brettanomyces custersianus upon the activity of Saccharomyces cerevisiae strains during the tumultuous phase of vinification

The aim of this work was to evaluate the influence of Brettanomyces custersianus on the metabolic activity of Saccharomyces cerevisiae during the tumultuous stage of wine production. The Cabernet Sauvignon grape must with the skin was inoculated with individual cultures of Sacch. cerevisiae and with mixed cultures of Sacch. cerevisiae and Br. custersianus. During the 6-day tumultuous phase of fermentation, the highest ethanol production and the highest sugar consumption were obtained with the strains without B. custersianus. Fermentations carried out with the addition of Brettanomyces metabolites, acetic acid and 4-ethylphenol, showed that only the former inhibited the growth of both Sacch. cerevisiae strains used. In some cases, Br. custersianus could affect the rate higher alcohols production and their final concentrations during the tumultuous phase of vinification.

Production of functional killer protein in batch cultures upon a shift from aerobic to anaerobic conditions

ABSTRACT The aim of this work was to study the production of functional protein in yeast culture. The cells of Saccharomyces cerevisiae Embrapa 1B (K + R + ) killed a strain of Saccharomyces cerevisiae Embrapa 26B (K - R - )in grape must and YEPD media. The lethal effect of toxin-containing supernatant and the effect of aeration upon functional killer production and the correlation between the products of anaerobic metabolism and the functional toxin formation were evaluated. The results showed that at low sugar concentration, the toxin of the killer strain of Sacch. cerevisiae was only produced under anaerobic conditions . The system of killer protein production showed to be regulated by Pasteur and Crabtree effects. As soon as the ethanol was formed, the functional killer toxin was produced. The synthesis of the active killer toxin seemed to be somewhat associated with the switch to fermentation process and with concomitant alcohol dehydrogenase (ADH) activity. Key words: Killer yeast, Crabtree effect, Pasteur effect, inhibition, mycocinogenic strain

MALDI-TOF MS Supplementary database for species identification employing the yeast diversity encountered on southern Brazil grapes

The study of grape microflora is of interest when autochthonous yeasts, which are related to typical wine characteristics, are intended to be used in winemaking. The election of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the first method for yeast identification was based on its accuracy and rapidity compared to alternative laboratory protocols for identification. The aims of this study are to consolidate the MALDI-TOF MS Supplementary database for environmental yeasts already constructed, to expand it through the addition of standard spectra of not included yeast species, and to discuss the grape microflora encountered in Southern Brazil. A total of 358 strains, isolated from grape berries, were submitted to protein profiling employing Biotyper and Supplementary database. Molecular biology techniques were used as alternatives to identify 6.4% of strains not promptly designated by protein profiling. These strains corresponded to the species Candida californica, Zygoascus meyerae, Candida akabanensis, Candida azyma, and Hanseniaspora vineae. The MALDI-TOF MS spectra of the identified species were added to Supplementary database. The presented results strengthen the need for further expansion of the mass spectra database to broaden its microbiological application.

Impact of Fungicide Residues on Polymerase Chain Reaction and on Yeast Metabolism

ABSTRACT The indiscriminate use of pesticides on grape crops is harmful for consumers´ healthin “in natura” consumption and in the ingestion of wine and grape juice. During winemaking, a rapid and efficient fermentation stage is critical to avoid proliferation of contaminating microorganisms and to guarantee the product´s quality. Polymerase chain reaction (PCR) has the advantage of detecting these contaminants in the early stages of fermentation. However,this enzymatic reaction may also be susceptible to specific problems, reducing its efficiency. Agricultural practices, such as fungicide treatments, may be a source of PCR inhibiting factors and may also interfere in the normal course of fermentation.The action of the pesticides captan and folpet on PCR and on yeast metabolism was evaluated, once these phthalimide compounds are widely employed in Brazilian vineyards. DNA amplification was only observed at 75 and 37.5 µg/mL of captan concentrations, whereas with folpet, amplification was observed only in the two lowest concentrations tested (42.2 and 21.1µg/mL).Besides the strong inhibition on Taq polymerase activity, phthalimides also inhibited yeast metabolism at all concentrations analyzed.Grape must containing captan and folpet residues could not be transformed into wine due to stuck fermentation caused by the inhibition of yeast metabolism. Non-compliance with the waiting period for phthalimide fungicides may result in financial liabilities to the viticulture sector.The use of yeasts with high fungicide sensitivity should be selected for must fermentation as a strategy for sustainable wine production and to assure that products comply with health and food safety standards.

Convivência Harmônica Entre Linhagens de Leveduras Killer e Sensíveis

Sheila Canossa, Bruna Agustini, Gildo Almeida da Silva.Convivencia Harmonica Entre Linhagens de Leveduras Killer e Sensiveis. In: Anais do 12o Congresso Latinoamericano de Microbiologia e Higiene de Alimentos MICROAL 2014 [= Blucher Food Science Proceedings, num.1, vol.1]. Sao Paulo: Editora Blucher, 2014. DOI 10.5151/foodsci-microal-019 Convivencia Harmonica Entre Linhagens de Leveduras Killer e Sensiveis