Comparative effects of high-pressure homogenization, ultrasound-assisted fermentation, and transglutaminase on a protein-rich pumpkin seed-based yogurt analogue

Plant-based yogurt analogues have emerged as one of the fastest-growing segments of the food industry. However, developing products with adequate gel structure and texture remains a major technological challenge. This study comparatively evaluated enzymatic (transglutaminase) and physical (high-pressure homogenization, and ultrasound-assisted fermentation) strategies for producing a pumpkin seed-based yogurt analogue enriched with pea protein. Among the evaluated strategies, homogenization was the most effective, increasing the consistency index by up to 176-fold and water-holding capacity by 41%, while reducing the fermentation time required to reach pH 4.60 ( p < 0.05). Based on these results, the non-homogenized control and the yogurt analogues homogenized at 30 and 150 MPa were selected for further characterization during 21 days of refrigerated storage. SEM micrographs revealed that homogenization promoted the formation of a more interconnected protein network, supporting the improvements observed in rheological properties and water-holding capacity. During storage, only slight changes were observed in acidity (+0.13% lactic acid) and pH (−0.11), whereas lactic acid bacteria remained stable at approximately 8.0 log CFU/mL. High-pressure homogenization also increased antioxidant activity by up to 24%, 25%, and 20% according to the FRAP, ABTS• + , and DPPH• assays, respectively, while maintaining these improvements throughout storage. Moreover, all yogurt analogues maintained cell viability above 90% in RAW 264.7 macrophages across the evaluated sample concentrations (100–500 μg mL −1 ) and storage periods (1 and 21 days), indicating no detectable in vitro cytotoxic effects under the tested conditions. Overall, high-pressure homogenization proved to be a promising clean-label strategy for producing protein-rich plant-based yogurt analogues with improved technological, functional, and storage stability properties.