Browsing by Author "Do DT"

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Heat-induced dissociation and association of proteins in hempseed protein bodies
(Elsevier Ltd, 2025-10) Do DT; Ye A; Singh H; Acevedo-Fani A
Protein bodies (PBs) are naturally occurring storage organelles in seeds. In hempseeds, the major storage proteins, including edestin (11S globulin) and albumin, are primarily located in the crystalloids and proteinaceous matrices of hemp protein bodies (HPBs), respectively. The retention of native PB structures in flours and dry-fractionated protein ingredients has important implications for protein functionality and digestibility, especially when heat treatment is applied during processing. While the thermal behaviour of hempseed proteins has been studied in protein isolate systems, to the best of our knowledge, it has not yet been explored in HPB systems. In this study, we isolated native HPBs using an enzymatic method. Aqueous suspensions of HPBs (4 % protein, w/w) were heated at selected temperatures (60–100 °C) and pH 7 for 20 min, followed by hydrolysis with trypsin at pH 7 and 37 °C for 120 min. The thermal aggregation of proteins in HPBs was characterised using confocal laser scanning microscopy (CLSM) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The hydrolysis of HPBs by trypsin was monitored over 120 min by measuring the degree of protein hydrolysis (DH) and analysing SDS-PAGE. Aggregation of edestin in HPBs, primarily driven by disulfide bond formation, occurred upon heating, most noticeably at temperatures above 80 °C. Heating increased DH and altered protein degradation patterns of both acidic and basic subunits of edestin. This may be related to conformational changes in the HPB structure resulting from heat-induced dissociation-association of multiple HPB protein fractions, including 11S edestin, 7S globulin, and 2S albumin. These findings contribute to our understanding of the structure-hydrolysis relationships of HPBs, potentially leading to their use as a new plant-based material for food applications.
Probing the Double-Layered Cotyledon Cell Structure of Navy Beans: Barrier Effect of the Protein Matrix on In Vitro Starch Digestion
(MDPI (Basel, Switzerland), 2023-01) Do DT; Singh J; Johnson S; Singh H; Bordoni A
The microstructure of legumes plays a crucial role in regulating starch digestion and postprandial glycemic responses. Starch granules are double encapsulated within the outer cell wall and the inner protein matrix of legume cotyledon cells. Despite progress in understanding the role of cell walls in delaying starch digestion, the role of the protein matrix has received little research attention. The aim of this study was to evaluate if the protein matrix and cell wall may present combined physical barriers retarding enzyme hydrolysis of intracellular starch. Intact cotyledon cells were isolated from navy beans and used to assess the barrier effect of the protein matrix on the digestion of starch under conditions simulating the upper gastrointestinal tract. The cells were pretreated with pepsin at 37 °C and pH 2.0 for 1, 4, or 24 h and without pepsin for 24 h (control) to facilitate removal of the intracellular protein matrix prior to cooking and simulated in vitro digestion. A longer pretreatment time resulted in a lower protein content of the cells and a higher initial rate and extent of starch hydrolysis. We suggest that in addition to the primary cell wall barrier, the protein matrix provides a secondary barrier restricting the accessibility of α-amylase to starch. This study provides a new fundamental understanding of the relationship between the structural organization of legume cotyledon cells and starch digestion that could inform the design of novel low glycemic index foods.
Protein bodies from hemp seeds: Isolation, microstructure and physicochemical characterisation
(Elsevier Ltd, 2024-04) Do DT; Ye A; Singh H; Acevedo-Fani A
Protein bodies are naturally occurring storage organelles in plant seeds. Although the microstructure of protein bodies has been studied, their physicochemical behaviour and stability under different environmental conditions remain poorly understood. In this study, hemp seed protein bodies (HPBs) were obtained using a sonication-assisted aqueous enzymatic extraction method. Then, their microstructures were characterised using various microscopic techniques. Next, the protein composition was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Lastly, the influence of pH (2–13) on the colloidal stability and structural integrity of aqueous HPB dispersions was investigated. Detailed microscopic examination showed that the HPBs exhibited spherical shape with an average diameter of about 4.6 μm. The structure consisted of a protein crystalloid and several phytin globoids, all surrounded by a proteinaceous matrix and a single membrane. Globulin edestin was the most abundant storage protein in the HPBs as revealed by SDS–PAGE. The HPB dispersions exhibited excellent colloidal stability only at neutral pH as opposed to their aggregation and/or solubilisation at other pH levels tested. The HPBs also showed irreversible structural changes in response to pH variation. Specifically, little to no swelling of the particles was observed at pH 5 (around the isoelectric point (pI) of the hemp protein). However, when the pH shifted away from the pI, swelling, rupture and eventual dissolution of the particles were pronounced under both extreme acidic and alkaline conditions. These physicochemical behaviours make the HPBs an interesting pH-sensitive material for food applications, which will be explored in subsequent studies.