Heat-induced aggregation kinetics of potato protein - Investigated by chromatography, calorimetry, and light scattering.

In this study, the heat-induced aggregation behavior of patatin rich potato protein isolate (PPI) was investigated by reversed-phase high-pressure liquid chromatography (RP-HPLC), differential scanning calorimetry (DSC), and dynamic light scattering. It could be shown that aggregation already occurs at low temperatures, despite low degrees of unfolding. The unfolding temperature, determined by DSC, coincided with a change in the reaction kinetics, which is determined by the unfolding step below a critical temperature up to the point, where the proteins are completely unfolded. The reaction rate k as a function of the absolute temperature T is then determined by diffusion of unfolded proteins forming aggregates. This change can be visualized in the Arrhenius diagram by a change of the slope of the relationship k âˆ¼ 1/T. A change in pH from 7 to 6 shifted the critical temperature towards higher values and resulted in larger aggregate sizes, due to reduced electrostatic repulsion.

Influence of pH, Temperature and Protease Inhibitors on Kinetics and Mechanism of Thermally Induced Aggregation of Potato Proteins.

Understanding aggregation in food protein systems is essential to control processes ranging from the stabilization of colloidal dispersions to the formation of macroscopic gels. Patatin rich potato protein isolates (PPI) have promising techno-functionality as alternatives to established proteins from egg white or milk. In this work, the influence of pH and temperature on the kinetics of PPI denaturation and aggregation was investigated as an option for targeted functionalization. At a slightly acidic pH, rates of denaturation and aggregation of the globular patatin in PPI were fast. These aggregates were shown to possess a low amount of disulfide bonds and a high amount of exposed hydrophobic amino acids (S0). Gradually increasing the pH slowed down the rate of denaturation and aggregation and alkaline pH levels led to an increased formation of disulfide bonds within these aggregates, whereas S0 was reduced. Aggregation below denaturation temperature (Td) favored aggregation driven by disulfide bridge formation. Aggregation above Td led to fast unfolding, and initial aggregation was less determined by disulfide bridge formation. Inter-molecular disulfide formation occurred during extended heating times. Blocking different protein interactions revealed that the formation of disulfide bond linked aggregation is preceded by the formation of non-covalent bonds. Overall, the results help to control the kinetics, morphology, and interactions of potato protein aggregation for potential applications in food systems.

Alteration of Intestinal Microbiome of Clostridioides difficile-Infected Hamsters during the Treatment with Specific Cow Antibodies.

Clostridioides difficile infection (CDI) often develops after pretreatment with antibiotics, which can lead to damage of the intestinal microbiome. The approach of this study was to use specific polyclonal antibodies isolated from the milk of immunized cows to treat CDI, in contrast to the standard application of nonspecific antibiotics. To gain a deeper understanding of the role of the microbiome in the treatment of CDI with bovine antibodies, stool and intestinal fluid samples of hamsters were collected in large quantities from various treatments (>400 samples). The results show that the regeneration of the microbiome instantly begins with the start of the antibody treatment, in contrast to the Vancomycin-treated group where the diversity decreased significantly during the treatment duration. All antibody-treated hamsters that survived the initial phase also survived the entire study period. The results also show that the regeneration of the microbiome was not an antibody-induced regeneration, but a natural regeneration that occurred because no microbiota-inactivating substances were administered. In conclusion, the treatment with bovine antibodies is a functional therapy for both the acute treatment and the prevention of recurrence in hamsters and could meet the urgent need for CDI treatment alternatives in humans.

Correlation between Physico-Chemical Characteristics of Particulated ß-Lactoglobulin and Its Behavior at Air/Water and Oil/Water Interfaces.

It is widely accepted that protein-based particles can efficiently stabilize foams and emulsions. However, it is not fully elucidated which particle properties are decisive for the stabilization of air/water and oil/water interfaces. To unravel this correlation, selected properties of nano-sized soluble ß-lactoglobulin particles were changed one at a time. Therefore, particles of (1) variable size but similar zeta potential and degree of cross-linking and (2) similar size but different further properties were produced by heat treatment under a specific combination of pH value and NaCl concentration and then analyzed for their interfacial behavior as well as foaming and emulsifying properties. On the one hand, it was found that the initial phase of protein adsorption at both the air/water and the oil/water interface was mainly influenced by the zeta potential, independent of the particle size. On the other hand, foam stability as resolved from the time-dependent evolution of mean bubble area negatively correlated with disulfide cross-linking, whereas emulsion stability in terms of oil droplet flocculation showed a positive correlation with disulfide cross-linking. In addition, flocculation was more pronounced for larger particles. Concluding from this, foam and emulsion stability are not linked to the same particle properties and, thus, explanatory approaches cannot be used interchangeably.

RP-HPLC method for simultaneous quantification of free and total thiol groups in native and heat aggregated whey proteins.

Disulfide formation of whey proteins during heat treatment via thiol oxidation is important with regard to techno-functional properties. Due to the formation of other oxidation products than disulfides, the decrease in free thiol concentration is not proportional to the disulfide formation. Thus, in order to evaluate thiol reactivity and disulfide concentration both parameters are required. Currently applied methods focus mainly on the loss of free thiols using the spectrophotometric Ellman's assay. Next to that, we improved an existing RP-HPLC assay using the thiol reagent 4,4'-Dithiodipyridine (DTDP) to quantify free thiols as well as total (free thiols and disulfide bonds) thiols of native and heat-treated whey proteins. Thereby, the sample preparation technique, the sample handling, and the analysis technique were optimized. Thus, the paper provides a simple RP-HPLC method for quantification of thiol oxidation reactions to determine heat-induced changes in the structure of whey proteins. In addition, the method should be applicable to other protein systems due to the method validation by proteins of different amounts of free and total thiols in their structure.•Simple RP-HPLC method for quantification of free and total thiols using 4,4'-Dithiodipyridine (DTDP).•High recovery rates for free and total thiols.•High stability within 24 h.

Comparison of the influence of pH on the selectivity of free and immobilized trypsin for ß-lactoglobulin hydrolysis.

Although immobilized trypsin is a viable alternative to the free one in solution for producing protein hydrolysates, the change of selectivity introduced by immobilization is unclear. In this study, we compared the selectivity of free and immobilized trypsin towards different cleavage sites of ß-lactoglobulin (ß-Lg) with a focus on the impact of environmental pH. Both free and immobilized trypsin exhibited greater accessibility to native ß-Lg at elevated pH (from pH 7.2 to 8.7). Additionally, free trypsin preferred to attack cleavage sites located at the C-terminus at pH 7.8, whereas an opposite preference for the N-terminus was observed at pH 8.7. Regarding the immobilized trypsin, the pH did not significantly influence its preference for the C- or N-terminus. Generally, immobilization of trypsin resulted in more focused cleavage within its specificity during the initial stage of hydrolysis, and some peptides were formed more rapidly by the immobilized trypsin.

Mechanical Properties of ZTO, ITO, and a-Si:H Multilayer Films for Flexible Thin Film Solar Cells.

The behavior of bi- and trilayer coating systems for flexible a-Si:H based solar cells consisting of a barrier, an electrode, and an absorption layer is studied under mechanical load. First, the film morphology, stress, Young's modulus, and crack onset strain (COS) were analyzed for single film coatings of various thickness on polyethylene terephthalate (PET) substrates. In order to demonstrate the role of the microstructure of a single film on the mechanical behavior of the whole multilayer coating, two sets of InSnOx (indium tin oxide, ITO) conductive coatings were prepared. Whereas a characteristic grain-subgrain structure was observed in ITO-1 films, grain growth was suppressed in ITO-2 films. ITO-1 bilayer coatings showed two-step failure under tensile load with cracks propagating along the ITO-1/a-Si:H-interface, whereas channeling cracks in comparable bi- and trilayers based on amorphous ITO-2 run through all constituent layers. A two-step failure is preferable from an application point of view, as it may lead to only a degradation of the performance instead of the ultimate failure of the device. Hence, the results demonstrate the importance of a fine-tuning of film microstructure not only for excellent electrical properties, but also for a high mechanical performance of flexible devices (e.g., a-Si:H based solar cells) during fabrication in a roll-to-roll process or under service.