Eduardo Mere Del Aguila

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

BackgroundPreparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target.ResultsWe investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65°C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step.ConclusionAlthough more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.

Purificação e caracterização da quitinase de uva (Vitis vinífera L. cv Red Globe) para a produção de quitosana a partir de quitina de camarão

Chitinase is produced by a wide variety of plants as a defense against peste attacks. In this study, grape chitinases were purified 16 times by fractionation in 80% ammonium sulfate followed by dialysis and filtration. Purified chitinases exhibited enzymatic activity toward chitin azure. The yield of purified chitinase was 229 mg/L with chitinase activity of 563 U/g. Chitinases had molecular masses of 24 and 30 kDa, as evaluated by SDS-PAGE 12.5%. Two pH optima were determined 3.0 and 6.0. The optimal temperature was 42 °C. Pre hydrolysis of crystalline shrimp chitin by chitinases caused in an increase in the deacetylation ratio triggered by chitin deacetylase producing chitooligosaccharides with DA (degree acetylation) of 58.8%.

Identification and molecular phylogeny of coagulase-negative staphylococci isolates from Minas Frescal cheese in southeastern Brazil: Superantigenic toxin production and antibiotic resistance.

Minas Frescal is a typical Brazilian fresh cheese and one of the most popular dairy products in the country. This white soft, semiskimmed, nonripened cheese with high moisture content is obtained by enzymatic coagulation of cow milk using calf rennet or coagulants, usually in industrial dairy plants, but is also manufactured in small farms. Contamination of Minas Frescal by several staphylococci has been frequently reported. Coagulase-negative staphylococci (CNS) strains are maybe the most harmful, as they are able to produce heat-stable enterotoxins with super antigenic activities in food matrices, especially in dairy products such as soft cheeses. The aim of the present study was to investigate the presence of CNS strains in Minas Frescal marketed in southeastern Brazil concerning the risk of staphylococci food poisoning by the consumption of improperly manufactured cheese and the possibility of these food matrices being a reservoir of staphylococcal resistance to antimicrobials. Ten distinct CNS strains were found in 6 cheeses from distinct brands. The most frequent species were Staphylococcus saprophyticus (40%), Staphylococcus xylosus (30%), Staphylococcus sciuri (20%), and Staphylococcus piscifermentans (10%). Three strains were identified to the Staphylococcus genera. Three major species groups composed of 3 refined clusters were grouped by phylogenetic analyses with similarities over to 90%. All CNS strains carried multiple enterotoxin genes, with high incidence of sea and seb (90 and 70%, respectively), followed by sec/see, seh/sei, and sed with intermediate incidence (60, 50, and 40%, respectively), and, finally, seg/selk/selq/selr and selu with the lowest incidence (20 and 10%, respectively). Real-time reverse transcription PCR and ELISA assays confirmed the enteroxigenic character of the CNS strains, which expressed and produced the enterotoxins in vitro. The CNS strains showed multiresistance to antimicrobial agents such as β-lactams, vancomycin, and linezolid, which have therapeutic importance in both human and veterinarian medicines. The risk of staphylococci food poisoning by the consumption of improperly manufactured Minas Frescal was emphasized, in addition to the possibility of these food matrices being a reservoir for antibiotic resistance. More effective control measures concerning the presence and typing of staphylococci in raw milk and dairy derivatives should be included to prevent the spread of pathogenic strains.

Physicochemical, nutritional, and sensory analyses of a nitrate-enriched beetroot gel and its effects on plasmatic nitric oxide and blood pressure

Background Beetroot (Beta vulgaris L.) is a dietary source of natural antioxidants and inorganic nitrate (NO3-). It is well known that the content of antioxidant compounds and inorganic nitrate in beetroot can reduce blood pressure (BP) and the risk of adverse cardiovascular effects. Objective The aim of the present study was to formulate a beetroot gel to supplement dietary nitrate and antioxidant compounds able to cause beneficial health effects following acute administration. Design and subjects A beetroot juice produced from Beta vulgaris L., without any chemical additives, was used. The juice was evaluated by physicochemical and microbiological parameters. The sample was tested in five healthy subjects (four males and one female), ingesting 100 g of beetroot gel. Results The formulated gel was nitrate enriched and contained carbohydrates, fibers, saponins, and phenolic compounds. The formulated gels possess high total antioxidant activity and showed adequate rheological properties, such as high viscosity and pleasant texture. The consumer acceptance test for flavor, texture, and overall acceptability of beetroot gel flavorized with synthetic orange flavor had a sensory quality score >6.6. The effects of acute inorganic nitrate supplementation on nitric oxide production and BP of five healthy subjects were evaluated. The consumption of beetroot gel increased plasma nitrite threefold after 60 min of ingestion and decreased systolic BP (−6.2 mm Hg), diastolic BP (−5.2 mm Hg), and heart rate (−7 bpm).

Expression of the yeast calcineurin subunits CNA1 and CNA2 during growth and hyper-osmotic stress

CNA1 and CNA2 encode isoforms of the catalytic subunit of calcineurin, a Ser/Thr-specific phosphoprotein phosphatase regulated by Ca(2+)/calmodulin. The relative abundance of both transcripts was evaluated during growth of Saccharomyces cerevisiae in glucose by reverse-transcription polymerase chain reaction using PDA1 mRNA as a novel internal standard. CNA1 and CNA2 were concomitantly transcribed with different average expression ratios at the exponential and stationary growth phases and both showed a remarkable drop in the expression at diauxie. Prolonged hyper-osmotic shock resulted in a moderate induction of CNA1, whereas CNA2 expression was not affected.

Safety Evaluation of the Coagulase-Negative Staphylococci Microbiota of Salami: Superantigenic Toxin Production and Antimicrobial Resistance

The risks of contracting staphylococci food poisoning by the consumption of improperly manufactured salami and the possibility of this food being reservoirs for antibiotic resistance were evaluated. Nineteen coagulase-negative staphylococci (CNS) strains were found in commercial and artisanal salami. The species in commercial salami were S. saprophyticus, S. sciuri, S. xylosus, and S. carnosus. Artisanal salami showed S. succinus, S. epidermidis, and S. hominis but no S. carnosus. Phylogenetic analyses grouped the strains into three major staphylococcal species groups, comprised of 4 refined clusters with similarities superior to 90%. Fifteen strains harbored multiple enterotoxin genes, with high incidence of seb/sec and sea, 57% and 50%, respectively, intermediate incidence of sed/seh/selm and sei/seln/tst-H, 33% and 27%, correspondingly, and low incidence of see/selj/selo and seg, of respectively 13% and 1%. Real time RT-PCR and enzyme-linked-immunosorbent assays confirmed the enterotoxigenicity of the strains, which expressed and produced enterotoxins in vitro. The CNS strains showed multiresistance to several antimicrobials of therapeutic importance in both human and veterinarian medicine, such as β-lactams, vancomycin, and linezolid. The effective control of undue staphylococci in fermented meat products should be adopted to prevent or limit the risk of food poisoning and the spread of antimicrobial-resistant strains.

Evaluating Physicochemical and Rheological Characteristics and Microbial Community Dynamics during the Natural Fermentation of Cassava Starch

The traditional fermentation of cassava starch was investigated by a polyphasic approach combining (i) microbial community identification using conventional and molecular techniques, (ii) analyses of organic acids, volatile compounds, fermentation products and spin-lattice relation time and (iii) evaluation of technological properties, such as pasting properties, water absorption and water solubility indexes. Cassava fermentation microbiota was dominated by bacteria and yeasts genera. Bacteria genera include Lactobacillus, Leuconostoc, Lactococcus and Enterococcus. Lactobacillus was the prevalent genera responsible for the acidification of cassava fermentation by the production of organic acids and also aromatic compounds. Yeast community was dynamically adjusted through the cassava fermentation Pichia kudriavzevii and Issatchenkia orientalis were succeeded by Geotrichum candidum, Clavispora lusitaniae and Rhodotorula mucilaginosa. Candida rugosa, C. pararugosa, C. akabenensis, Cryptococcus albidus, Neurospora crassa and N. intermedia were found exclusively in sour cassava. The acidification of sour cassava was due to the production of acetic, lactic and succinic acids. Volatile compounds, including aliphatic and aromatic hydrocarbons, esters and terpenes contribute to the aroma and correspond to 23% of compounds found after fermentation and sun-drying treatment. The acidification and fermentation process reduced the peak viscosity, paste viscosity, breakdown viscosity and set back viscosity in cassava starch. Solid-state NMR relaxometry measures were associated to the expansion ability and indicated that the fermented and sun-dried products were more inclined to expansion. Loaf expansion ability and pasting temperatures were increased in sour cassava (fermented and sun-dried). The results showed here should be useful to standardize the manufacturing of cassava starch in Brazil, providing homogeneous and high quality products.

Evaluation of the efficiency of deterioration of aromatic hydrocarbons by bacteria from wastewater treatment plant of oil refinery

Three bacterial strains were isolated from the activated sludge system of petroleum refinery wastewater, identified by partial sequencing of 16S rDNA, and classified as Acinetobacter genomospecies 3, Bacillus pumilus, and Bacillus flexus. The degradation efficiency of aromatic hydrocarbons was evaluated by gas chromatography with a flame ionization detector. In a mineral medium containing anthracene and phenanthrene and the consortium of microorganisms, the removal efficiency was 96% and 99%, respectively, after 30 days. The good rate of hydrocarbon degradation proves the operational efficiency of the microbial consortium in treating effluents containing these compounds.

Polyphenols from Root, Tubercles and Grains Cropped in Brazil: Chemical and Nutritional Characterization and Their Effects on Human Health and Diseases

Throughout evolution, plants have developed the ability to produce secondary phenolic metabolites, which are important for their interactions with the environment, reproductive strategies and defense mechanisms. These (poly)phenolic compounds are a heterogeneous group of natural antioxidants found in vegetables, cereals and leguminous that exert beneficial and protective actions on human health, playing roles such as enzymatic reaction inhibitors and cofactors, toxic chemicals scavengers and biochemical reaction substrates, increasing the absorption of essential nutrients and selectively inhibiting deleterious intestinal bacteria. Polyphenols present in some commodity grains, such as soy and cocoa beans, as well as in other vegetables considered security foods for developing countries, including cassava, taro and beetroot, all of them cropped in Brazil, have been identified and quantified in order to point out their bioavailability and the adequate dietary intake to promote health. The effects of the flavonoid and non-flavonoid compounds present in these vegetables, their metabolism and their effects on preventing chronic and degenerative disorders like cancers, diabetes, osteoporosis, cardiovascular and neurological diseases are herein discussed based on recent epidemiological studies.

Analysis of the cocobiota and metabolites of Moniliophthora perniciosa-resistant Theobroma cacao beans during spontaneous fermentation in southern Brazil: Dynamic cocobiota in the spontaneous fermentation of bean-pulp mass in Brazil

BACKGROUND Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, yeasts, lactic acid, and acetic acid bacteria. The spontaneous fermentation of cocoa beans by Theobroma cacao TSH565 clonal variety, a highly productive hybrid resistant to Moniliophthora perniciosa and Phytophthora spp., was investigated. The natural cocobiota involved in the spontaneous fermentation of this hybrid in southern Brazil, was investigated by using both a culture-dependent microbiological analysis and a molecular analysis. The changes in the physicochemical characteristics and the kinetics of substrate utilization and metabolite production during fermentation were also evaluated. RESULTS Yeasts (178) and bacteria (244) isolated during fermentation were identified by partial sequencing of the ITS and 16S rDNAs, respectively. After 144 h of fermentation, the indigenous yeast community was composed of Hanseniaspora spp., Saccharomyces spp., and Pichia spp. The bacterial population comprised Lactococcus spp., Staphylococcus spp., Acetobacter spp. and Lactobacilli strains. The kinetics of substrate transformation reflected the dynamic composition of the cocobiota. Substrates such as glucose, fructose, sucrose, and citric acid, present at the beginning of fermentation, were metabolized to produce ethanol, acetic acid, and lactic acid. CONCLUSION The results described here provide new insights into microbial diversity in cocoa bean-pulp mass fermentation and the kinetics of metabolites synthesis, and pave the way for the selection of starter cultures to increase efficiency and consistency to obtain homogeneous and best quality cocoa products.