Trypsin

Polyploid rice protein (PRP) has the advantage of high nutritional value, but its functional properties are minimal due to its poor solubility. This work aims to improve the solubility of PRP through enzymatic hydrolysis and assess the effect of hydrolysis time (5-330 min) and protease type (Alcalase, Neutrase, and Trypsin) on the structural, functional, and antioxidant properties of PRP hydrolysates (PRPHs). Compared to PRP, PRPHs exhibited significantly decreased free sulfhydryl content and surface hydrophobicity and improved structural flexibility, regardless of the protease used. With increasing time, the nitrogen solubility index of the hydrolysates increased by 25.01 %, which was attributed to the reduction in molecular weight (< 15 kDa). The highest emulsifying activity (48.81 m²/g) and hydroxyl radical scavenging activity (IC₅₀ of 5.49 mg/mL) were observed from Neutrase hydrolysates at 210 min and 330 min, respectively. Trypsin hydrolysate at 210 min demonstrated the lowest IC₅₀ (0.17 mg/mL) in ABTS⁺. Moreover, compared to diploid rice protein hydrolysates (DRPHs) obtained under the same conditions, PRPHs by all proteases exhibited superior functional and antioxidant properties and richer amino acid content. This study showed the potential of PRPHs applied to functional foods with favorable functional and antioxidant properties.

Although the eye has traditionally been considered an immune-privileged organ, progressive studies have re-evaluated the role of the immune response in retinopathy. The application of flow cytometry for immunophenotyping analysis of retinal single-cell suspensions has gradually attracted attention.

We dissociated the retinal tissue using trypsin digestion, papain digestion, mechanical grinding treatment and liberase + DNase Ⅰ digestion, respectively. Subsequently we assessed the quality of cell suspension (clumping rate, concentration and viability) with two different dyes, Trypan Blue (TPB) and Acridine Orange/Propidium Iodide (AO/PI). We distinguished T cells and their sub-populations by flow cytometry. Furthermore, we analyzed their immune responses to evaluate those four methods for preparing retinal single-cell suspension.

The AO/PI staining method enables a rapid and precise evaluation of cell quality of retinal single cell suspensions, while TPB staining has limitations. Flow cytometric analysis showed that single cell suspensions dispersed with papain and trypsin exhibited reduced cell adhesion. However, trypsin digestion may affect antibody binding. The mechanical grinding treatment reduced cell yield and was prone to double-positivity. The number of cells within the cell-circle gate is significantly limited in the Liberase + DNase I digestion method.

The AO/PI staining method was employed to assess the quality of retinal single-cell suspensions. T cells and their subpopulations in retinal tissues were analyzed by flow cytometry. These results were integrated to evaluate the optimal preparation protocol for retinal single-cell suspensions.

Papain digestion is a superior method for preparing retinal single-cell suspensions.