Exploiting Salt Induced Microphase Separation To Form Soy Protein Microcapsules or Microgels in Aqueous Solution

Self-assembly of native glycinin at room temperature was investigated as a function of the pH and the NaCl concentration. Microphase separation leading to the formation of dense protein microdomains was observed by confocal laser scanning microscopy. Depending on the conditions, the microdomains coalesced into a continuous protein rich phase or associated into large clusters. Addition of β-conglycinin inhibited phase separation and reduced the pH range in which it occurred. Microdomains of glycinin that were formed in the presence of 0.1 M NaCl transformed into hollow stable cross-linked microcapsules when heated above 60 °C with diameters between 3 and 30 μm depending on the protein concentration and a shell thickness between 1.0 and 1.4 μm. The microcapsules were stable to dilution in salt free water, whereas microdomains formed at room temperature redispersed. Microdomains formed in mixtures with β-conglycinin did not transform into microcapsules, but they became stable cross-linked homogeneous microgels

Stable and pH-Sensitive Protein Nanogels Made by Self-Assembly of Heat Denatured Soy Protein

In this study, we examined the possibility of preparing stable soy protein nanogels by simply heating homogeneous soy protein dispersion. The protein nanogels formed were characterized by <i>z</i>-average hydrodynamic diameter, polydispersity index, turbidity, ζ-potential, morphology, and their stability to pH and ionic strength change. Soy protein dispersion (1% w/v) was homogeneous around pH 5.9 where it had the lowest polydispersity index (∼0.1). Stable and spherical nanogels were formed by heating soy protein dispersion at pH 5.9 under 95 °C. They sustained constantly low polydispersity index (∼0.1) in the investigated pH range of 6.06–7.0 and 2.6–3.0. The nanogels were pH-sensitive and would swell with pH change. They were stable at 0–200 mM NaCl concentration. Denaturation of soy glycinin was the prerequisite for the formation of stable nanogels. Soy protein nanogels had a core–shell structure with basic polypeptides and β subunits interacting together as the hydrophobic core; and acid polypeptides, α′, and α subunits locating outside the core as hydrophilic shell. The inner structure of soy protein nanogels was mainly stabilized by disulfide bonds cross-linked network and hydrophobic interaction. Soy protein nanogels made in this study would be useful as functional ingredients in biotechnological, pharmaceutical, and food industries

Evaluation of the Hydrolysis Specificity of an Aminopeptidase from <i>Bacillus licheniformis</i> SWJS33 Using Synthetic Peptides and Soybean Protein Isolate

The substrate specificity of aminopeptidases has often been determined against aminoacyl-<i>p</i>-nitroanilide; thus, its specificity toward synthetic peptides and complex substrates remained unclear. The hydrolysis specificity of an aminopeptidase from <i>Bacillus licheniformis</i> SWJS33 (BLAM) was evaluated using a series of synthetic peptides and soybean protein isolate. The aminopeptidase showed high specificity for dipeptides with Leu, Val, Ala, Gly, and Phe at the N-terminus, and the specificity was significantly affected by the nature of the penultimate residue. In the hydrolysis of soy protein isolate, BLAM preferred peptides with Leu, Glu, Gly, and Ala at the N-terminus by free amino acid analysis and preferred peptides with Leu, Ala, Ser, Trp, and Tyr at the N-terminus by UPLC-MS/MS. The introduction of complex substrates provides a deeper understanding of the aminopeptidase’s specificity, which can instruct the application of the enzyme in protein hydrolysis

Development of a Sono-Assembled, Bifunctional Soy Peptide Nanoparticle for Cellular Delivery of Hydrophobic Active Cargoes

Soy proteins are prone to aggregate upon proteolysis, hindering their sustainable development in food processing. Here, a continuous work on the large insoluble peptide aggregates was carried out, aiming to develop a new type of soy peptide-based nanoparticle (SPN) for active cargo delivery. Sono-assembled SPN in spherical appearance and core–shell structure maintained by noncovalent interactions was successfully fabricated, exhibiting small particle size (103.95 nm) in a homogeneous distribution state (PDI = 0.18). Curcumin as a model cargo was efficiently encapsulated into SPN upon sonication, showing high water dispersity (129.6 mg/L, 10⁴ higher than its water solubility) and storage stability. Additionally, the pepsin-resistant SPN contributed to the controlled release of curcumin at the intestinal phase and thus significantly improved the bioaccessibility. Encapsulated curcumin was effective in protecting glutamate-induced toxicity in PC12 cells, where the matrix SPN can simultaneously reduce lipid peroxidation and elevate antioxidant enzymes levels, innovatively demonstrating its bifunctionality during cellular delivery

Effusanin E induced apoptosis.

<p>(<b>A</b>) Effusanin E (EFE) induced NPC cells apoptosis. Apoptosis was analyzed using TUNEL-based, fluorescence-activated cell sorter analysis and was represented by the relative percentages of TUNEL-positive cells versus those of DMSO-treated cells. (<b>B</b>) The percentages of apoptotic cells were calculated. (<b>C</b>) Western blotting of cleaved caspase-3, caspase-9 and PARP proteins in NPC cells. Total proteins isolated from the indicated cells were blotted with antibodies as labeled, and GAPDH was used as a control for sample loading.</p

Effusanin E affects the activity of the NF-κB pathway.

<p>(<b>A</b>) Effusanin E inhibited the expression of p50 and p65. NPC cells were treated with DMSO (basal) or LPS (2 µg/ml) for 8 hours followed by effusanin E treatment at125 µM or 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours. At the indicated time points, the cells were detected by Western blot analysis. (<b>B</b>) The binding of p50 and p65 NF-κB to the biotin-labeled, COX-2 promoter probe was analyzed by streptavidin-agarose pulldown assays, and the levels of p50 and p65 protein expression were detected by Western blot analysis. (<b>C</b>) The effect of effusanin E was inhibited by an inhibitor of NF-κB in NPC cells. NPC cells were treated with PDTC (100 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) treatment for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. (<b>D</b>) The effect of effusanin E was blocked by an activator of NF-κB. NPC cells were treated with LPS (2 µg/ml) or ammonium pyrrolidinedithiocarbamate (PDTC) (100 µM) for 8 hours followed by effusanin E at125 µM or 250 µM (EFE 125 µM or EFE 250 µM) treatment for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. (<b>E</b>) Analysis of the reduced nuclear translocation of NF-κB p65 by immunofluorescence imaging (IFI). CNE1 and CNE2 cells were treated with effusanin E at 125 µM (EFE) or PDTC (100 µM) or PDTC (100 µM) for 8 hours followed by effusanin E at 125 µM (PDTC + EFE), and NF-κB nuclear translocation in CNE1 and CNE2 cells was determined by immunofluorescence imaging analysis. * represent P<0.05.</p

Effusanin E affects activity of the COX-2 pathway.

<p>(<b>A</b>) NPC cells were pretreated with lipopolysaccharides (LPS) (2 µg/ml) for 8 hours then treated with effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM). At 24 hours after treatment, COX-2 protein expression was determined by western blotting. (<b>B</b>) NPC cells were transfected with a luciferase expression vector containing a COX-2 5-flanking fragment for 8 hours and treated with effusanin E (EFE) at the indicated doses. At 24 hours after treatment, COX-2 promoter activities were determined. (<b>C</b>) The effect of effusanin E was inhibited by an inhibitor of COX-2 in NPC cells. NPC cells were treated with celecoxib (CB) (20 µM or 50 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. The data are presented as the mean ± S.D. of three separate experiments. * represent P<0.05.</p

Effusanin E inhibits proliferation of NPC cells in vitro.

<p>(<b>A</b>) The cell morphology changes of human CNE1 and CNE2 cells after treatment with effusanin E (EFE) at 125 µM or 250 µM. (<b>B</b>) Colony formation assay of human CNE1 and CNE2 cells treated with effusanin E (EFE) at 3.1–25.0 µM. Cells were cultured as indicated in 6-well plates and treated with effusanin E for 10 days. After treament, the cells were dyed with crystal violet and took pictures. (<b>C</b>) The number of colony formation assay. The grown colonies of colony formation assay were scored. The data are presented as the mean ± SD of three separate experiments. * represent P<0.05.</p