Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable

Abstract

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.

Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure

“…In this context, the resistance to digestion is in relation to foods consumed. As such, the current practice to evaluate resistance to pepsin digestion is a relevant consideration in the allergenicity risk assessment of novel proteins that should be taken along, mostly as an exposure assessment in the weight-of-evidence approach [ 28 ]. The addition of a sequential pancreatin (duodenal) phase to follow the pepsin digestion phase does not improve the power to discriminate allergens from non-allergens.…”

Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

“…Additionally, no obvious peptide fragments of Hb were observed in pepsin digestion under optimal conditions. Under sub-optimal conditions, pH 5 PPR 0.1, there was no change in the intensity of the intact Hb band nor were additional bands observed (Fig 2), indicating that pepsin was unable to degrade Hb under these conditions as [16]. Pepsin was barely visible on the gel due to the amount being 1% of what was in the optimal pepsin condition.…”

“…Additionally, analysis of digestion reactions by staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was outlined in the standardized pepsin resistance assay, has also been criticized for lacking the ability to detect peptides smaller than 3 kDa. The allergenicity predictability of physiological digestion test conditions that cover the portion of the human population who have impaired digestive conditions (such as people who had gastric bypass surgery or are taking proton inhibitor drugs) remains uncertain because proteins that are readily digested by pepsin using the standardized protocol remain intact or partially intact after incubation of pepsin under sub-optimal conditions [15,16]. These results indicate that under sub-optimal conditions, high pH and low pepsin-to-protein ratios (PPR), the predictive value of the assay is not improved because both allergens and non-allergens are resistant to pepsin digestion.…”

Effect of soybean processing on cell wall porosity and protein digestibility

“…The band around 36 kDa, for instance, might belong to pepsin, while the bands 51-54 kDa, 38 kDa and 23-27 kDa might correspond to pancreatin enzymes including trypsin, amylase, lipase, ribonuclease, and protease. 29,30 Nonetheless, the protein profiles of the remaining pellet of cells after digestion (lane 6-8), showed no visible differences between BC, BCF, and GBC.…”

“…The band of molecular weights around 21 kDa was previously identified as trypsin inhibitor subunits. 28,29 Moreover, the increased intensities of oligopeptides with molecular weight <14 kDa in BCF (lane 4) could be the product of partial protein hydrolysis by proteolytic enzymes produced during fermentation. The new bands at 26 kDa in GBC might originate from the proteolysis of soybean storage proteins during germination.…”