Comparison between different enzymatic inhibition methods in the standardized gastrointestinal model (INFOGEST): a case study of Pleurotus ostreatus mushroom biomass

Pleurotus ostreatus (PO) is one of the most cultivated and consumed mushrooms worldwide, playing crucial roles in human health (e.g., prebiotic and immunomodulatory)1. Mushroom biomass (MB) combines the mycelium and young fruiting bodies1,2. Both parts possess numerous bioactive compounds such as α- and β-glucans1. A possible synergetic effect between their distinct macromolecules has been proposed2. The authors characterized the PO biomass composition for the first time. The results suggested an interesting nutritional profile, where the main differences between PO biomass and fruiting body are the higher α-glucans (77.49%) content and lower concentration of β-glucans (2.96%), free sugars (0.34%), protein (4.62%) and ashes (1.36%). However, it is imperative to assess the influence of the gastrointestinal tract (GIT) on their bioactive compounds, especially α-glucans. Accordingly, the standardized GIT model (INFOGEST) was used3. Samples were taken from oral, gastric, and intestinal phases to better assess the impact of different GIT phases. The results indicated a loss of approximately 19 and 44% of α-glucans in the oral and intestinal phases. However, we identified some issues, mainly linked to the difficulty in collecting homogenous samples (high variability and time-demanding) and non-enzymatic inactivation. During the INFOGEST protocol, enzymatic inactivation may overestimate the enzymatic activity, playing a key role in the bioactive compound quantification. In this context, we tested different enzymatic inhibition approaches to select the method that allows a more realistic macromolecule quantification. We selected temperature and pH for all enzymes and specific inhibitors (enzymes or other specific reagents) for each GIT enzyme (oral: α-amylase inhibitor from Triticum aestivum; gastric: pepstatin A and orlistat; intestinal: 4-bromophenylboronic acid, pefabloc SC and Na2CO3). Oral, gastric and intestinal phases were analyzed with independent GIT simulations to minimize heterogeneity. The influence of the freezing or freeze-drying process was also evaluated. The impact on GIT α-amylases, proteases and lipases inhibition was monitored by analyzing α-glucans content, fatty acid, protein and peptide profiles. Overall, the results corroborated the importance of enzymatic inactivation. The protein and peptide profiles did not suggest key differences between inhibition methods. A significantly higher concentration of α-glucans was quantified, but no significant differences were generally found between different inactivation methods in this bioactive molecule. In some GIT phases, freeze-drying seems to have a negative impact on α-glucan content and especially on total phenolics (TP) and antioxidant activity. pH showed the most negative effect on TP and antioxidant activity, while temperature usually appears not to influence these parameters.