High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans

Abstract

Background: Industrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability.

Methods: In Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a "dry" blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion.

Results: Protein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0-150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions.

Conclusions: These data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein. This trial was registered at clinicaltrials.gov as NCT05584358.

Keywords: INFOGEST; amino acids; bioavailability; digestion; extrusion; mycoprotein; pea protein.

FIGURE 1

Protocol schematic of the experimental visit. GI, gastrointestinal; VAS, visual analog scale.

FIGURE 2

Time course (A) and incremental area under the curve (B) (incremental area under the curve [iAUC]; calculated as above t = 0) of serum insulin concentrations for a 5-h postprandial period. Experimental beverage consisting of 39%/61% mycoprotein/pea protein (31.9 g of dry blend containing 25 g protein [CON] or 31.4 g of extruded blend containing 25 g protein [EXT]) consumed at t = 0. Time course data were analyzed using a 2-way repeated measures analysis of variance (condition × time) with Sidak post hoc tests to detect differences at individual time points. iAUC data were analyzed using a paired t test. Time effect; P < 0.05. Condition effect; P > 0.05. Condition × time interaction; P < 0.001. Values are mean ± standard error of the mean.

FIGURE 3

Time course and incremental area under the curve (incremental area under the curve [iAUC]; calculated as above t = 0) of plasma total amino acids (TAAs) (A, B), essential amino acids (EAAs) (C, D), nonessential amino acids (NEAAs) (E, F), and branched-chain amino acids (BCAAs) (G, H) over a 5-h postprandial period. iAUCs are displayed for early (0–150 min), late (150–300 min), and total (0–300 min) postprandial periods. Experimental beverage consisting of 39%/61% mycoprotein/pea protein (31.9 g of dry blend containing 25 g protein [CON] or 31.4 g of extruded blend containing 25 g protein [EXT]) consumed at t = 0. Time course data were analyzed using a 2-way repeated measures analysis of variance (condition × time) with Sidak post hoc tests to detect differences at individual time points. iAUC data were analyzed using a paired t test. ∗ denotes individual differences between groups at that time point and a difference between conditions on the bar graphs (P < 0.05). Time effect; all P < 0.05. Condition effect; all P > 0.05. Condition × time interaction; all P < 0.001). Values are mean ± standard error of the mean.

FIGURE 4

Time course and incremental area under the curve (iAUC; calculated as above t = 0) of plasma leucine (A, B), isoleucine (C, D), valine (E, F), phenylalanine (G, H), methionine (I, J), tyrosine (K, L), lysine (M, N), glycine (O, P), and threonine (Q, R) over a 5-h postprandial period. Experimental beverage consisting of 39%/61% mycoprotein/pea protein (31.9 g of dry blend containing 25 g protein [CON] or 31.4 g of extruded blend containing 25 g protein [EXT]) consumed at t = 0. Time course data were analyzed using a 2-way repeated measures analysis of variance (condition × time) with Sidak post hoc tests to detect differences at individual time points. iAUC data were analysed using a paired t test. ∗ denotes individual differences between groups at that time point and a difference between conditions on the bar graphs (P < 0.05). Time effect; all P < 0.05. Condition effect; all P > 0.05 with the exception of tyrosine, phenylalanine, and lysine (P < 0.05). Condition × time interaction; all P < 0.05 with the exception of glycine and methionine (P > 0.05). Values are mean ± standard error of the mean.

FIGURE 5

Time course of protein concentrations in the digesta measured via BCA assay for CON and EXT (both 0.3 g protein powder assimilated in 4.4 mL of ddH₂O) during the INFOGEST static in vitro digestion (2 h gastric and 2 h intestinal phase). Data were analyzed using a 2-way ANOVA (condition × time) with Sidak post hoc tests to detect differences at individual time points. Time × condition interaction (P < 0.0001) during the gastric phase. Condition effect (P < 0.001) during the intestinal phase. Values are mean (n = 6) ± SEM. BCA, bicinchoninic acid; CON, dry blend; EXT, extruded blend; SEM, standard error of the mean.

FIGURE 6

Optical micrographs obtained using fluorescence microscopy with fast green (FG; stained as pink/red) and calcofluor white (CW; stained as a brilliant blue) stains for protein and cell wall, respectively, in CON and EXT conditions. Images are presented from before (A, D) and after gastric (B, E) and intestinal (C, F) INFOGEST static in vitro digestion. Protein is visibly more abundant in the CON compared with EXT condition in the undigested sample. The darker red and more clustered protein structures in the undigested EXT sample indicate dense aggregate structures. This likely underpins the rapid and greater protein availability observed with CON measured both in vivo and in vitro. The free protein that existed in the CON sample has been digested by the end of the gastric phase, leaving only the protein that is found within the hyphal structure of mycoprotein, which is liberated from the cell wall and digested during the intestinal phase. Conversely, dense protein aggregates can still be observed in the EXT sample following intestinal digestion, supporting the impaired digestibility of the EXT. CON, dry blend; EXT, extruded blend.