Charge Repulsion Research Articles

Impact of operating conditions on chromatographic column performance: experimental studies on adsorption of high-value minor whey proteins

Over the last decade, ion-exchange chromatography (IEC) has been extensively explored for protein purification at both small and large scales. Despite several IEC columns are commercialized, the physical phenomena underlying the adsorption of proteins on ion-exchange columns performance has not been thoroughly investigated. In this work, the influence of operating conditions on the adsorption of lactoperoxidase (LP) and lactoferrin (LF) on cation exchange chromatography adsorbent is experimentally studied in order to understand fundamental pertaining to underlying mechanism. Analysis was carried out in columns with different IDs (7.7 and 16 mm), packed for 100 mm with 90 μm particle size polymer-grafted cation exchanger. The flow distribution was measured using acetone as a non-binding tracer. An evaluation of van Deemter plots was done as well as LP breakthrough curves at different flow rates and LP loading concentrations. The results were compared with two columns in terms of efficiency and the LP binding capacity. The dynamic binding capacity at 10% breakthrough was found to be independent of the applied flow rate. Surprisingly for both systems, LP breakthrough takes place later at higher loading concentrations, which is in contrast to IEC. The results propose a major presence of non-ideal effects as steric shielding and charge repulsion of protein in the adsorption. In addition, the accessibility of binding sites for protein at higher concentrations seems more available than sodium counter-ions in buffer.

Implications of humic acid, inorganic carbon and speciation on fluoride retention mechanisms in nanofiltration and reverse osmosis

The impact of pH and humic acid (HA) on the retention of fluoride (F) and inorganic carbon (IC) by nanofiltration (NF) and reverse osmosis (RO) membranes was determined. Synthetic waters were prepared using realistic ranges of F, IC and HA for carbonaceous waters found for example in the fluoride rich waters in Tanzania. These waters were filtered using NF270 and BW30 membranes to determine retention mechanisms.IC changes speciation with pH. The dominant species at pH<6, 6–10 and >10 are H2CO3, HCO3− and CO32− respectively. This results in changes in charge and size of the hydrated ion radius. The mechanism for IC retention by the NF270 and BW30 membranes are charge repulsion and size exclusion, respectively. F retention increases with pH. IC influenced F retention at pH>10 where IC exists as divalent CO32− and is retained more easily than the monovalent F−.HA enhances the retention of F by NF/RO membranes under certain conditions. The enhancement effect is more pronounced at neutral than at acidic pH and basic pH. The mechanism for the enhancement is attributed to the change in surface charge of the membranes by adsorption of HA. At high HA concentration the F retention enhancement is annulled by deposit formation on the membrane.The results obtained in this study indicate the complexity of retention in real surface- and ground waters that can alter significantly in pH as well as IC and HA content. The research is situated in the context of developing membrane technologies for autonomous systems in remote locations where water quality is variable and mechanisms of membrane performance are poorly understood.

Disruption of a putative intersubunit electrostatic bond enhances agonist efficacy at the human α1 glycine receptor

Partial agonists have lower efficacies than compounds considered ‘full agonists’, eliciting submaximal responses even at saturating concentrations. Taurine is a partial agonist at the glycine receptor (GlyR), a member of the cys-loop ligand-gated ion channel superfamily. The molecular mechanisms responsible for agonism are not fully understood but evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a residue located near the human α1 glycine binding site (aspartate-97; D97) that, when mutated to arginine (D97R), results in GlyR channels opening spontaneously with a high open probability, mimicking the effects of saturating glycine concentrations on wildtype GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit, stabilizing the channel in a shut state. Here we demonstrate that the disruption of this putative bond increases the efficacy of partial agonists including taurine, as well as two other β-amino acid partial agonists, β-aminobutyric acid (β-ABA) and β-aminoisobutyric acid (β-AIBA). Even the subtle charge-conserving mutation of D97 to glutamate (D97E) markedly affects partial agonist efficacy. Mutation to the neutral alanine residue in the D97A mutant mimics the effects seen with D97R, indicating that charge repulsion does not significantly affect these findings. Our findings suggest that the determination of efficacy following ligand binding to the glycine receptor may involve the disruption of an intersubunit electrostatic interaction occurring near the agonist binding site.

Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics

BackgroundCellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far. MethodsTo investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used. ResultsHDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation. ConclusionsTransition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH. General significanceThe results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells.

PH-Controlled Formation of a Stable β-Sheet and Amyloid-like Fibers from an Amphiphilic Peptide: The Importance of a Tailor-Made Binding Motif for Secondary Structure Formation.

The new amphiphilic peptide 1 is composed of alternating cyclohexyl side chains and guanidiniocarbonyl pyrrole (GCP) groups. In contrast to analogue 2, which contains lysine instead of the GCP groups and only exists as a random coil owing to charge repulsion, peptide 1 forms a stable β-sheet at neutral pH in aqueous medium. The weakly basic GCP groups (pKa ≈7) are key for secondary structure formation as they stabilize the β-sheet through mutual interactions (formation of a "GCP zipper"). The β-sheets further aggregate into left-handed helically twisted fibers. However, β-sheet formation is completely reversible as a function of pH. At low pH (ca. 4), peptide 1 is unstructured (random coil) as all GCP units are protonated. Only round colloidal particles are observed. The amyloid nature of the fibers formed at neutral pH was confirmed by staining experiments with Congo Red and thioflavin T. Furthermore, at millimolar concentrations, peptide 1 forms a stable hydrogel.

PH‐Controlled Formation of a Stable β‐Sheet and Amyloid‐like Fibers from an Amphiphilic Peptide: The Importance of a Tailor‐Made Binding Motif for Secondary Structure Formation

AbstractThe new amphiphilic peptide 1 is composed of alternating cyclohexyl side chains and guanidiniocarbonyl pyrrole (GCP) groups. In contrast to analogue 2, which contains lysine instead of the GCP groups and only exists as a random coil owing to charge repulsion, peptide 1 forms a stable β‐sheet at neutral pH in aqueous medium. The weakly basic GCP groups (pKa≈7) are key for secondary structure formation as they stabilize the β‐sheet through mutual interactions (formation of a “GCP zipper”). The β‐sheets further aggregate into left‐handed helically twisted fibers. However, β‐sheet formation is completely reversible as a function of pH. At low pH (ca. 4), peptide 1 is unstructured (random coil) as all GCP units are protonated. Only round colloidal particles are observed. The amyloid nature of the fibers formed at neutral pH was confirmed by staining experiments with Congo Red and thioflavin T. Furthermore, at millimolar concentrations, peptide 1 forms a stable hydrogel.

Binary Fe and Mn Oxide Nanoparticle Supported Polymeric Anion Exchanger for Arsenic Adsorption: Role of Oxides, Supported Materials, and Preparation Solvent

In this work, binary Fe/Mn oxide nanoparticles were incorporated onto the matrix of anion exchange resin, resulting in hybrid polymeric/inorganic nanoadsorbent named as A502P-Fe/Mn. During synthesis process, effects of various types of metal oxides, preparation solvent, supporting materials, and loading cycles were also investigated. To reduce the charge repulsion force between cationic Fe3+ and Mn4+ ions and fixed-positively charged quaternary amine (R4N+) functional groups of the anion exchange support, mixed solution containing DI/ethanol was introduced to dissolve metal salts during the preparation process. The data obtained by equilibrium batch test indicated that the A502P-Fe/Mn prepared from mixed 50:50 of DI and ethanol exhibited the highest As (V) sorption capacity. The synthesized material was further characterized by using scanning electron microscope (SEM) equipped with energy dispersive X ray spectroscopy (EDX) to verify the existence and distribution of elemental Fe, Mn, and As inside the polymeric beads. Equilibrium As (V) sorption isotherm, effect of solution pH, and point of zero charge of material were also evaluated. This A502P-Fe/Mn can have a promising potential for arsenic removal applications.

Use of electrospinning to directly fabricate three-dimensional nanofiber stacks of cellulose acetate under high relative humidity condition

Unique structure-controllable three-dimensional (3D) nanofiber stacks of cellulose acetate (CA) were fabricated successfully by simply increasing relative humidity (RH) during the electrospinning process. It is found that once the RH exceeding 60 %, 3D flocculent nanofiber stacks would grow on the flat plate collector toward the needle tip without using special assisting apparatus or preparing special electrospinning solution. Compared with those obtained at low RH, the as-prepared nanofibers fabricated under high RH condition exhibited similar nanofiber diameter, density and porosity, and more importantly, 3D flocculent structures instead of typical two-dimensional (2D) electrospun non-woven mats, which would contribute to a significant improvement on the hydrophilicity. It is believed that rapid solidification of CA during the jet process and strong charge repulsion among CA nanofibers play important roles in the formation of 3D nanofibrous structure. Furthermore, these 3D flocculent nanofiber scaffolds exhibited better cytocompatibilities with human MG-63 cells than common 2D nanofibrous mats. Thus a facile and effective approach was presented to prepare 3D nanofiber stacks with tunable and reproducible properties for biodegradable scaffold applications.

Recognition-Mediated Hydrogel Swelling Controlled by Interaction with a Negative Thermoresponsive LCST Polymer.

Most polymeric thermoresponsive hydrogels contract upon heating beyond the lower critical solution temperature (LCST) of the polymers used. Herein, we report a supramolecular hydrogel system that shows the opposite temperature dependence. When the non‐thermosesponsive hydrogel NaphtGel, containing dialkoxynaphthalene guest molecules, becomes complexed with the tetra cationic macrocyclic host CBPQT4+, swelling occurred as a result of host–guest complex formation leading to charge repulsion between the host units, as well as an osmotic contribution of chloride counter‐ions embedded in the network. The immersion of NaphtGel in a solution of poly(N‐isopropylacrylamide) with tetrathiafulvalene (TTF) end groups complexed with CBPQT4+ induced positive thermoresponsive behaviour. The LCST‐induced dethreading of the polymer‐based pseudorotaxane upon heating led to transfer of the CBPQT4+ host and a concomitant swelling of NaphtGel. Subsequent cooling led to reformation of the TTF‐based host–guest complexes in solution and contraction of the hydrogel.

Recognition‐Mediated Hydrogel Swelling Controlled by Interaction with a Negative Thermoresponsive LCST Polymer

AbstractMost polymeric thermoresponsive hydrogels contract upon heating beyond the lower critical solution temperature (LCST) of the polymers used. Herein, we report a supramolecular hydrogel system that shows the opposite temperature dependence. When the non‐thermosesponsive hydrogel NaphtGel, containing dialkoxynaphthalene guest molecules, becomes complexed with the tetra cationic macrocyclic host CBPQT4+, swelling occurred as a result of host–guest complex formation leading to charge repulsion between the host units, as well as an osmotic contribution of chloride counter‐ions embedded in the network. The immersion of NaphtGel in a solution of poly(N‐isopropylacrylamide) with tetrathiafulvalene (TTF) end groups complexed with CBPQT4+ induced positive thermoresponsive behaviour. The LCST‐induced dethreading of the polymer‐based pseudorotaxane upon heating led to transfer of the CBPQT4+ host and a concomitant swelling of NaphtGel. Subsequent cooling led to reformation of the TTF‐based host–guest complexes in solution and contraction of the hydrogel.

Separation of xylose and glucose using an integrated membrane system for enzymatic cofactor regeneration and downstream purification

Mixtures of xylose, glucose and pyruvate were fed to a membrane bioreactor equipped with a charged NF membrane (NTR 7450). Value-added products were obtained in the reactor via enzymatic cofactor-dependent catalysis of glucose to gluconic acid and pyruvate to lactic acid, respectively. The initial cofactor (NADH) concentration could be decreased to 10% of the stoichiometric value (relative to glucose) without compromising process time and substrate conversion via i) efficient cofactor regeneration and ii) high retention of cofactor (R=0.98) in the membrane bioreactor. Furthermore, accumulation of xylose (R<0.1), lactic acid (R=0.38) and gluconic acid (R=0.63) was minimized. After separation of the cofactor in the membrane bioreactor, xylose, lactic acid and gluconic acid were separated based on charge repulsion and size exclusion in a sequence of two NF steps. Broad substrate specificity of glucose dehydrogenase (EC 1.1.1.47) (GDH) lead to partial conversion of xylose to xylonic acid, causing some loss of xylose, but the results obtained nevertheless showed that it is possible to build a robust system for conducting enzyme reactions by sequentially regenerating the cofactor and at the same time obtaining valuable products of high purity.

Rapid, Surfactant-Free, and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA under Physiological pH and Its Application in Molecular Beacon-Based Biosensor.

The controlled attachment of thiolated DNA to gold nanoparticles (AuNPs) dictates many applications. This is typically achieved by either "aging-salting" processes or low-pH method, where either Na+ or H+ is used to minimize charge repulsion and facilitate attachment of thiolated DNA onto AuNPs. However, the "aging-salting" process takes a long time, and is prone to aggregation when used with larger AuNPs. Surfactants are needed to precoat and thereby enhance the stability of AuNPs. The low-pH method can disrupt the structural integrity of DNAs. We report here an oligoethylene glycol (OEG) spacer-assisted method that enables quantitative and instantaneous attachment at physiological pH without the need for surfactants. The method is based on our finding that an uncharged OEG spacer as short as six EG units can effectively shield against repulsion between AuNPs and DNAs, substantially enhancing both the adsorption kinetics and thermodynamics of thiolated DNAs. We applied this to thiolated DNAs of various lengths and thiol modification positions and to large AuNPs. Importantly, our method also allows for the direct immobilization of thiolated molecular beacons (MB), and avoids particle aggregation due to intermolecular hydrogen bonding. The prepared MB-AuNPs were successfully used for the fluorescent detection of target DNA at nanomolar concentrations. The OEG spacer appears to offer a highly effective parameter for tuning DNA adsorption kinetics and thermodynamics besides pH and salt, providing a novel means for highly controllable and versatile functionalization of AuNPs.

PH-controlled doxorubicin anticancer loading and release from carbon nanotube noncovalently modified by chitosan: MD simulations

In the present study, we describe here the pH condition activating doxorubicin (DOX) anticancer drugs loading and release over single-wall carbon nanotube (SWNT) non-covalently wrapped with chitosan (CS). The possibility of drug displacement on DOX/CS/SWNT nanocarrier was investigated using molecular dynamics simulations. The drug loading and release were monitored via displacement analysis and binding energy calculations. The simulated results clearly showed that the drugs well interacted with the CS/SWNT at physiological pH (pH 7.4), where CS was in the deprotonated form. Contrastingly, in weakly acidic environments (pH 5.0–6.5) which is a pH characteristics of certain cancer environments, the protonated CS became loosen wrapped around the SWNT and triggered drugs release as a result of charge–charge repulsion between CS and drug molecules. The obtained data fulfil the understanding at atomic level of drug loading and release controlled by pH-sensitive polymer, which might be useful for further cancer therapy researches.

Cyclic Voltammetry Studies of a Redox-Responsive 4 H-Bond Ureidopyrimidinone Alkyl-Pyridinium Capable of Self-Dimerization

The design of stimuli-responsive systems in which supramolecular structure changes in response to external signals, such as the change in voltage of an electrode, is important for many applications, for example, self-healing polymers and gels, triggered release of entrapped molecules for drug delivery, and “smart” materials. In this study, a four H-bond ureidopyrimidinone (UPy) array with an alkyl-pyridinium, (RP), redox center has been synthesized, UPy(RP), shown below. This array prefers the tautomer that presents an ADAD H-bond motif in the starting oxidation state. Due to electrostatic repulsions and unfavorable secondary H-bond interactions, this motif would form a dimer with relatively weak H-bonding. Upon 2e- reduction, where 1e-is gained per R-pyridinium redox center, the H-bond strength should increase due to the loss of the repulsive charges, making the nitrogen a stronger hydrogen acceptor. Because the nitrogen is now a better hydrogen acceptor, there could be a possibility of an intermolecular proton transfer. This would encourage the tautomer to have an AADD motif that will make the H-bonding stronger by increasing the favorable secondary H-bond interactions. UPy dimers can exist in two tautomeric forms called pyrimidinol and pyrimidinone. In order to more efficiently study the electrochemistry of the two forms, two compounds, 4-acetylpyridinium (AcP) and N-methyl-4,4’-bipyridinium (MeV+), were used as model compounds. AcP resembles the pyrimidinone tautomer and MeV+ resembles the pyrimidinol tautomer. The cyclic voltammetry (CV) scans for the two model compounds in CH2Cl2 and CH3CN show two widely spaced, reversible redox waves. UPy(RP) is insoluble in CH2Cl2, but soluble in CH3CN. The concentration dependent NMR spectra suggest that UPy(RP) is a monomer in acetonitrile. The CV’s of UPy(RP) in CH3CN show three reductions. The first occurs at a potential very similar to that seen with simple model compounds. The second is considerably positive of that observed for the model compounds and the third is very close to the second reduction peak of the model compounds. The first reduction is reversible if the scan direction is switched immediately after the peak, but irreversible if the scan direction is switched after the second reduction peak. As the scan rate increases, the second and the third reduction peaks disappear and a new peak emerges in between. Going through the second and third reductions also leads to the appearance of new oxidation waves at more positive potentials. DFT calculations suggest the preferred tautomer changes upon reduction, and this may explain the unexpected voltammetry observed in acetonitrile where the compound is not dimerized. Further studies to help elucidate what is actually happening in this system will include using a new counter anion in the electrolyte to help the solubility of the compound in CH2Cl2. Figure 1

Proton conductive and low methanol permeable PVA-based zwitterionic membranes

A desirable membrane for direct methanol fuel cell (DMFCs) must both conduct protons and function as a methanol barrier. However, it remains challenge to balance the proton conduction and methanol permeability. As a commonly used pervaporation membrane, PVA has high selectivity for water over methanol, but its proton conductivity is too low for DMFCs applications. To improve conductivity, efforts have been predominantly focused on direct incorporation of strongly acidic groups, such as sulfonated or phosphoric groups on the PVA chain. This strategy can result in improvement in the proton conduction. However, the performance of acid modified PVA is compromised due to the strong charge–charge repulsion of attached groups, which results in the increased fraction free volume that facilitates methanol permeation. We attempt to minimize the tradeoff between proton conduction and methanol permeability by modifying PVA with zwitterionic groups. This is achieved by functionalizing PVA with pendent zwitterionic groups (PVA-ZW-n) at controllable substitution degree (5 < n < 20). The investigation of their 1H NMR spectra and glass transition temperatures (Tg) suggests that there is strong electrostatic interaction in the PVA-ZW-20 polymer, as confirmed by the observed proton spatial distribution in NMR, and increased Tg along with the increase of substitution degree in DSC. The synthesized PVA-ZW-20 can be easily casted into mechanically stable membranes with modest proton conductivity. After incorporation with (3-Mercaptopropyl)trimethoxysilane (MPTMS) and successive oxidation, the synthesized PVA-ZW-20-Si exhibits lower methanol permeability and reasonably higher proton conductivity compared to Nafion 117. A series of control experiments revealed that the superior performance of PVA-ZW-20-Si could be related to the presence of zwitterionic groups, which result in enhanced packing density of polymer chain and more efficient proton dissociation.

Topological phases and transport properties of screened interacting quantum wires

We study theoretically the effects of long-range and on-site Coulomb interactions on the topological phases and transport properties of spin–orbit-coupled quasi-one-dimensional quantum wires imposed on a s-wave superconductor. The distributions of the electrostatic potential and charge density are calculated self-consistently within the Hartree approximation. Due to the finite width of the wires and charge repulsion, the potential and density distribute inhomogeneously in the transverse direction and tend to accumulate along the lateral edges where the hard-wall confinement is assumed. This result has profound effects on the topological phases and the differential conductance of the interacting quantum wires and their hybrid junctions with superconductors. Coulomb interactions renormalize the gate voltage and alter the topological phases strongly by enhancing the topological regimes and producing jagged boundaries. Moreover, the multicritical points connecting different topological phases are modified remarkably in striking contrast to the predictions of the two-band model. We further suggest the possible non-magnetic topological phase transitions manipulated externally with the aid of long-range interactions. Finally, the transport properties of normal–superconductor junctions are further examined, in particular, the impacts of Coulomb interactions on the zero-bias peaks related to the Majorana fermions and near zero-energy peaks.

Development of a positively charged nanofiltration membrane for use in organic solvents

In this work, a positively charged nanofiltration membrane was developed by chemical modification of a P84 copolyimide ultrafiltration membrane using branched polyethylenimine. The effect of reaction temperature on membrane morphology, surface charge, and separation performance was investigated. A reaction temperature of 70°C was optimal as the membrane showed best performance in terms of salt rejections and water fluxes when Na2SO4, NaCl and CaCl2 aqueous solutions were used as feeds. Such membrane had a molecular weight cut off (MWCO) of 226Da indicating its effectiveness for separation of organics. Rejections to three dyes in methanol solutions were in the order of methyl orange (negative, MW: 327Da)<disperse red (neutral, MW: 314Da)<safranine O (positive, MW: 351Da). The results suggest that the positive charge on membrane surface induce higher rejection to positive organic molecules due to the charge repulsion effect. Additionally, the membrane had very high stability in organic solvents; no significant changes in membrane performance and membrane structure were observed after being immersed in dimethyl formamide (DMF) for 1 month.

Tuning the backbones and side chains of cationic meta-linked poly(phenylene ethynylene)s: Different conformational modes, tunable light emission, and helical wrapping of multi-walled carbon nanotubes

We have an ongoing interest in the design and control of helical conformation of water-soluble meta-linked poly(phenylene ethynylene)s (PPEs) and the further development of novel functional materials. Four cationic meta-linked PPEs (P1′-P4′) were synthesized to study the influence of differences in the backbone and side chain structure on their conformations. For P1′ and P4′ without side chains on the para-phenylene units, fluorescence spectroscopic investigations indicated obvious intramolecular helical folding, whereas for P2′ and P3′ with nonpolar and polar side chains on the para-phenylene units respectively, a significantly different conformational mode, namely, cofacial intermolecular aggregation was suggested. The side chains on the para-phenylene units of P2′ and P3′ may be located in the interior cavity of helix and induce steric effect on the formation of helix. However, the introduction of 2,1,3-benzothiadiazole (BT), a low energy gap unit, into the backbone of P4′ showed little influence on the formation of helix despite its larger and more rigid structure than the phenylene unit. Thus, the light emission of these polymers can be tuned in the range from blue, green to yellow with the changes of conformational modes. Moreover, the functionalization of multi-walled carbon nanotubes (MWCNTs) by P4′ in methanol and water, and by using its neutral precursory polymer P4 in tetrahydrofuran was explored through transmission electron microscopy and fluorescence spectroscopy. P4′ was directly observed to individualize MWCNT by forming a monolayer helical wrapping on the nanotube surface, which may be attributed to the backbone flexibility of meta-linked PPE and the strong π−π interactions between the PPE backbone and the CNT surface. Moreover, P4′ served as a better dispersing agent for MWCNT than P4, suggesting that the cationic side groups may act as solubilizing groups which also separated the individual nanotubes because of charge repulsion.

Effect of tetrasodium pyrophosphate concentration and cooking time on the physicochemical properties of process cheese

Tetrasodium pyrophosphate (TSPP) is widely used as an emulsifying salt (ES) in process cheese. Previous reports have indicated that TSPP exhibits some unusual properties, including the gelation of milk proteins at specific ES concentrations. We studied the effect of various concentrations (0.25–2.75%) of TSPP and cooking times (0–20min) on the rheological, textural, and physical properties of pasteurized process Cheddar cheese using a central composite rotatable experimental design. Cheeses were made with a constant pH value to avoid pH as a confounding factor. Modeling of the textural properties of process cheese made with TSPP exhibited complex behavior, with polynomial models (cubic) giving better predictions (higher coefficient of determination values) than simpler quadratic models. Meltability indices (degree of flow from the UW MeltProfiler (University of Wisconsin–Madison), loss tangent value at 60°C from rheological testing, and Schreiber melt area) initially decreased with increasing TSPP concentrations, but above a critical ES concentration (~1.0%) meltability increased at higher TSPP concentrations. The storage modulus values measured at 70°C for process cheese initially increased with increasing TSPP concentration, but above a concentration of 1% ES, the storage modulus values decreased. Cooking time had little effect on the various melting or rheological properties. With an increase in TSPP concentration, the insoluble Ca and P contents increased, suggesting that TSPP addition resulted in the formation of insoluble calcium pyrophosphate complexes; some of which were likely associated with caseins. A portion of the added TSPP remained in the soluble phase. The acid-base buffering profiles also indicated that calcium pyrophosphate complexes were formed in cheese made with TSPP. In milk systems, low levels of TSPP have been shown to induce protein crosslinking and gelation, whereas at higher TSPP concentrations milk gelation was inhibited due to excessive charge repulsion from these calcium pyrophosphate complexes. We hypothesized that a similar phenomenon was occurring in our process cheese, resulting in the initial reduction in meltability with TSPP addition due to protein crosslinking, but at higher TSPP levels meltability increased due to excessive charge repulsion.

Quantitative Label-Free Listeria Analysis Based On Aptamer Modified Nanoporous Sensor

In this Letter, we proposed a nanoporous sensor to realize a quick and sensitive detection of Listeria monocytogenes with the application of porous anodic aluminum oxide membrane combined with a specific aptamer. On the basis of electrochemical detection, it can be known that the reduction current response upon the diffusion flux of electrochemical probe is affected by the charge repulsion and steric blockage of Listeria monocytogenes. A rapid bacteria detection can be achieved within 10 min, with a high sensitivity of 100 CFU/mL and a good linear range between 100 to1250 CFU/mL. Moreover, it has a high specificity for Listeria monocytogenes even exposed in 108 CFU/mL Staphylococcus aureus or Escherichia coli.