Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5′,5″-trifluor-d,l-leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. “Cachaça” produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in “cachaça” production.     

Use of selected indigenous Saccharomyces cerevisiae strains for the production of the traditional cachaça in Brazil

AIMS: To test indigenous Saccharomyces cerevisiae as starters to produce cacha?a in large-scale in a traditional distillery, establishing the period in which, each strain predominates in the vats, chemical composition and sensory attributes of the beverage, and to compare these data with vats prepared by spontaneous fermentation. METHODS AND RESULTS: Strains were evaluated for kinetic fermentation parameters, permanence in vats, volatile compound production, and sensory attributes for the cacha?as produced. In general the vats in which starter strains were used, no difference in restriction mitochondrial DNA (mtDNA) profiles of isolates was observed. In the vats in which spontaneous fermentation occurred, different mtDNA restriction profiles were observed. Most of the non-Saccharomyces species isolated could be regarded as contaminants of fermentation. All cacha?as produced, despite being recently distilled and with differences in their chemical composition, were well accepted by the judges. CONCLUSIONS: It was possible to detect the differences in the fermentation capacities of S. cerevisiae strains, in their relative abundances at different time periods, and in the chemical compositions and sensory attributes of the resulting beverages. SIGNIFICANCE AND IMPACT OF THE STUDY: The indigenous strains utilized to prepare cacha?a have shown potential to be used as starters of this traditional fermentation process.     

Self‐selection of photoperiod as a technique for studying endogenous rhythms in fish

Abstract

Drawing on work with passerine birds, investigations on the self‐selection of photoperiod in a constant environment by pumpkinseed sunfish (Lepomis gibbosus) capable of controlling their lighting have been carried out. Each aquarium with lighting equipment was set up in a separate, light‐tight compartment in a quiet, constant‐temperature room. Under one treatment, an overhead fluorescent light could be turned on and off by breaking a photobeam. Under two further treatments, breaking the photobeam turned the light on for three minutes. Fish were fed daily at a regular hour. The latter two treatments, but not the first, resulted in data containing diurnal rhythmic components as analyzed by autocorrelation techniques. The results are discussed in the context of different methods of measuring “internal clock” mechanisms.     

The Production of Volatile Compounds by Yeasts Isolated from Small Brazilian cachaça distilleries

Summary The production of volatile compounds by 24 strains of Saccharomyces cerevisiae and one strain each of Candida apicola, C. famata, C. guilliermondii, Hanseniaspora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe was evaluated with respect to the production of cacha?a. They were isolated from small cacha?a distilleries (27), industrial cacha?a distilleries (2) and one sugarcane alcohol distillery, and tested in synthetic medium for the production of acetaldehyde, ethyl acetate, propanol, isobutanol, isoamyl alcohol, acetic acid and glycerol. The Saccharomyces strains showed a narrow range of variation in the production of such compounds, near 50% of the average of each volatile compound concentration. Principal component analysis showed the separation of the strains into six groups, and acetic acid production was the variable of greatest impact in the differentiation of the strains. The strains of S. pombe formed a distinct group (Group 2), and the strains of C. apicola and H. occidentalis formed a joint group (Group 6) as did Sc13 and Sc4 (Group 4). Group 1 was formed exclusively of S. cerevisiae. The closest non-Saccharomyces strains were C. apicola and H. occidentalis, with a similarity index of about 0.95. The strain P. subpelliculosa showed general characteristics more similar to those of the S. cerevisiae strains than to the non-Saccharomyces strains.     

Genetic diversity of Saccharomyces cerevisiae strains during the 24 h fermentative cycle for the production of the artisanal Brazilian cachaça

AIMS: Characterization of yeast populations and genetic polymorphism of Saccharomyces cerevisiae strains collected during the short fermentative cycles from the spontaneous fermentations during the artisanal cacha?a production. METHODS AND RESULTS: The prevalent S. cerevisiae strains were analysed by PFG and RAPD-PCR using primers EI1 and M13. The molecular analysis have showed a high degree of genetic polymorphism among the strains within a 24 h fermentative cycle. CONCLUSION: The genetic diversity observed in the S. cerevisiae strains may be occurring due to the existence of a large number of individual genotypes within the species. The unique characteristics of the cacha?a fermentation process probably allows for a faster detection of molecular polymorphisms of yeast strains than other types of fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Spontaneous fermentations to produce cacha?a, due to their characteristics, are an excellent model for the study of molecular diversity of S. cerevisiae strains during the production of fermented beverages.     

Validation of criteria for the selection of AFLP markers to assess the genetic variation of a breeders’ collection of evergreen azaleas

Fluorescent AFLP and automated data analysis were employed to assess the genetic conformity within a breeders’ collection of evergreen azaleas. The study included 75 genotypes of Belgian pot azaleas (Rhododendron simsii Planch. hybrids), Kurume and Hirado azaleas and wild ancestor species from the Tsutsusi subgenus. Fluorescent detection and addition of an internal size standard to each lane enabled the automated scoring of each fragment arising from a single AFLP primer combination (PC). The use of three PCs generated an initial data set with a total of 648 fragments ranging from 70 bp to 450 bp. Different marker selection thresholds for average fluorescent signal intensity and marker frequency were used to create eight extra restricted data subsets. Pairwise plant genetic similarity was calculated for the nine data sets using the Simple Matching coefficient (symmetrical, including double-zeros) and Jaccard coefficient (asymmetrical, excluding double zeros). The averages, the ranges and the correlation to one other (Mantel analysis) were compared for the obtained similarity matrices. This revealed the sensitivity of ordinations obtained by both similarity coefficients for the presence of weak or intensive markers or for the degree of polymorphism of the markers. For 34 cultivars, pedigree information (at maximum to the fifth ancestor generation) was available. Genetic similarity by descent (kinship coefficient) was turned into a genetic distance and correlated to the genetic conformity, as revealed by the different selections of AFLP markers (Mantel analysis). Use of a Simple Matching coefficient with no or moderate selection to signal intensity and excluding rare and abundant markers gave the best correlation with pedigree. Finally, the ordination of the studied genotypes by means of dendrograms and principal co-ordinate analysis was confronted with known or accepted relationships based on geographical origin, parentage and morphological characters. Genotypes could be assigned to three distinct groups: pot azaleas, Kurume azaleas and Hirado azaleas. Wild ancestor species appeared to be more related to the Japanese azaleas. Intermediate cultivars could be typified as crossings with Kurume or Hirado azaleas or with wild species. Received: 3 September 1998 / Accepted: 25 March 1999     

The influence of different yeasts on the fermentation,composition and sensory quality of cachaça

Summary Cachaça (sugarcane wine) was produced using different yeast strains, six being strains of Saccharomyces cerevisiae and one each of Candida apicola, Hanseniaspora occidentalis, Pichia subpelliculosa and Schizosaccharomyces pombe. The ethanol yields (%) of the non-Saccharomyces strains were similar to those of the Saccharomyces strains. The following determinations were carried out on the cachaça: acetaldehyde, ethyl acetate, propanol, isobutyl alcohol, isoamyl alcohol, volatile acidity. The cachaças showed variations in the levels of secondary compounds, but these variations did not result in differences (P ≤ 0.05) in the sensory attributes of aroma and flavour and overall impression. Of the volatile compounds quantified in the cachaças, only propanol showed a positive correlation (P ≤ 0.05) with the flavour attributes and overall impression. The S. pombe strain was considered inadequate for the production of cachaça. The cachaças were classified into five groups in an exploratory Hierarchical Cluster Analysis as a function of the volatile compounds. Principal Component Analysis showed that 93% of the variation (PC 1) occurred among the samples, and was explained by the individual volatile compounds and the total secondary compounds, with the exception of isoamyl alcohol only 7% (PC 2) was associated with the volatile acidity. The negative correlations shown between the volatile compounds of the cachaças and the ethanol content of the sugarcane wine, with the exception of acetaldehyde, showed that the variation in ethanol content of the sugarcane wine is an important factor for standardization of the ethanol/volatiles ratio and the beverage quality.     

Chemoradiotherapy or chemotherapy as adjuvant treatment for resected gastric cancer: should we use selection criteria?

BackgroundThe management of gastric adenocarcinoma is essentially based on surgery followed by adjuvant treatment. Adjuvant chemotherapy (CT) as well as chemoradiotherapy (CTRT) have proven their effectiveness in survival outcomes compared to surgery alone. However, there is little data comparing the two adjuvant approaches. This study aimed to compare the prognosis and survival outcomes of patients with gastric adenocarcinoma operated and treated by adjuvant radio-chemotherapy or chemotherapyMaterials and methodsWe retrospectively evaluated 80 patients with locally advanced gastric cancer (LGC) who received adjuvant treatment. We compared survival outcomes and patterns of recurrence of 53 patients treated by CTRT and those of 27 patients treated by CT.ResultsAfter a median follow-up of 38.48 months, CTRT resulted in a significant improvement of the 5-year PFS (60.9% vs. 36%, p = 0.03) and the 5-year OS (55.9% vs. 33%, p = 0.015) compared to adjuvant CT. The 5-year OS was significantly increased by adjuvant CTRT (p = 0.046) in patients with lymph node metastasis, and particularly those with advanced pN stage (p = 0.0078) and high lymph node ratio (LNR) exceeding 25% (p = 0.012). Also, there was a significant improvement of the PFS of patients classified pN2–N3 (p = 0.022) with a high LNR (p = 0.018). CTRT was also associated with improved OS and PFS in patients with lymphovascular and perineural invasion (LVI and PNI) compared to chemotherapy.ConclusionThere is a particular survival benefit of adding radiotherapy to chemotherapy in patients with selected criteria such as lymph node involvement, high LNR LVI, and PNI.     

The evolution of peer review as a basis for scientific publication: directional selection towards a robust discipline?

Peer review is pivotal to science and academia, as it represents a widely accepted strategy for ensuring quality control in scientific research. Yet, the peer‐review system is poorly adapted to recent changes in the discipline and current societal needs. We provide historical context for the cultural lag that governs peer review that has eventually led to the system's current structural weaknesses (voluntary review, unstandardized review criteria, decentralized process). We argue that some current attempts to upgrade or otherwise modify the peer‐review system are merely sticking‐plaster solutions to these fundamental flaws, and therefore are unlikely to resolve them in the long term. We claim that for peer review to be relevant, effective, and contemporary with today's publishing demands across scientific disciplines, its main components need to be redesigned. We propose directional changes that are likely to improve the quality, rigour, and timeliness of peer review, and thereby ensure that this critical process serves the community it was created for.     

Identification of kinectin as a novel Behçet's disease autoantigen

There has been some evidence that Beh?et's disease (BD) has a significant autoimmune component but the molecular identity of putative autoantigens has not been well characterized. In the initial analysis of the autoantibody profile in 39 Chinese BD patients, autoantibodies to cellular proteins were uncovered in 23% as determined by immunoblotting. We have now identified one of the major autoantibody specificities using expression cloning. Serum from a BD patient was used as a probe to immunoscreen a lambdaZAP expression cDNA library. Candidate autoantigen cDNAs were characterized by direct nucleotide sequencing and their expressed products were examined for reactivity to the entire panel of BD sera using immunoprecipitation. Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sj?gren's syndrome. Six independent candidate clones were isolated from the cDNA library screen and were identified as overlapping partial human kinectin cDNAs. The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%) BD patient sera by immunoprecipitation of the in vitro translation products. Sera from controls showed no reactivity. The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed.     

Bacterial cellulose–alginate composite sponge as a yeast cell carrier for ethanol production

A bacterial cellulose–alginate (BCA) sponge, fabricated by a freeze-drying process, was successfully used as a yeast cell carrier for ethanol fermentation. The BCA sponge exhibited several advantageous properties, such as high porosity, appropriate pore size, strong hydrophilicity and high mechanical, chemical and thermal stabilities. BCA has an asymmetric structure, with a thin, dense outer layer covering an interior of interconnected macropores that are distributed throughout the sponge, which is effective for yeast immobilization. At 48 h of the fermentation, the maximum ethanol concentration produced by the immobilized culture (IC) in the BCA carrier was about 100 g/L, which was approximately 13% and 45% higher than that from the suspended culture (SC) and from IC in Ca-alginate matrix, respectively. Repeated-batch ethanol productions using IC in BCA carriers were also more stable than those using SC or IC in Ca-alginate matrix. The results of a 15 cycle repeated batch operation demonstrated that the system with IC in BCA exhibited superior long-term stability for ethanol fermentation with the average ethanol productivity at 1.9 g/L h and the immobilized yield at 86%. The improved ethanol fermentation performance was mainly due to the water uptake ability and properly interconnected pore structure, which help to overcome limiting mass transfer.     

Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil

In this work, 74 Saccharomyces cerevisiae strains isolated from cachaça fermentation of six different geographic regions in Brazil were characterized by mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) and by their ability to grow on stress conditions occurring during the cachaça fermentation process. Cachaça S. cerevisiae strains showed high mtDNA-RFLP polymorphism with the occurrence of 32 different molecular patterns. The S. cerevisiae strains presenting prevalent mtDNA were able to grow better in the stress conditions than strains represented by infrequent patterns. The principal coordinate analysis on 17 stress conditions revealed that the major source of growth variation were high ethanol concentrations and low temperatures. These results indicate that the stress conditions occurring in the fermentation process influence the prevalence of the most adapted S. cerevisiae strains in each distillery. The physiological tests used in our study can be used as a criterion for rapidly selecting autochthonous yeast strains for further purposes such as the selection of fermentative starters of S. cerevisiae strains.     

Evaluation of a novel Wave Bioreactor® cellbag for aerobic yeast cultivation

The Wave Bioreactor is widely used in cell culture due to the benefits of disposable technology and ease of use. A novel cellbag was developed featuring a frit sparger to increase the system's oxygen transfer. The purpose of this work was to evaluate the sparged cellbag for yeast cultivation. Oxygen mass transfer studies were conducted in simulated culture medium and the sparged system's maximum oxygen mass transfer coefficient (kLa) was 38 h(-1). These measurements revealed that the sparger was ineffective in increasing the oxygen transfer capacity. Cultures of Saccharomyces cerevisiae were successfully grown in oxygen-blended sparged and oxygen-blended standard cellbags. Under steady state conditions for both cellbag designs, kLa values as high as 60 h(-1) were obtained with no difference in growth characteristics. This is the first report of a successful cultivation of a microbe in a Wave Bioreactor comparing conventional seed expansion in shake flasks and stirred tank bioreactors.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

δ15N as a cultivar selection tool for differentiating alfalfa varieties under biosaline conditions

Plant and Soil - The potential of “biosaline agriculture” relies on easy-to-apply tools to select plant genotypes that are best adapted to saline conditions. We aimed to determine the...     

An Evaluation of β-Propiolactone for the Sterilization of Fermentation Media

Twenty-five bacterial species were cultured in basal broth plus 1 of 19 different carbohydrates which were sterilized by Seitz filtration, autoclaving (112 C, 10 min), or exposure to 0.2% β-propiolactone (BPL). No significant differences were found either in the visual observations for acid and gas, pH, or titrable acidity determinations after 3 days of incubation with any of the three preparations tested. An effort was made to further determine the effect of BPL and heat on carbohydrates by assaying for glucose before and after treatment. Results indicated that glucose was not degraded by 0.2% BPL, however, it was shown that autoclave temperatures caused extensive degradation. Statistical treatment of the results from Warburg studies indicated that BPL-treated glucose showed no appreciable toxic effects, although the actual oxygen uptake was not as great as with Seitz- or autoclave-treated glucose. The application of the BPL sterilization process was discussed.