A Comparative Functional Analysis of Pea Protein and Grass Carp Protein Mixture via Blending and Co-Precipitation

Currently, the application of protein mixture derived from plants and animals is of great interest to the food industry. However, the synergistic effects of isolated protein blends (BL) are not well established. Herein, the development of a more effective method (co-precipitation) for the production of protein mixtures from pea and grass carp is reported. Pea protein isolate (PPI), grass carp protein isolate (CPI), and pea–carp protein co-precipitates (Co) were prepared via isoelectric solubilization/precipitation using peas and grass carp as raw materials. Meanwhile, the BL was obtained by blending PPI with CPI. In addition, the subunit composition and functional properties of Co and BL were investigated. The results show that the ratios of vicilin to legumin α + β and the soluble aggregates of Co were 2.82- and 1.69-fold higher than that of BL. The surface hydrophobicity of Co was less than that of BL, PPI, and CPI (p < 0.05). The solubility of Co was greater than that of BL, PPI, and CPI (p < 0.05), and the foaming activity was higher than that of BL and CPI (p < 0.05) but slightly lower than that of PPI. In addition, based on the emulsifying activity index, particle size, microstructure, and viscosity, Co had better emulsifying properties than BL, PPI, and CPI. The study not only confirmed that co-precipitation was more effective than blending for the preparation of mixed protein using PPI and CPI but also provided a standard of reference for obtaining a mixture of plant and animal proteins.

High Purity of α-Lactalbumin from Binary Protein Mixture by Charged UF Membrane Far from the Isoelectric Point to Limit Fouling

Separation and high recovery factor of proteins similar in molecular mass is a challenging task, and heavily studied in the literature. In this work, a systematic study to separate a binary protein mixture by charged ultrafiltration membranes without affecting membrane performance was carried out. α-lactalbumin (ALA, 14.4 kDa) and β-lactoglobulin (BLG, 18.4 kDa) were used as a binary model system. These two proteins are the main proteins of whey, a very well-known byproduct from the dairy industry. Initially, a systematic characterization of individual proteins was carried out to determine parameters (protein size and aggregation, zeta potential) which could influence their passage through a charged membrane. Then, the influence of operating parameters (such as initial protein concentration, pH, and critical pressure) on the UF process was investigated, so as to identify conditions that limit membrane fouling whilst maximizing protein recovery factor and purity. The study permitted to identify process conditions able to fully separate ALA from BLG, with high purity (95%) and recovery factor (80%), in a single UF step. Compared to studies reported in literature, here, the main approach used was to carry out a charged UF process far from proteins isoelectric point (pI) to limit protein aggregation and membrane fouling.

Rapid Separation of Human Hemoglobin on a Large Scale From Non-clarified Bacterial Cell Homogenates Using Molecularly Imprinted Composite Cryogels

The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions.

Protein primary structure correlates with calcium oxalate stone matrix preference

Despite the apparent importance of matrix proteins in calcium oxalate kidney stone formation, the complexity of the protein mixture continues to elude explanation. Based on a series of experiments, we have proposed a model where protein aggregates formed from a mixture containing both strongly charged polyanions and strongly charged polycations could initiate calcium oxalate crystal formation and crystal aggregation to create a stone. These protein aggregates also preferentially adsorb many weakly charged proteins from the urine to create a complex protein mixture that mimics the protein distributions observed in patient samples. To verify essential details of this model and identify an explanation for phase selectivity observed in weakly charged proteins, we have examined primary structures of major proteins preferring either the matrix phase or the urine phase for their contents of aspartate, glutamate, lysine and arginine; amino acids that would represent fixed charges at normal urine pH of 6–7. We verified enrichment in stone matrix of proteins with a large number of charged residues exhibiting extreme isoelectric points, both low (pI<5) and high (pI>9). We found that the many proteins with intermediate isoelectric points exhibiting preference for stone matrix contained a smaller number of charge residues, though still more total charges than the intermediate isoelectric point proteins preferring the urine phase. While other sources of charge have yet to be considered, protein preference for stone matrix appears to correlate with high total charge content.

Comparative two-dimensional fluorescence gel electrophoresis

Comparative two-dimensional gel electrophoresis (CoFGE) is a special version of two-dimensional polyacrylamide GE (2D-PAGE) and related to difference GE (2D-DIGE). It provides reproducibility and standardisation for 2D-PAGE by introducing a reference to the experiment. CoFGE uses different fluorescent labels to distinguish analyte and a marker protein mixture. The method allows in silico correction of the assignment of gel-separated proteins based on the co-run references, which form a grid of landmarks across the entire gel. The variability of spot coordinates is reduced to ~1% error and data can thus be compared to results generated independently with the same method. In this way, searchable repositories of gel-separated proteins become feasible. In addition, the CoFGE experimental principle can be used for protein quantification by applying the proteins of the marker grid in different concentrations. Here we present the protocol for conducting a CoFGE experiment, which takes about 2 days to complete for a technician skilled in GE.

The Effects of Hydrocolloids-Protein Mixture as a Fat Replacer on Physicochemical Characteristics of Sugar-Free Muffin Cake: Modeling and Optimization

The purpose of this study was to evaluate the hydrocolloids–protein mixture as a fat replacer in sugar-free low-fat muffin cakes. In this study, a hydrocolloids mixture including konjac and guar gums and soy protein isolate (SPI) was applied to the cake. The combination of gums and SPI was named as mixture of stabilizers (MOS), and the treatments were designed using Design-Expert software and the response surface methodology (RSM) in order to optimize and reduce the oil content of muffin cakes by replacing it with MOS. Evaluation of treatments characteristics were investigated on days 1 and 15 of their production. The dependent variables were moisture content, water activity, specific volume, porosity, hardness, cohesiveness, springiness, chewiness and crumb color of cakes. The results show that increasing the percentage of MOS has positive effects on the final products in comparison to oil. In other words, an increase in the MOS content resulted in an increase in the moisture content, water activity, specific volume, height, springiness, cohesiveness, chewiness and L* (lightness) values, but negative effect on hardness, a* (redness) and b* (yellowness) values. As a result of optimizing using RSM, the usage of 4.08% oil and 0.31% MOS resulted in a 62.9% reduction in oil content in comparison with the control sample. The panelists assigned the lowest score to hardness and crumb color and the highest score to overall acceptability and cohesiveness to the optimized muffin.

Mechanism of autonomous synchronization of the circadian KaiABC rhythm

The cyanobacterial circadian clock can be reconstituted by mixing three proteins, KaiA, KaiB, and KaiC, in vitro. In this protein mixture, oscillations of the phosphorylation level of KaiC molecules are synchronized to show the coherent oscillations of the ensemble of many molecules. However, the molecular mechanism of this synchronization has not yet been fully elucidated. In this paper, we explain a theoretical model that considers the multifold feedback relations among the structure and reactions of KaiC. The simulated KaiC hexamers show stochastic switch-like transitions at the level of single molecules, which are synchronized in the ensemble through the sequestration of KaiA into the KaiC–KaiB–KaiA complexes. The proposed mechanism quantitatively reproduces the synchronization that was observed by mixing two solutions oscillating in different phases. The model results suggest that biochemical assays with varying concentrations of KaiA or KaiB can be used to test this hypothesis.