Mechanism of Resistance of Saccharomyces bailii to Benzoic, Sorbic and Other Weak Acids Used as Food Preservatives

Saccharomyces bailii grows in the presence of high concentrations of sorbic, benzoic and other short-chain monocarboxylic acids commonly used as preservatives. Starved cells concentrate these acids intracellularly, approximately as expected from the pH of the ceil and the p K _a of the acid. On addition of glucose, the intracellular content of preservative is considerably reduced. The glucose effect is sensitive to metabolic inhibitors, and anaerobic respiration is stimulated by the preservatives. The ability to maintain a low intracellular concentration of any of the preservatives tested is induced by growth in the presence of sorbic or benzoic acid and less effectively by butyric or acetic acid. Both induced and uninduced cells are permeable to benzoic and butyric acids. Benzoate and sorbate are not metabolized at a rate significant with respect to the permeation rate. Resistance to these preservatives apparently results primarily from an inducible, energy requiring system which transports preservative from the cell.     

Oxalic acid overproduction by copper-tolerant brown-rot basidiomycetes on southern yellow pine treated with copper-based preservatives

Accumulation of oxalic acid (OA) by brown-rot fungi and precipitation of copper oxalate crystals in wood decayed by copper-tolerant decay fungi has implicated OA in the mechanism of copper tolerance. Understanding the role of OA in copper tolerance is important due to an increasing reliance on copper-based wood preservatives. In this study, four copper-tolerant brown-rot fungi were evaluated for decay capacity and OA production in early stages of exposure to four waterborne copper-based wood preservatives (ammonical copper quat type B and D, ammonical copper citrate, and chromated copper arsenate, type C) and one oilborne copper-based wood preservative (oxine copper) in southern yellow pine blocks. Weight losses were less than 14% during the 4-week incubation. The presence of copper in waterborne preservatives uniformly stimulated OA production by the test fungi within 2 weeks of exposure of the treated blocks to test fungi; 66% to 93% more OA was produced in treated blocks than untreated controls. Oxine copper, a nickel-containing oilborne preservative, prevented both weight loss and OA production in all fungi tested.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

The complexation of aqueous metal ions relevant to biological applications. 2. Reactions of copper(II) citrate and copper(II) succinate with selected amino acids

Abstract

Addition of amino acids, glycine, alanine, and serine, to poorly soluble copper(II) salts [copper(II) citrate and copper(II) succinate] all increase solubility of the copper(II) salts. Relative increases in solubility follow the polarity trend in the selected amino acids, with serine creating the greatest increase in solubility. Simultaneous equilibria calculations indicate the formation of mixed-ligand complexes in the copper(II) succinate–amino acid systems, the first time such mixed-ligand complexes have been observed. In contrast, mixed-ligand complexes are not predicted in the copper(II) citrate–amino acid systems. Potential bioavailability of copper(II) appears to be increased by the inclusion of amino acids in solution, roughly in parallel with the increase in solubility of the copper(II) salt. Therefore, measurement of the change in solubility caused by addition of amino acids to aqueous solution gives qualitative insight to the potential increase in bioavailability of the metal ion.     

Addition of magnetic field capability to existing extremely-low-frequency electric field exposure systems

Magnetic field systems were added to existing electric field exposure apparatuses for exposing cell suspensions in vitro and small animals in vivo. Two horizontally oriented, rectangular coils, stacked one directly above the other, have opposite electric currents. This configuration minimizes leakage fields and allows sham- and field-exposure systems to be placed in the same room or incubator. For the in vitro system, copper plates formed the loop-pair, with up to 900 A supplied by a 180:1 transformer. Electric fields were supplied via electrodes at the ends of cell-culture tubes, eight of which can be accommodated by each exposure system. Two complete systems are situated in an incubator to allow simultaneous sham and field exposure up to 1 mT. For the in vivo system, four pairs of 0.8 x 2.7-m coils made of copper bus bar are employed. This arrangement is energized from the power grid via a 30:1 transformer; horizontal magnetic flux densities up to 1 mT can be generated. Pairs of electrode plates spaced 30.5 cm apart provide electric field exposure of up to 130 kV/m. Four systems with a capacity of 48 rats each are located in one room. For both the in vitro and in vivo systems, magnetic exposure fields are uniform to within +/- 2.5%, and sham levels are at least 2,500-fold lower than exposure levels. Potential confounding factors, such as heating and vibration, were examined and found to be minimal.     

Investigating a simplified method for noninvasive genetic sampling in East African mammals using silica dried scat swabs

Swabbing scat has proved to be an effective noninvasive method to collect DNA from mammals in the field. Previously, this method has relied on preservative liquids or freezing to preserve the DNA collected on swabs. In this study, we determine the effectiveness of using silica to simply dry the swab in field as an alternative way to prevent DNA degredation. Four species were included in the study; reticulated giraffe, impala, fringe‐eared oryx, and lion. Swabs were taken at multiple time points for giraffe and impala scat samples, with the lion and oryx sampled opportunistically. Mitochondrial DNA was successfully amplified and sequenced from scat swabs from all species; however, effectiveness varied between species, with 81.8% amplification success rate from swabs taken from impala scat compared to 25% amplification success rate in giraffe. This variation in success rate was overcome by taking multiple swabs, thus increasing the probability of a successful amplification. The true merit of this method is in its simplicity and cheapness; no preservative liquids were required to be brought into the field, at no stage in the 2 weeks of field sampling were samples frozen, and no commercial kits were used for DNA extraction.     

Loss of effectiveness of preservative systems of mascaras with age.

The preservative systems of unused, anhydrous mascaras were challenged periodically with microorganisms, using a modified membrane procedure. Shelf life of preservative activity against Pseudomonas aeruginosa varied for different brands from as little as 1 month to over 36 months. Generally, P. aeruginosa grew in mascaras after the mascaras were challenged first with Staphylococcus epidermidis or Candida albicans.     

Fine steps of electrocatalytic oxidation and sensitive detection of some amino acids on copper nanoparticles

The electrocatalytic oxidation of five amino acids—glycine, aspartic acid, cysteine, glutamic acid, and tyrosine—on two copper-based electrodes comprising copper microparticle-modified carbon paste electrode (m-CPE) and copper nanoparticle-modified CPE (n-CPE) was investigated. In the voltammograms recorded using m-CPE, a single anodic peak related to the oxidation of amino acids appeared and was related to the electrocatalytic oxidation of the amino acids via the electrogenerated Cu(III) species. Using n-CPE, however, two overlapped anodic peaks in the voltammograms appeared and were related to two fine tunable steps of the oxidation process. The currents of the two peaks were controlled by diffusion and were confirmed by chronoamperometric measurements. The amino acids were oxidized on n-CPE at higher rates and at lower potentials compared with m-CPE. This was attributed to the nanosize of copper nanoparticles. Some primary linear-chain amines and primary branched-chain amines were oxidized on the copper-based electrodes as markers. The catalytic rate constants, the transfer coefficients, and the diffusion coefficients for the amino acids are reported. Simple, sensitive, and time-saving sensing procedures in both batch and flow systems were developed for the analysis of the amino acids, and the corresponding analytical parameters are reported.     

Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality

Lactic acid bacteria (LAB) have been used for centuries in the fermentation of a variety of dairy products. The preservative ability of LAB in foods is attributed to the production of anti-microbial metabolites including organic acids and bacteriocins. Bacteriocins generally exert their anti-microbial action by interfering with the cell wall or the membrane of target organisms, either by inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death. The incorporation of bacteriocins as a biopreservative ingredient into model food systems has been studied extensively and has been shown to be effective in the control of pathogenic and spoilage microorganisms. However, a more practical and economic option of incorporating bacteriocins into foods can be the direct addition of bacteriocin-producing cultures into food. This paper presents an overview of the potential for using bacteriocin-producing LAB in foods for the improvement of the safety and quality of the final product. It describes the different genera of LAB with potential as biopreservatives, and presents an up-to-date classification system for the bacteriocins they produce. While the problems associated with the use of some bacteriocin-producing cultures in certain foods are elucidated, so also are the situations in which incorporation of the bacteriocin-producer into model food systems have been shown to be very effective.     

Degradation of Wood Preservatives by Fungi

Wood-inhabiting fungi, not necessarily responsible for major decay, are shown to be capable of degrading a toxic compound into a less potent form, thus rendering it less effective in protecting wood from decay by less-tolerant basidiomycetous wood-destroyers. Sweetgum or pine sapwood blocks treated with preservatives (ammoniacal copper arsenate, fluor-chrome-arsenate-dinitrophenol, a creosote or pentachlorophenol) were exposed progressively to two different wood-inhabiting fungi with sterilization between the first and second exposure. The fungus in the first exposure was usually an Ascomycete or a Fungi Imperfecti-Chaetomium globosum, Phoma, Orbicula, Graphium, Pestalozzia, or Trichoderma species, isolated from wood below the ground. In one experiment, the fungus in the first exposure was a basidiomycete, Lenzites trabea or Polyporus versicolor. The second fungus, a prominent Basidiomycete-Coniophora puteana, Lentinus lepideus, or Lenzites trabea-was the bioassay fungus, since its purpose was to show whether the first fungus had degraded the preservative. Generally, the treated block, except where exposed to another fungus, remained virtually untouched by the bioassay fungus. Clearly, therefore, the first fungus had rendered the preservative ineffective but without appreciably decaying the wood itself Chemical analyses of treated blocks indicated that in the first exposure the fungi had substantially depleted sodium arsenate and pentachlorophenol.     

Effects of iron,copper, and chromate ions on the oxidative degradation of cellulose model compounds

Iron(II) and iron(III) ions promote the degradation of the cellulose model 1,5-anhydrocellobiitol by oxygen or hydrogen peroxide; copper and chromate ions have marked and different effects on the iron catalysis. With starch, iron promotes the hydrogen peroxide-induced reaction and copper and chromate ions further enhance the reaction rate. The tensile strength of paper board is reduced by the action of hydrogen peroxide and iron(II) salts, and mixtures of copper, chromate, and arsenate salts (CCA, a timber preservative) also promote degradation in the presence or absence of iron ions. The oxidation of 1,5-anhydrocellobiitol by oxygen in the presence of iron ions is strongly inhibited by CCA and by cetyltrimethylammonium chloride, and is accelerated by phenols and related compounds.     

A comparison of methionine, histidine and cysteine in copper(I)-binding peptides reveals differences relevant to copper uptake by organisms in diverse environments

The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu(i) binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu(i) binding affinity and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu(i) affinity was examined using an ascorbic acid oxidation assay, an electrospray ionization mass spectrometry technique, and spectrophotometric titration with a competitive Cu(i) chelator. The relative affinities of the peptides with Cu(i) reveal a trend whereby Cys > His > Met at pH 7.4 and Cys > Met > His at pH 4.5. Ligand geometry and metric parameters were determined with X-ray absorption spectroscopy. Susceptibility of the peptides to oxidation by hydrogen peroxide and copper-catalyzed oxidative conditions was evaluated by mass spectrometry. These results support hypotheses as to why certain Cu(i) binding amino acids are preferred over others in proteins expressed at different pH and exposed to oxidative environments. The results also have implications for interpreting site-directed mutagenesis studies aimed at identifying copper binding amino acids in copper trafficking proteins.     

Evaluation of decay and termite resistance of wood treated with copper in combination with boron and N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na)

This study evaluated the relative ability of various combinations of copper sulfate with either boric acid or calcium-precipitating agent, N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na), to inhibit fungal degradation and attack by Formosan subterranean termites (Coptotermes formosanus Shiraki). Wood specimens were treated with either 1%, 0.5%, or 0.1% concentrations of copper sulfate, boric acid, NHA-Na, copper sulfate + boric acid, or copper sulfate + NHA-Na mixtures. Treated specimens were subjected to laboratory decay-resistance tests by using petri dishes inoculated with the Basidiomycetes fungi Tyromyces palustris and Trametes versicolor for 12 weeks. Treated wood specimens were also subjected to termite-resistance tests under laboratory conditions. Increased efficacy of copper sulfate against the brown-rot fungus T. palustris was observed when either boric acid or NHA-Na was added. The most effective treatments against the fungi tested were NHA-Na only treatments at 1% and 0.5% concentration levels. Boric acid treatments were not able to protect wood against decay after leaching because of excessive leaching of boron. Similar results were obtained in termite-resistance tests in comparison with decay-resistance tests. These results indicate that the efficacy of the treatments in preventing fungal and termite attack is a function of the type of preservative.     

The effect of copper and nitrogen on the amino acid composition of oat straw

Summary The effect of fertilization with nitrogen and copper on the amino acid composition of oat straw has been studied.The plants (Avena sativa cv Yielder) were grown in peat with a very low copper content and supplied with two levels of nitrogen (NH₄ or NO₃) and three levels of copper sulphate.The higher level of nitrogen stimulated growth only when copper was added, whereas, without copper, it had an adverse effect on growth and prevented grain formation altogether. The higher level of nitrogen increased the nitrogen content of the straw at all levels of copper, but particularly in plants receiving no copper.Total amino acids in the straw hydrolysate of copper sufficient oats accounted for about 50% of the total N and was about 20% higher in copper-deficient tissues. The addition of copper caused a decrease in the amounts of all amino acids. The relative proportions of most of the amino acids to glycine remained fairly constant. Threonine, serine, alanine, iso-leucine, histidine and arginine showed small significant differences with copper treatment, whereas valine, tyrosine, phenylalanine, proline, lysine and cysteic acid (derived from cysteine and cystine) showed no differences. The proportion of aspartic acid relative to glycine in the straw hydrolysate was greatly increased in copper deficient plants supplied with the higher level of nitrogen, particularly as ammonium. The proportion of glutamic acid was also increased by the higher level of nitrogen, but showed no effect of added copper. Most of the difference in aspartic acid could be accounted for as free asparagine. The possible reasons for higher proportions of asparagine are discussed in relation to the metabolism of the oat plant.     

Assessing the relative bioavailability of copper to fungi degrading treated wood

Copper is ubiquitous as a biocide component in wood preservatives. Some fungi detoxify copper by immobilizing copper ions with oxalate, decreasing its physiological availability (bioavailability). Decreases in copper bioavailability may also occur during wood treatment. To date, however, copper retention in wood has been measured as overall weight-to-volume concentration without an estimate of its bioavailability and without assessment of its relative contribution to preservative efficacy. Here, we gauge the bioavailability of copper ions in treated wood by using oxalate to pre-treat wood prior to colonization by a moderately copper-tolerant fungus. Copper-treated wood was treated with a gradient of sodium oxalate concentrations, rinsed thoroughly, and exposed in soil-block jars to an isolate of Serpula himantioides. Wood treated with copper ethanolamine was extremely effective in preventing decay by S. himantioides, but toxicity could repeatedly be overcome above a threshold level of oxalate pretreatment. In agar plates, copper-treated wood stimulated oxalate production by S. himantioides, but levels were less than those needed (>10 mM) to overcome copper in soil-block jars. This capacity to overcome copper using an oxalate pretreatment was absent in commercially available samples treated with co-biocide(s). Results demonstrate a useful relative measure of copper bioavailability, with potential to be modified for specific quantification.     

A simple protocol to study blue copper proteins by NMR.

In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues. The proposed protocol differs from previous approaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form. This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiring a specific expertise in the field. The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.     

Phenols and antioxidative status of Raphanus sativus grown in copper excess

Raphanus sativus L. cv. Rimbo was grown for 10 days after emergence in hydroponic culture containing 0.12 (control), 5, 10 and 15 µ M copper. The seeds were germinated in the presence of the copper solution. The Cu contents increased with the treatment in both shoots and roots, maintaining in the roots a value eight- to ten-fold higher than in the shoots. With the treatment both shoots and roots underwent growth inhibition and an increase in the percentage of dry weight. Membrane damage and lipid peroxidation increased and glutathione was oxidized as the copper concentration increased, indicating an acceleration of oxidative processes. Control shoots had high contents of reduced glutathione and low contents of phytochelatin-SH whereas roots showed an opposite pattern, suggesting an utilization of reduced glutathione for phytochelatin synthesis. In both parts phytochelatin-SH content reached the maximum at 5 µ M copper and then decreased, reaching at 15 µ M copper the control value in the roots and a value five-fold higher than the control value in the shoots. The main phenolic acids represented in R. sativus were chlorogenic, vanillic, caffeic, siringic, p-coumaric and ferulic acids whereas the least represented were gallic, protocatechuic and p-hydroxybenzoic acids. The phenolic acids as well as the total and reduced ascorbate contents increased with the intensification of copper treatment. Notwithstanding these changes, total ascorbate remained 35% higher in the shoots than in the roots. The reduced ascorbate was thus able to replenish reducing equivalents to phenoxyl radicals thus explaining the increase in the phenolic compounds.     

Intensification of biosorption of copper ions from solution by the yeast Saccharomyces cerevisiae in magnetic field

We studied the possibility of intensification of biosorption of copper ions by the yeast Saccharomyces cerevisiae 1968 from a copper sulfate solution by immersing a metal 80-rod headpiece into the solution and applying an external magnetic field. The field was parallel or perpendicular to the axes of headpiece rods. It was shown that intensification of extraction of copper ions at various geometries of the system differed insignificantly and that copper ions were extracted from the solution via biosoprtion and cementation at the metal headpiece.     

Variations in the microbiome due to storage preservatives are not large enough to obscure variations due to factors such as host population,host species,body site,and captivity

The study of the primate microbiome is critical in understanding the role of the microbial community in the host organism. To be able to isolate the main factors responsible for the differences observed in microbiomes within and between individuals, confounding factors due to technical variations need to be removed. To determine whether alterations due to preservatives outweigh differences due to factors such as host population, host species, body site, and habitat, we tested three methods (no preservative, 96% ethanol, and RNAlater) for preserving wild chimpanzee (fecal), wild lemur (fecal), wild vervet monkey (rectal, oral, nasal, otic, vaginal, and penile), and captive vervet monkey (rectal) samples. All samples were stored below ? 20°C (short term) at the end of the field day and then at ? 80°C until DNA extraction. Using 16S rRNA gene sequencing, we show a significant preservative effect on microbiota composition and diversity. Samples stored in ethanol and RNAlater appear to be less different compared with samples not stored in any preservative (none). Our differential analysis revealed significantly higher amounts of Enterococcaceae and Family XI in no preservative samples, Prevotellaceae and Spirochaetaceae in ethanol and RNAlater preserved samples, Oligosphaeraceae in ethanol‐preserved samples, and Defluviitaleaceae in RNAlater preserved samples. While these preservative effects on the microbiome are not large enough to remove or outweigh the differences arising from biological factors (e.g., host species, body site, and habitat differences) they may promote misleading interpretations if they have large enough effect sizes compared to the biological factors (e.g., host population).     

湖北江汉平原农田生态系统钢的循环与平衡

对湖北江汉平原农田生态系统8种种植模式中Cu的输入、输出和平衡进行了分析。结果表明,农田Cu的输出主要是作物收获,其输出量占总输出量的85.9%-95.1%,流失和淋溶输出很少,农田Cu的输入主要是有机肥输入和降雨输入,其次是降尘、作物自然归还和灌溉,而种子种苗和无机肥输入占很少的比例。农田Cu的平衡分析表明,按照当地施肥习惯,菜田和水田Cu盈余较多,而旱地4种种植模式基本平衡。