The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10(4) cells were 4.66 at 30 degrees C, 4.36 at 10 degrees C and 4.19 at 5 degrees C. These limits were salt dependent, low (4-6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4.66 at 30 degrees C but this was increased to 4.83 at 10 degrees C. At 5 degrees C growth occurred at pH 7.0 but not at pH 5.13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10 degrees C and 30 degrees C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19

The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence-based method for assaying CIRV p19:siRNA binding using Ni(2+)-NTA coated 96-well plates. Using this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent pK(a) values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6.     

The influence of pH on the G-quadruplex binding selectivity of perylene derivatives

Three new perylene derivatives with branched ionizable side chains were synthesized, and their G-quadruplex binding specificities were compared by spectroscopic and electrophoretic analysis with two well-studied G-quadruplex ligands: PIPER and TmPyP4. The value of pH and consequent charge formation and self-aggregation of these perylene derivatives influences not only the type of G-quadruplex formation, but also the G-quadruplex binding selectivity.     

The influence of salt concentration on CD spectra of tRNA and aminoacyl-tRNA synthetase

The CD spectra of serine tRNA or seryl-tRNA synthetase were measured. The [theta] values at 210 nm were minimum at 50mM - 0.2M NaCl, at that concentration the velocity of aminoacylation was maximum. This results suggest that A . U and G . C base pairs loosened. The [theta] values at 200 nm decreased according to the decreasing of salt concentration, suggesting the decomposition of A . U base pairs. The CD spectra of seryl-tRNA synthetase at 210-240 nm were not changed in the range of 10mM-0.3M NaCl but the spectra at 260-290 nm showed minimum in the range between 50mM-0.2M NaCl. These results suggest that the influence of salt concentration on the velocity of aminoacylation depends on both the conformational changes of tRNA and seryl-tRNA synthetase.     

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

The chemistry of human transcortin. The effects of pH, urea, salt, and temperature on the binding of cortisol and progesterone.

The interaction of cortisol and progesterone with pure transcortin was investigated. The temperature dependence of cortisol and progesterone binding is a result of the predominantly negative enthalpy of binding which suggests a very good fit between ligand and protein such that the bonds formed are of the van der Waals type. The optimal pH of cortisol (8.0) and progesterone (8.5) binding suggests involvement of cysteine, histidine, and/or tyrosine residues in the binding process. Transcortin is irreversibly denatured at pH 4.0. The effect of sodium chloride on the binding of both steroids is small. At lower sodium chloride concentrations (less than 0.15 m), binding decreases somewhat with decreasing salt concentration. Urea produces a progressive decrease in the association constants of both steroids which is completely reversible up to 2.0 m and 30% reversible at 3.0 m. Scatchard analysis of cortisol binding in the presence of a constant amount of progesterone and vice versa confirms earlier data obtained on plasma that cortisol and progesterone do not bind at two independent sites. It is not possible, however, to decide whether they bind at the same site or at two interdependent (interacting) sites.     

The effect of changes in salt concentration and pH on the interaction between glycosaminoglycans and cationic polypeptides.

Circular dichroism (CD) spectroscopy has been used to investigate the effects of changes in salt concentration and pH on the interactions between basic polypeptides and connective tissue glycosaminoglycans in dilute aqueous solution. The polypeptides undergo conformation-directing interactions in the presence of glycosaminoglycans, which are subject to transitions as the ionic strength and pH are varied. For poly(L -lysine), the conformational change due to interaction breaks down as the ionic strength (monovalent ions) is increased. Based on the ionic strength at which disruption occurs, the glycosaminoglycans can be placed in order of increasing strength of interaction: chondroitin 6-sulfate, hyaluronic acid, chondroitin 4-sulfate, heparin, and dermatan sulfate. Prior to the conformational transition, scattering effects are observed, indicating the development of larger aggregates. Each glycosaminoglycan induces α-helicity for poly(L -arginine), which does not break down as the ionic strength is increased, indicating a stronger interaction for this polypeptide. The pH-induced transitions are in the pH range 2.5–3.8 and are probably related to deionization of carboxyl groups. For poly(L -lysine) the conformational effect is disrupted at low pH. For poly(L -arginine), the transitions are not complete, but appear to correspond to an increase in scattering.     

The effect of salt concentration and pH on the heat resistance of Escherichia coli in tryptic soy broth

The effect of the acid and the osmotic stress on the heat resistance of Escherichia coli (EC1 and EC2) was studied at 63 degrees C in tryptic soy broth adjusted to various pHs (2.5, 4.5 and 6) and various NaCl concentrations (2, 4 and 8%). In the second study, the effect of pretreatment on thermotolerance of E. coli cells was determined. The heat resistance of both strains was low at pH 2.5, but strain EC1 was more resistant than strain EC2. On the contrary, the heat resistance increased with increasing the pH values. Addition of NaCl (2%) to TSB medium, was involved in the protection of cells against heat inactivation, this protective effect was, however, not observed by increasing the NaCl concentration up to 8%. The combined effect of the pH and NaCl on the thermal resistance of both strains was significantly lower at pH 2.5 and NaCl 8%, the number of viable cells decreased from approximately 10(8) CFU/ml to an undetectable number within 20 min for strain EC1 and 15 min for strain EC2, respectively. This study indicates that heat resistance of strain EC1 was enhanced after acid or thermal adaptation. Heat resistance of strain EC2 was, however, enhanced only after thermal adaptation. For both strains no relationship was found between salt adaptation and the ability to resist thermal stress.     

Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor

The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-alphaMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 degrees C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.     

Vibrio anguillarum: influence of temperature, pH, NaCl concentration and incubation time on growth

An analysis of the effect of growth conditions on the growth (O.D. values) of five Vibrio anguillarum strains showed that the optima were as follows: pH 7, temperature 25 degrees C, NaCl concentration 2%, and O.D. estimates increased with the incubation time. The independent parameters, as well as their interactions significantly influenced the growth of Vibrio anguillarum (P less than 0.0001). Only the strain-salinity interaction was not always statistically significant. A restriction of the parameters to a level relevant for Danish marine recipients showed that pH and NaCl concentration (salinity) might be of minor importance while the temperature was always of high significance. The possible impact of these observations on local conditions is discussed.     

Effects of pH and salt concentration on the formation and properties of chitosan-cellulose nanocrystal polyelectrolyte-macroion complexes

This study examines the effects of pH and salt concentration on the formation and properties of chitosan-cellulose nanocrystal (CNC) polyelectrolyte-macroion complexes (PMCs). The components' pK values, determined by potentiometric titration, were 6.40 for chitosan and 2.46 for the CNCs. The turbidity of PMC particle suspensions was measured as a function of chitosan-CNC ratio, pH, and salt concentration. The maximum turbidity values in titrations of a chitosan solution with a CNC suspension and vice versa occurred at charge ratios of 0.47 ± 0.11 (SO(3)(-)/NH(3)(+)) and 1.16 ± 0.06 (NH(3)(+)/SO(3)(-)), respectively. A pH increase caused a turbidity decrease due to shrinking of the PMC particles upon changes in their components' degrees of ionization. An increase in salt concentration caused a decrease in turbidity due to charge-screening-related shrinking of the PMC particles. The effects of pH and salt concentration on particle size were confirmed by scanning electron microscopy.     

Effects of temperature, pH, and salt concentration on beta-lactoglobulin deposition kinetics studied by optical waveguide lightmode spectroscopy

Deposition kinetics of beta-lactoglobulin at a solid-liquid interface was studied with optical waveguide lightmode spectroscopy (OWLS) over a range of temperatures between 61 and 83 degrees C. A new temperature-controlled cell for OWLS measurements allows fast, on-line monitoring of the deposit formation at elevated temperatures. Primary protein layers were deposited at 25 degrees C in order to precondition and stabilize the waveguide surface. Sustained deposition lasting from a few minutes (around 80 degrees C) to hours (below 70 degrees C) resulted in multilayer deposits up to several tens of nanometers thick. The measured deposition rates were strongly influenced by temperature, pH, and NaCl concentration. Deposition rates decreased with increasing pH from 5.5. to 7.4, in a trend similar to that for noncovalent aggregation of beta-lactoglobulin in solution. Activation energies for deposition rates decreased with increasing pH, from 340 kJ/mol at pH 5.5 to 230 kJ/mol at pH 7.4 and were similar to the activation energies for denaturation of beta-lactoglobulin in solution.     

Influence of pH and salt concentration on the cross-flow microfiltration of BSA through a ceramic membrane

In this paper, the influence of pH in the 4–8 interval and NaCl concentration up to 25 mM on the cross-flow microfiltration of BSA was investigated. A tubular ceramic membrane with a pore size of 0.14 μm was employed and its point of zero charge was calculated. The evolution of permeate flow and BSA transmission with time was determined at 45 °C, a cross-flow velocity of 3.5 m/s and a transmembrane pressure of 100 kPa. The curves of permeate flow were explained according to the resistances in series model. Maximum protein transmission was obtained at the isoelectric point of BSA (4.9), with significant transmission also at the point of zero charge of the membrane and null transmission at pH 4 and 8. The highest permeate flow was observed at pH 7 and the lowest at 4.9. Finally, the addition of salt resulted to some extent in an improvement of both protein transmission and permeate flow.     

The influence of serum proteins and pH on the effective amino acid concentration in tissue culture medium

Amino acid binding by calf serum proteins in Dulbecco's modified Eagle medium has been studied by an ultrafiltration method. For some of the amino acids studied, up to 20 % may be unavailable to the cell because of non-specific, reversible binding to serum proteins. The extent of binding varies over the pH range ordinarily encountered during cell growth. A method is presented by which the degree of binding may be determined. The greatest degree of binding is noted for tryptophan, arginine and lysine.     

The influence of calcium ions on fibrin polymerization

The influence of calcium ions on the polymerization induced in fibrinogen solutions by thrombin and by Reptilase has been investigated by meansof static and dynamic light scattering in combination with measurements of the release of the fibrinopeptide A. The calcium concentration was varied in the range between 0.3 and 103 calcium ions per fibrinogen molecule. The enzyme concentration was chosen sufficiently low so that it was possible to make quantitative observations as a function of time, in particular, beforethe onset of gelation. Likewise, the influence of calcium ions on the enzymatically induced polymerization of fragment X was studied. The results indicate that there are at least three mechanisms by which calcium can influence the evolution of the polymer system on the path to gelation and clotting. Which mechanism dominates depends upon the calcium concentration.