Enzymatic hydrolysis of soy and chickpea protein with Alcalase and Flavourzyme and formation of hydrogen bond mediated insoluble aggregates.

Food applications involving plant proteins require modification of their functionality to mimic the unique properties of animal proteins. Enzymatic hydrolysis is commonly used to alter the functionality of plant proteins, particularly to improve their solubility near the isoelectric point. Current methodological approaches mostly indicate improved solubility upon hydrolysis. However, published methods include the removal of insoluble material before analysis, and calculations are based on only the solubilized material as a percentage of the filtered protein. This approach artificially increases solubility estimation and gives an incorrect assessment of the efficacy of hydrolysis. By using the total amount of protein, this study aims to determine the effect of two microbial proteases, Flavourzyme and Alcalase, on the solubility and structural and thermal properties of soy and chickpea proteins. Protein isolates were first extracted from soy and chickpea flour and hydrolyzed from 0 to 3 h. Then, their degree of hydrolysis and solubility at a range of pHs were determined using the o-phthaldialdehyde (OPA) and Lowry methods, respectively. Proteins' electrophoretic mobility, protein-protein interactions, thermal properties, and protein secondary structures were also determined. Solubility decreased over time though the solubility of the hydrolysate improved near the isoelectric point. Soy Flavourzyme hydrolysates remained the most soluble and chickpea Flavourzyme hydrolysates showed the least solubility. Thermal data suggested that Alcalase reduced the protein denaturation temperature, leading to a loss of solubility upon thermal enzyme inactivation. The loss of solubility of hydrolysates was strongly associated with hydrogen bonding, which may result from the formation of polar peptide termini. These results challenge commonly accepted beliefs that hydrolysis inevitably improves solubility of plant proteins. Instead, it is shown that hydrolysis causes structural changes that result in aggregation, thus potentially limiting the application of enzymatic hydrolysis without the addition of further processing methods.

Pulse protein processing: The effect of processing choices and enzymatic hydrolysis on ingredient functionality.

Plant-based protein ingredients are an emerging solution to the environmental and health issues associated with animal-based proteins. Pulses have become a promising source of these plant-based ingredients. In order to produce functional proteins from pulse grains, extensive processing must be conducted to extract their proteins. These processing steps have consequential effects on the composition and structure of the resulting proteins which may modify their functional properties. This study reviews the most prominent options for each unit operation of pulse protein processing such as extraction, isolation, and drying. It also emphasizes the benefits and drawbacks of such methods and their effects on the pulse protein functionality. Furthermore, enzymatic hydrolysis is discussed as an optional processing step that is thought to counteract loss of functionality associated with pulse protein isolation. However, review of enzymatic hydrolysis literature reveals methodological issues in which insoluble and nonfunctional fractions of pulse protein hydrolysates are removed before analysis. This literature may draw into question the validity of the conventional wisdom that enzymatic hydrolysis is always beneficial to protein functionality.

Transcriptional Dynamics of Cultured Human Villous Cytotrophoblasts.

During human pregnancy, cytotrophoblasts (CTBs) play key roles in uterine invasion, vascular remodeling, and anchoring of the feto-placental unit. Due to the challenges associated with studying human placentation in utero, cultured primary villous CTBs are used as a model of the differentiation pathway that leads to invasion of the uterine wall. In vitro, CTBs emulate in vivo cell behaviors, such as migration, aggregation, and substrate penetration. Although some of the molecular features related to these cell behaviors have been described, the underlying mechanisms, at a global level, remain undefined at midgestation. Thus, in this study, we characterized second-trimester CTB differentiation/invasion in vitro, correlating the major morphological transitions with the transcriptional changes that occurred at these steps. After plating on Matrigel as individual cells, CTBs migrated toward each other and formed multicellular aggregates. In parallel, using a microarray approach, we observed differentially expressed (DE) genes across time, which were enriched for numerous functions, including cell migration, vascular remodeling, morphogenesis, cell communication, and inflammatory signaling. DE genes encoded several molecules that we and others previously linked to critical CTB function in vivo, suggesting that the novel DE molecules we discovered played important roles. Immunolocalization confirmed that CTBs in situ gave a signal for two of the most highly expressed genes in vitro. In summary, we characterized, at a global level, the temporal dynamics of primary human CTB gene expression in culture. These data will enable future analyses of various types of in vitro perturbations-for example, modeling disease processes and environmental exposures.