Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis

The defense mechanism by which cells combat metal stress remains poorly understood. By utilizing a newly developed technique - the differential gel electrophoresis (DIGE) - we evaluated the biological alterations of metal stress on Saccharomyces cerevisiae at its translational level. By simultaneously comparing the differential expression profiles of thousands of proteins as results of 15 different metal treatments, we were able to closely examine the response of a large number of proteins within the yeast proteome towards individual metals, as well as the response of the same proteins towards different metals. This, to our knowledge, is the first case which demonstrates the potential of DIGE as a high-throughput tool for large-scale proteome analysis. From our studies, where yeast cells were exhaustively treated with exogenous metals, 20-30% of all proteins detected showed statistically significant changes. According to different effects (up-/downregulation) of protein expression levels observed, we were able to tentatively divide the 15 metals into three groups. By mass spectrometric analysis, more than 50 protein spots were positively identified, both quantitatively and qualitatively. One of the proteins was identified to be Cu/Zn superoxide dismutase (SOD1), and its expression levels as a result of 15 different metal treatments was further examined in greater details. Significant changes in SOD1 expression were observed throughout all 15 DIGE gels.     

Indigenous Saccharomyces cerevisiae Could Better Adapt to the Physicochemical Conditions and Natural Microbial Ecology of Prince Grape Must Compared with Commercial Saccharomyces cerevisiae FX10

Indigenous Saccharomyces cerevisiae, as a new and useful tool, can be used in fermentation to enhance the aroma characteristic qualities of the wine-production region. In this study, we used indigenous S. cerevisiae L59 and commercial S. cerevisiae FX10 to ferment Prince (a new hybrid variety from Lion Winery) wine, detected the basic physicochemical parameters and the dynamic changes of fungal communities during fermentation, and analyzed the correlations between fungal communities and volatile compounds. The results showed that the indigenous S. cerevisiae L59 could quickly adapt to the specific physicochemical conditions and microbial ecology of the grape must, showing a strong potential for winemaking. Compared with commercial S. cerevisiae FX10, the wine fermented by indigenous S. cerevisiae L59 contained more glycerol and less organic acids, contributing to a rounder taste. The results of volatile compounds indicated that the indigenous S. cerevisiae L59 had a positive effect on adding rosy, honey, pineapple and other sweet aroma characteristics to the wine. Overall, the study we performed showed that selection of indigenous S. cerevisiae from the wine-producing region as a starter for wine fermentation is conducive to improving the aroma profile of wine and preserving the aroma of the grape variety.     

Production of single cell protein (SCP) from food and agricultural waste by using Saccharomyces cerevisiae

Food waste is the single-largest component of the waste stream, in order to protect and safeguard the public health, useful and innovative recycling methods are investigated. The conversion of food wastes in value-added products is becoming a more economically viable and interesting practice. Food waste, collected in the distribution sector and citrus industries, was characterised for its potential as a raw material to use in fermentation processes. In this study, the production of single-cell protein (SCP) using food waste as a substrate was investigated. The purpose of this study has been to produce SCP from mixtures of food waste using Saccharomyces cerevisiae. The main fermentation test was carried out using a 25 l bioreactor. The utilisation of food waste can allow us to not only to reduce environmental pollution, but also to obtain value-added products such as protein supply for animal feed.     

光散射法快速灵敏测定和表征啤酒酵母

研究了啤酒酵母的光散射光谱. 结果表明， 在波长308.0 nm处, 光散射强度与啤酒酵母浓度在2.0×104-2.0×106 Cell/mL 范围内呈线性关系, 检出限(3σ)为4.94×102 Cell/mL. 将此方法成功地应用于培养液中啤酒酵母含量的快速、 灵敏的测定, 并对其散射光谱进行了表征.     

Profile of Volatile Compounds of On-Farm Fermented and Dried Cocoa Beans Inoculated with Saccharomyces cerevisiae KY794742 and Pichia kudriavzevii KY794725

This study aimed to identify the volatile compounds in the fermented and dried cocoa beans conducted with three distinct inoculants of yeast species due to their high fermentative capacity: Saccharomyces cerevisiae, Pichia kudriavzevii, the mixture in equal proportions 1:1 of both species, and a control fermentation (with no inoculum application). Three starter cultures of yeasts, previously isolated and identified in cocoa fermentation in the municipality of Tomé-Açu, Pará state, Brazil. The seeds with pulp were removed manually and placed in wooden boxes for the fermentation process that lasted from 6 to 7 days. On the last day of fermentation, the almonds were packaged properly and placed to dry (36 °C), followed by preparation for the analysis of volatile compounds by GC-MS technique. In addition to the control fermentation, a high capacity for the formation of desirable compounds in chocolate by the inoculants with P. kudriavzevii was observed, which was confirmed through multivariate analyses, classifying these almonds with the highest content of aldehydes, esters, ketones and alcohols and low concentration of off-flavours. We conclude that the addition of mixed culture starter can be an excellent alternative for cocoa producers, suggesting obtaining cocoa beans with desirable characteristics for chocolate production, as well as creating a product identity for the producing region.     

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 ${\mathrm{PO}}_3^{-}$monomers with toluidine blue or 4′,6-diamidino-2-phenylindole. PolyP chain lengths longer than 62${\mathrm{PO}}_3^{-}$monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich “Type 45”, estimated mean length of 45 ${\mathrm{PO}}_3^{-}$ monomers), trimetaphosphate (trimetaP: 3 ${\mathrm{PO}}_3^{-}$ ring), tripolyphosphate (tripolyP), pyrophosphate (PP_i), and inorganic orthophosphate (o-P_i) were visualized after separation by an in situ hydrolytic degradation process to o-P_i that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol–chloroform extraction was confirmed with the Friedman test. ³¹P diffusion–ordered NMR spectroscopy confirmed that extraction also reduced PP_i diffusivity by <10%. Linear regression between the R_f peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 ${\mathrm{PO}}_3^{-}$monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-P_i, but no PP_i.     

Efficient and sample-specific interpretation of ToF-SIMS data by additional postprocessing of principal component analysis results

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool for surface analysis, but fragmentation of molecular species during the SIMS process may lead to complex mass spectra. While the fragmentation pattern is typically characteristic for each compound, industrial samples are engineered materials, and, thus, may contain a mixture of many compounds, which may result in a variety of overlapping peak patterns in ToF-SIMS spectra. Consequently, the process of data evaluation is challenging and time-consuming. Principal component analysis (PCA) can be used to simplify data analysis for complex sample systems. Especially, correlation loadings were observed as an ideal tool to identify relevant signals in PCA results, which induce the separation of different sample groups. This is because correlation loadings show the relevance of signals independent from their intensity in the raw data. In correlation loadings, however, fragmentation patterns are no longer observed and the identification of peaks' sum formulas is challenging. In this study, a new approach is presented, which simplifies peak identification and assignment in ToF-SIMS spectra after PCA is performed. The approach uses a mathematical transformation that projects PCA results, in particular loadings and correlation loadings, in the direction of specific sample groups. The approach does not change PCA results but rather presents them in a new way. This method allows to visualize characteristic spectra for specific sample groups that contain only relevant signals and, additionally, visualize fragmentation patterns. Data analysis is simplified and helps the user to focus on data interpretation rather than processing.     

Modelling of chemical fractionation patterns of metals in soils by two-way and three-way principal component analysis

The ‘pseudo-total’ contents and the chemical fractionation pattern of eight toxic elements (As, Cd, Co, Cu, Cr, Ni, Pb and Zn) have been determined in 12 soil samples collected around a coal-fuelled power plant (Velilla del Río Carrión, Spain) by using, respectively, the US-EPA 3051 norm and the modified BCR (SM&T) chemical fractionation procedure. The ‘pseudo-total’ dataset has been analyzed by classical two-way principal component analysis (PCA) finding a PC accounting for the metal ‘pollution’ of the area. On the other hand, the three-dimensional (samples × metals × fractions) X array obtained after application of the modified BCR SM&T procedure, has been studied by matrix augmentation (MA-PCA) and three-way principal component analysis (3-PCA) using PARAFAC and TUCKER3 models. Whereas both MA-PCA and PARAFAC originated two-factor models biased towards the different chemical fractionation of the samples, the best TUCKER3 model [1, 2, 2] takes into account simultaneously both the ‘pseudo-total’ contents and the chemical fractionation of the soil samples. Therefore, the TUCKER3 originated a better representation of the global environmental impact caused by the power plant, and the plotting of the soil samples loadings, A1, in the physical space allowed to locate the most potentially hazardous areas.     

Evaluation of Italian wine by the electronic tongue: recognition, quantitative analysis and correlation with human sensory perception

The electronic tongue based on a sensor array comprising 23 potentiometric cross-sensitive chemical sensors and pattern recognition and multivariate calibration data processing tools was applied to the analysis of Italian red wines. The measurements were made in 20 samples of Barbera d’Asti and in 36 samples of Gutturnio wine. The electronic tongue distinguished all wine samples of the same denomination and vintage, but from different vineyards. Simultaneously the following quantitative parameters of the wines were measured by the electronic tongue with precision within 12%: total and volatile acidity, pH, ethanol content, contents of tartaric acid, sulphur dioxide, total polyphenols, glycerol, etc. The electronic tongue is sensitive to multiple substances that determine taste and flavour of wine and, hence, the system was capable of predicting human sensory scores with average precision of 13% for Barbera d’Asti wines and 8% for Gutturnio wines.     

Structural and mechanical properties of alkali hydrides investigated by the first-principles calculations and principal component analysis

The structural and mechanical properties of alkali hydrides (LiH, NaH, KH, RbH, and CsH) were investigated via first-principles calculations which cover the optimized structural parameters. The density functional theory in combination with the generalized gradient approximation (GGA) were used in this study. From the present study, one could note that alkali hydrides are brittle materials and mechanically stable. It was found that stiffness and shear resistance are greater in LiH than in other hydrides. It is more brittle in nature, and comparatively harder than the other materials under study; it also presents a high degree of anisotropy. The results were then investigated and analyzed with principal component analysis (PCA), which is one of the most common techniques in multivariate analysis, was used to explore the correlations among material properties of alkali hydrides and to study their trends. The alkali hydrides obtained by the first-principles calculations were also compared with the alkaline-earth metal hydrides (BeH₂, MgH₂, CaH₂, SrH₂, and BaH₂) and discussed in this work.     

三波长融合指纹图谱结合6组分定量和主成分分析评价银翘解毒片的质量

建立了三波长融合高效液相色谱指纹图谱,并结合6组分定量和主成分分析(PCA)评价25批银翘解毒片的质量一致性。采用反相高效液相色谱法(RP-HPLC)分别于230、279、327 nm下检测。运用多波长融合指纹图谱技术建立银翘解毒片三波长融合指纹图谱,采用系统指纹定量法对其进行定性和整体定量评价。结果鉴别出25批银翘解毒片样品完全合格且区分良好。同时测定6组分含量,与指纹图谱结合,从整体和局部角度评价银翘解毒片质量。此外,运用PCA法对融合指纹图谱进行分析,通过主成分得分图可以明显区分来自两个厂家的25批银翘解毒片样品。方法综合性较强且有效,为科学评价与有效控制银翘解毒片的质量提供了可靠的参考。     

Analysis of methylglyoxal in water and biological matrices by capillary zone electrophoresis with diode array detection

We describe a new method for the determination of methylglyoxal in water and biological matrices, using o-phenylenediamine as derivatizing agent and solid-phase extraction followed by capillary zone electrophoresis with diode array detection. 25 mM sodium phosphate running buffers at pH 2.2, 30 kV, and 25 degrees C allowed the best instrumental conditions for the optimum separation of methylglyoxal in a suitable analytical time (< 10 min), using an uncoated fused-silica capillary of 75 microm inner diameter and an effective length of 45.1 cm with an extended light path and the wavelength set to 200 nm. Under optimized instrumental conditions, good reproducibility of the migration time (< 1.1%), precision (< 5%), an excellent linear dynamic range from 0.1 to 3.6 mg/L (r(2) = 0.9997), and low limits of detection (7.2 microg/L) were obtained for methylglyoxal measurements, using the internal standard methodology. Assays on laboratory-spiked tap and ground water samples allowed a remarkable accuracy, presenting yields of 95.0 +/- 4.3 and 94.0 +/- 1.1%, respectively, and good performance to determine methylglyoxal in beer and yeast cells suspensions matrices was also obtained at trace level. The present methodology is a cost-effective alternative for routine quality control analysis, showing to be reliable, sensitive, and with a low sample volume requirement to monitor methylglyoxal in water and biological matrices.     

Study for developing an electronic tongue to discriminate three different classes of waters,by using common sensors and the principal component analysis

A basic research has been carried out using four probes to develop an electronic tongue able to discriminate three different kinds of natural waters, i.e. rain, river and groundwater. Several natural water samples were analysed to this purpose using a pH glass electrode, a digital (thermistor) thermometer, an amperometric gaseous diffusion oxygen sensor for measuring the dissolved oxygen and a direct methanol fuel cell enzymatic probe for measuring the concentration of alcoholic traces. Data were processed by means of principal component analysis, obtaining three well differentiated clusters, that demonstrate how the use of only four different sensors can discriminate in an inexpensive and effective way three different classes of natural waters.     

Usefulness of chemometrics and mass spectrometry-based electronic nose to classify Australian white wines by their varietal origin

A combination of mass spectrometry-based electronic nose (MS e_nose) and chemometrics was explored to classify two Australian white wines according to their varietal origin namely Riesling and unwooded Chardonnay. The MS e_nose data were analysed using principal components analysis (PCA), discriminant partial least squares (DPLS) and linear discriminant analysis (LDA) applied to principal components scores and validated using full cross validation (leave one out). DPLS gave the highest levels of correct classification for both varieties (>90%). LDA classified correctly 73% of unwooded Chardonnay and 82% of Riesling wines. Even though the conventional analysis provides fundamental information about the volatile compounds present in the wine, the MS e_nose method has a series of advantages over conventional analytical techniques due to simplicity of the sample-preparation and reduced time of analysis and might be considered as a more convenient choice for routine process control in an industrial environment. The work reported here is a feasibility study and requires further development with considerably more commercial samples of different varieties. Further studies are needed in order to improve the calibration specificity, accuracy and robustness, and to extend the discrimination to other wine varieties or blends.     

Comparison of shikimic acid determination by capillary zone electrophoresis with direct and indirect detection with liquid chromatography for varietal differentiation of red wines

Two capillary zone electrophoretic (CZE) methods for determination of shikimic acid in Chilean red wine were developed and compared with a HPLC method. Both electrophoretic methods were carried out by using a reversed electroosmotic flow induced by trimethyl(tetradecyl)ammoniumbromide (TTAB) with indirect detection at 260 nm using p-aminobenzoic acid as a UV-absorbing co-ion or by direct detection at 213 nm. In both cases, the separation was carried out in a 50 microm I.D. uncoated capillary with an effective length of 48 cm, a negative power supply of 30 kV, using a buffer based on bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane (Bis-Tris), pH 7.0 or 7.5 and hydrodynamic injection. The chromatographic separations were carried out on a C-18 reversed phase column followed by a sulfonyl-styrene-divinylbenzene (S-DVB) ion exclusion column at 70 degrees C with H2SO4 0.02 M as isocratic mobile phase and a flow rate of 0.5 mL min(-1). The three methods allowed the quantification of shikimic acid with quantification limits between 1.0 and 12.0 mg L(-1) and precision between 7.3 and 10.1%, however, only the concentrations obtained by CZE with direct detection were statistically similar to those of HPLC. This parameter was evaluated as analytical tool to verify varietal authenticity of red wines. In all cases, the Cabernet Sauvignon wines presented higher concentrations of shikimic acid, compared with Merlot or Carmenère wines.     

Adsorption of genetically engineered proteins studied by time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS). Part A: data acquisition and principal component analysis (PCA)

Recent progress in the adaptation of combinatorial biology selection protocols to materials science has created a new class of polypeptides with specific affinity to inorganics. Here, we use one of the genetically engineered proteins, a gold binding protein (GBP‐1), to assess quantitatively its binding specificity to Au, Ag and Pd surfaces by using time‐of‐flight secondary ion mass spectroscopy (TOF‐SIMS). The GBP‐1, originally selected using cell‐surface display techniques, consisting of 14 amino acids with a sequence of MHGKTQATSGTIQS, was used in this study. Three‐repeat and single‐repeat forms of GBP‐1 were prepared. In earlier studies, GBP‐1 was shown to bind to Au particles and self‐assemble on flat Au surfaces. Through the fingerprint analysis of these specific peptides, their role in binding can be investigated in terms of their contribution to surface interaction possibly forming the right molecular architecture for binding. To achieve this purpose, a large‐sized data matrix produced by TOF‐SIMS must be properly treated for analysis. In Part A, we use principal component analysis (PCA) to visualize the spectral variations for a variety of adsorption conditions and suggest possible contribution of the specific types of amino acids (binding site) to the interactions. Copyright © 2007 John Wiley & Sons, Ltd.     

Characterization of 1,3-alternate calix[4]arene-silica bonded stationary phases and their comparison to selected commercial columns by using principal component analysis

Twelve calix[4]arene stationary phases in 1,3-alternate conformation, synthesized in the authors’ laboratory, were characterized in terms of their surface coverage, hydrophobic selectivity, aromatic selectivity, shape selectivity, hydrogen bonding capacity and ion-exchange capacity. The set of tests commonly used for evaluation of commercially available stationary phases was applied to assess fundamental chromatographic properties of the calixarene phases. The new calixarene phases were compared to each other, to Caltrex and LiChrosorb C-18 columns. Principal component analysis has been used to provide comparison between 1,3-alternate calix[4]arene phases and commercially available phenyl, fluorophenyl and fluoroalkyl columns.     

高效阴离子交换色谱-积分脉冲安培检测法同时测定黄酒中的单糖和低聚糖及其指纹图谱的构建

建立了黄酒中葡萄糖、果糖、异麦芽糖、异麦芽三糖、麦芽糖、潘糖、麦芽三糖共7种糖类的高效阴离子交换色谱分离-积分脉冲安培检测的方法。以氢氧化钠和醋酸钠为淋洗液进行梯度洗脱,CarboPac^TM10阴离子交换柱进行分离。该方法可在26 min内实现黄酒中7种糖类的定量分析,在0.5~50 mg/L浓度范围内线性关系良好,样品定量限为0.1 g/L,加标回收率为76.5%~108.4%,精密度(RSD)为3.02%~8.23%。将该法用于不同厂家不同批次的绍兴加饭酒中各种糖类含量的检测,基于所得的检测结果,利用中位数法构建了标准指纹图谱,并采用夹角余弦法评价了各个样品与标准指纹图谱之间的相似度。结果初步显示,不同厂家加饭酒之间的相似度差别较大,该法可作为区别不同厂家酒的依据之一。     

Simultaneous determination of iridoids, phenolic acids, flavonoids, and saponins in Flos Lonicerae and Flos Lonicerae Japonicae by HPLC-DAD-ELSD coupled with principal component analysis

A method, HPLC coupled with diode-array and evaporative light scattering detectors (HPLC-DAD-ELSD), was newly developed to evaluate the quality of Flos Lonicerae (FL) and Flos Lonicerae Japonicae (FLJ), through a simultaneous determination of multiple types of bioactive components. By employing DAD, the detection wavelengths were set at 240 nm for the determination of iridoids, 330 nm for phenolic acids, and 360 nm for flavonoids, respectively. While ELSD, connected in series after DAD, was applied to the determination of saponins. This assay was fully validated with respect to precision, repeatability, and accuracy. Moreover, principal component analysis (PCA) was used for the similarity evaluation of different samples, and it was proven straightforward and reliable to differentiate FL and FLJ samples from different origins. For PCA, two principal components have been extracted. Principal component 1 (PC1) influences the separation between different sample sets, capturing 54.598% variance, while principal component 2 (PC2) affects differentiation within sample sets, capturing 12.579% variance. In conclusion, simultaneous quantification of bioactive components by HPLC-DAD-ELSD coupled with PCA would be a well-acceptable strategy to differentiate the sources and to comprehensively control the quality of the medicinal plants FL and FLJ.     

Solid-phase extraction combined with headspace single-drop microextraction of chlorophenols as their methyl ethers and analysis by high-performance liquid chromatography-diode array detection

Solid-phase extraction (SPE) of phenol and chlorophenols, their derivatization to methyl ethers, headspace single-drop microextraction (HS-SDME) of methyl ethers using 1-butanol as extraction solvent, and direct transfer of the drop into the injector for high performance liquid chromatography with diode array detection (HPLC-DAD) have been reported. A flanged-end polytetrafluoroethylene sleeve, 3 mm × 0.5 mm, placed at the tip of the syringe needle, allowed the use of 10 μL solvent drop for extraction. The procedure has been optimized for variables involved in SPE and HS-SDME. A rectilinear relationship was obtained between the amount of chlorophenols and peak area ratio of their methyl ethers/internal standard (4-methoxyacetophenone) in the range 0.01-10 mg L⁻¹, correlation coefficient in the range 0.9956-0.9996, and limit of detection in the range 1.5-3.9 μg L⁻¹ when HS-SDME alone was used for sample preparation. When using coupled SPE and HS-SDME, the linear range obtained was 0.1-500 μg L⁻¹, correlation coefficient in the range 0.9974-0.9998, and the limit of detection in the range 0.04-0.08 μg L⁻¹. Spiked real samples have been analyzed with adequate accuracy, and application of the method has been demonstrated for the analysis of chlorophenols formed upon bamboo pulp bleaching.