The lack of adequate guidance and control of the extraction conditions as well as the gap between bench- and industrial-scale production, contributes to the poor functionality of commercial pea protein isolate (cPPI). Therefore, pea protein extraction conditions were evaluated and scaled up to maximize protein purity and yield, while maintaining structural integrity, following mild alkaline solubilization with isoelectric precipitation and salt solubilization coupled with membrane filtration. Both extraction methods resulted in high protein yield (>64%) and purity (>87%). Structure-function characterization illustrated the preserved structural integrity of PPI samples and their superior solubility, gelation, and emulsification properties compared to cPPI. Results confirmed, for the first time, that double solubilization at mild pH (7.5) can replace single solubilization at high alkalinity and achieve a similar yield while preserving structural integrity. Additionally, this study demonstrated, the scalability of the benchtop salt extraction coupled with ultrafiltration/diafiltration. Scaling up the production eliminated some structural and functional differences between the salt-extracted PPI and pH-extracted PPI. Scaling-up under mild and controlled conditions resulted in partial denaturation and a low degree of polymerization, coupled with the superior functionality of the produced isolates compared to cPPI. Results of this work can be used as a benchmark to guide the industrial production of functional pea protein ingredients.

A clean-label process to endogenously glycate and purify pea protein was investigated. The production of maltodextrin from pea starch with a specific dextrose equivalent (DE) was optimized. The produced maltodextrin (14.6 DE) was used to initiate a limited and controlled Maillard-induced glycation of pea protein. The partially glycated pea protein (PG-PP) was subjected to hydrophobic interaction chromatography to remove unreacted carbohydrate, followed by characterization of the purified product. The extent of Maillard-induced glycation was monitored by assessing changes in color, free amino groups, and protein/glycoprotein profiles. The purified PG-PP was evaluated for thermal denaturation, surface properties, protein secondary structure, protein solubility, thermal stability, and digestibility. Maillard-induced glycation was limited to initial stages and resulted in a moderate blockage of amine groups (∼30%). The purified PG-PP had a relatively low surface hydrophobicity, a markedly enhanced protein solubility (∼90%) at pH 3.4, and a nonimpacted protein in vitro digestibility (∼100%). This work provided the impetus needed for future scale-up and process optimization for the production of value-added pea protein ingredient intended for high protein beverage applications.

The discovery of functional amyloids in bacteria dates back several decades, and our understanding of the Escherichia coli curli biogenesis system has gradually expanded over time. However, due to its high aggregation propensity and intrinsically disordered nature, CsgA, the main structural component of curli fibrils, has eluded comprehensive structural characterization. Recent advancements in cryo-electron microscopy (cryo-EM) offer a promising tool to achieve high-resolution structural insights into E. coli CsgA fibrils. In this study, we outline an approach to addressing the colloidal instability challenges associated with CsgA, achieved through engineering and electrostatic repulsion. Then, we present the cryo-EM structure of CsgA fibrils at 3.62 Å resolution. This structure provides new insights into the cross-β structure of E. coli CsgA. Additionally, our study identifies two distinct spatial arrangements within several CsgA fibrils, a 2-CsgA-fibril pair and a 3-CsgA-fibril bundle, shedding light on the intricate hierarchy of the biofilm extracellular matrix and laying the foundation for precise manipulation of CsgA-derived biomaterials. IMPORTANCE: The visualization of the architecture of Escherichia coli CsgA amyloid fibril has been a longstanding research question, for which a high-resolution structure is still unavailable. CsgA serves as a major subunit of curli, the primary component of the extracellular matrix generated by bacteria. The support provided by this extracellular matrix enables bacterial biofilms to resist antibiotic treatment, significantly impacting human health. CsgA has been identified in members of Enterobacteriaceae , with pathogenic E. coli being the most well-known model system. Our novel insights into the structure of E. coli CsgA protofilaments form the basis for drug design targeting diseases associated with biofilms. Additionally, CsgA is widely researched in biomaterials due to its self-assembly characteristics. The resolved spatial arrangements of CsgA amyloids revealed in our study will further enhance the precision design of functional biomaterials. Therefore, our study uniquely contributes to the understanding of CsgA amyloids for both microbiology and material science.

Hemp (Cannabis sativa L.) is increasingly gaining traction as a novel and sustainable source of plant protein. Accordingly, the aim of this study was to investigate the effectiveness of two protein extraction methods, alkaline extraction coupled with isoelectric precipitation (AE-IEP) and salt extraction coupled with ultrafiltration (SE-UF) in producing hemp protein isolates (pH-HPI and salt-HPI) with high purity and yield. Structural characterization as impacted by extraction method and cultivar was performed and related to functional performance and nutritional quality. Both extraction methods, with carefully selected parameters, resulted in HPI with high purity (86.6–88.1% protein) and protein extraction yields (81.6–87.3%). All HPI samples had poor solubility (∼9–20%) at neutral pH compared to commercial soy protein and pea protein isolates (cSPI, cPPI). A relatively high surface hydrophobicity and low surface charge contributed to such poor solubility of HPI. However, HPI demonstrated similar solubility at acidic pH (50–67%) and comparable gel strength (up to 24 N) to cSPI. Comparing experimental amino acid composition to the theoretical amino acid distribution in hemp protein provided insights to the functional performance of the protein isolates. While pH-HPI demonstrated better functionality than salt-HPI, minimal structural, functional, and nutritional differences were noted among the pH-HPI samples extracted from four different cultivars. Overall, results from this work could be used to guide future attempts to further develop successful protein extraction processes, and to provide valuable insights to propel breeding efforts that target enhanced hemp protein characteristics for food applications.

Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, belonging to the genus beta-CoV, have caused outbreaks or pandemics. SARS-CoV-2 has evolved into many variants with increased resistance to the current vaccines. Spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are important vaccine targets; however, the RBD of the SARS-CoV-2 Omicron variant is highly mutated, rending neutralizing antibodies elicited by ancestral-based vaccines targeting this region ineffective, emphasizing the need for effective vaccines with broad-spectrum efficacy against SARS-CoV-2 variants and other CoVs with pandemic potential. This study describes a pan-beta-CoV subunit vaccine, Om-S-MERS-RBD, by fusing the conserved and highly potent RBD of MERS-CoV into an RBD-truncated SARS-CoV-2 Omicron S protein, and evaluates its neutralizing immunogenicity and protective efficacy in mouse models. Om-S-MERS-RBD formed a conformational structure, maintained effective functionality and antigenicity, and bind efficiently to MERS-CoV receptor, human dipeptidyl peptidase 4, and MERS-CoV RBD or SARS-CoV-2 S-specific antibodies. Immunization of mice with Om-S-MERS-RBD and adjuvants (Alum plus monophosphoryl lipid A) induced broadly neutralizing antibodies against pseudotyped MERS-CoV, SARS-CoV, and SARS-CoV-2 original strain, as well as T-cell responses specific to RBD-truncated Omicron S protein. Moreover, the neutralizing activity against SARS-CoV-2 Omicron subvariants was effectively improved after priming with an Omicron-S-RBD protein. Adjuvanted Om-S-MERS-RBD protein protected mice against challenge with SARS-CoV-2 Omicron variant, MERS-CoV, and SARS-CoV, significantly reducing viral titers in the lungs. Overall, these findings indicated that Om-S-MERS-RBD protein could develop as an effective universal subunit vaccine to prevent infections with MERS-CoV, SARS-CoV, SARS-CoV-2, and its variants. IMPORTANCE: Coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, the respective causative agents of coronavirus disease 2019, SARS, and MERS, continually threaten human health. The spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are critical vaccine targets. Nevertheless, the highly mutated RBD of SARS-CoV-2 variants, especially Omicron, significantly reduces the efficacy of current vaccines against SARS-CoV-2 variants. Here a protein-based pan-beta-CoV subunit vaccine is designed by fusing the potent and conserved RBD of MERS-CoV into an RBD-truncated Omicron S protein. The resulting vaccine maintained effective functionality and antigenicity, induced broadly neutralizing antibodies against all of these highly pathogenic human CoVs, and elicited Omicron S-specific cellular immune responses, protecting immunized mice from SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV infections. Taken together, this study rationally designed a pan-beta-CoV subunit vaccine with broad-spectrum efficacy, which has the potential for development as an effective universal vaccine against SARS-CoV-2 variants and other CoVs with pandemic potential.