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.
Highlights
The fate of most dietary proteins is degradation into small peptides and amino acids that are subsequently absorbed and predominantly used for new protein synthesis and energy [1]
Results pH dependence of degradation of proteins known to be pepsin susceptible To assess the influence of pH on the degradation of proteins by pepsin, three proteins known to be susceptible to pepsin degradation, ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), and hemoglobin (Hb) [23, 26,27,28], were incubated at a constant pepsin-to-substrate protein ratio (10 U:1 μg substrate protein) following a standardized protocol [23] but using a range of pH values from 1.2–6.0 for a duration of 2 minutes
The proteins selected for this analysis included three proteins known to be susceptible to pepsin degradation [ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (Rubisco Large Subunit (LS)), horseradish peroxidase (HRP), hemoglobin (Hb)] and two known to be pepsin-resistant [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]
Summary
The fate of most dietary proteins is degradation into small peptides and amino acids that are subsequently absorbed and predominantly used for new protein synthesis and energy [1] This process is facilitated by the proteolytic enzyme pepsin, which is secreted by gastric chief cells as the precursor protein pepsinogen [2]. Study-to-study variations in the pH, purity and activity of pepsin, pepsin-to-substrate protein ratio, purity and conformation of the substrate protein, and incubation time have made it difficult to directly compare their results and determine if a true correlation between pepsin resistance and allergenicity exists [6, 15,16,17,18,19,20] Consistent with this observation, an extensive review of in vitro digestibility studies identified a lack of harmonized test conditions as a confounding factor towards enabling comparison of results across studies [19]. This pattern is similar to what has previously been observed in humans [22]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
