High-pressure processing (HPP) is a non-thermal food processing technology offering an alternative to heat treatment, minimizing detrimental effects on sensory and nutritional qualities. HPP alters food polymer structures by disrupting non-covalent interactions, leading to textural and quality improvements. This study focuses on the impact of HPP on pea protein concentrate (PPC), a valuable plant protein source gaining increasing interest. A challenge with pulse proteins like those from peas is their lower protein quality compared to animal proteins, stemming from limited amino acid profiles and low digestibility due to inaccessible enzymatic cleavage sites and antinutritional factors such as trypsin inhibitors. Understanding HPP's effects on pulse protein digestibility and trypsin inhibitor activity is crucial for improving protein quality. Previous research on HPP-treated pulse or soy protein has yielded inconsistent digestibility results, often due to low protein concentrations or incomplete enzyme sets in the studies. PPC also contains starch, and pressure-treated starch exhibits greater granule structure and potentially increased resistance to digestion compared to heat-treated starch. This study aims to compare the effects of HPP and heat treatment on the in vitro digestibility of proteins and starch, as well as trypsin inhibitor activity in PPC, using two protein concentrations (5% and 15% w/w) relevant to various food applications. The HPP parameters (600 MPa/5 °C/4 min) are commonly used in the food industry, while heat treatment (95 °C/15 min) is used for comparison, given its known impact on pea protein gel formation.

The literature review section extensively covers existing research on HPP's effects on various food proteins and starches. Studies cited include those investigating the effects of high hydrostatic pressure on the in vitro digestion of soy protein, meat and lupin proteins, and chickpea and lentil proteins. The review also delves into the impact of HPP and heat treatment on the functional and nutritional properties of soy protein isolate, and the varying effects of pressure on starch digestibility observed in different studies. Previous work on the digestibility of thermally-induced plant protein gel networks is also reviewed, highlighting the novelty of this study in addressing the digestibility of pressure-induced plant protein networks. The literature review also addresses the role of antinutritional factors like trypsin inhibitors in affecting the digestibility of pulse proteins.

Two in vitro digestion models were employed: the COST INFOGEST static model and the TIM-1 dynamic model. Pea protein concentrate (PPC) samples with 5% (5P) and 15% (15P) protein were prepared and subjected to HPP (600 MPa/5 °C/4 min) or heat treatment (95 °C/15 min). The static model involved sequential oral, gastric, and intestinal phases, with sampling at the end of the gastric (SG) and intestinal (SI) phases. The dynamic model (TIM-1) simulated the entire gastrointestinal tract, with samples collected from the jejunum and ileum at various time points (20, 40, 60, 90, 120, 150, 180, 210, and 240 min). Digesta were analyzed using several methods: SDS-PAGE to assess protein and peptide fractions, BCA assay for peptides (<10 kDa), OPA assay for small peptides (<6 kDa) and free amino acids, and glucose assay for starch digestibility. Trypsin inhibitor activity was determined using a modified BAPNA method. Statistical analysis involved linear models with fixed and random effects to account for treatment, time, and replicate variation. The detailed methodologies for each assay are thoroughly described, including sample preparation, reagent details, and analytical procedures. The static and dynamic models are explained and compared. The activity levels of all digestive enzymes used were assayed to ensure uniform activity across all digestions. All relevant information, including sample sizes, replicates and statistical methods are discussed.

HPP-treated PPC exhibited significantly higher gastric proteolysis compared to untreated and heat-treated PPC in both static and dynamic models (p<0.05), particularly during the initial 20 minutes of jejunal and total digestion for 5P. However, the overall in vitro protein digestibility was not significantly different among treatments once the digestions were completed. Differences in protein digestibility during dynamic digestion were significant only in the initial stages (first 20 min for 5P jejunal, ileal, and total digestion, and first 60 min for 15P ileal digestion). Neither static nor dynamic starch digestibility varied significantly between treatments (p > 0.05). Heat treatment significantly reduced trypsin inhibitor activity by approximately 70% (p < 0.05), while HPP did not show any significant effect. SDS-PAGE revealed different peptide patterns between treatments, reflecting treatment-dependent pepsin cleavage dynamics. The BCA and OPA assays both indicated that HPP increased the extent of protein hydrolysis compared to the untreated and heat-treated samples. The proportions of rapidly digestible, slowly digestible, and resistant starch in the 15P samples were analyzed, however, there were no statistically significant differences in digestible starch content across treatment types (p>0.05). The 15P samples showed a sigmoidal curve in the dynamic digestion models.

The findings suggest that HPP-induced structural changes in pea proteins enhance gastric proteolysis, likely by exposing more cleavage sites to pepsin. This initial enhancement in proteolysis does not translate to significantly altered overall protein digestibility. The differences in digestibility between HPP and heat treatment are likely due to the nature of aggregates formed; HPP-induced aggregates, primarily held by weaker physical bonds, are more readily broken down than heat-induced aggregates crosslinked by covalent bonds. The study also highlights the influence of protein concentration on digestibility, with 15P samples showing less susceptibility to enzymatic attack due to increased protein-protein interactions and gel network formation. Discrepancies between static and dynamic models are likely due to differences in sampling points, enzyme:substrate ratios, and shear forces. HPP's lack of effect on trypsin inhibitor activity contrasts with heat treatment's significant reduction. The lack of significant impact on starch digestibility might result from the relatively low starch concentration in PPC and the high enzyme:starch ratio in the digestions.

This study demonstrates that HPP does not negatively affect the overall in vitro digestibility of pea protein or starch but can enhance initial gastric proteolysis. The ability of HPP to create desirable textural changes without compromising digestibility is a valuable finding for food applications. Future research could explore the in vivo digestibility of HPP-treated pea protein, investigate the specific peptide profiles generated by HPP, and examine the long-term effects of HPP on protein quality and nutritional value. Further research could also explore the effects of HPP at higher starch concentrations.

The study's limitations include the use of in vitro digestion models, which may not perfectly replicate the complex in vivo environment. The relatively low starch content in PPC may have limited the ability to observe significant differences in starch digestibility between treatments. Further studies are needed to validate these in vitro findings in humans.