Exploring genotypic diversity and processing effects on protein quality for nutritional and functional enhancement in pigeon pea (Cajanus cajan L.)

Pigeon pea protein isolate (PpPI) offers a sustainable and high-quality plant-based protein alternative. This study investigated the in-vitro protein digestibility of thirty pigeon pea genotypes, identifying two contrasting lines—Pusa Arhar 2018–4 (low digestibility) and ICP 1452 (high digestibility). The cytotoxicity, amino acid bioavailability, and gene expression modulation induced by PpPI hydrolysates, along with the impact of thermal processing, were evaluated using Caco-2 cells. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay confirmed that PpPI hydrolysates were non-cytotoxic in both control and autoclaved samples. The amino acid bioavailability assay revealed that neutral and polar amino acids (glycine, alanine, serine, proline, and leucine) had higher transport efficiency (17.43-27.90%) than charged amino acids (9.08-18.76%). Autoclaving significantly improved amino acid transport, with bioavailability ranging from 3.18-21.93% in Pusa Arhar 2018–4 and 4.38-34.84% in ICP 1452. Gene expression analysis using Caco2 cell line showed upregulation of the peptide transporter gene PepT1 (1.04-1.87-fold in Pusa Arhar 2018–4; 1.07-1.89-fold in ICP 1452), with significantly higher expression in autoclaved samples (p <0.05). SREBP2, a key cholesterol metabolism regulator, was downregulated in both genotypes, though not significantly affected by thermal processing. These findings highlight the potential of autoclaved PpPI hydrolysates to improve amino acid bioavailability and intestinal gene expression, providing key insights for selecting pigeon pea genotypes with superior protein quality to guide breeding for enhanced nutritional functionality.