In Vitro Protease Digestion Affects Protein-Derived Bioactive Compound Content in Functional Ingredients

Background: Proteins in food are degraded by proteases in the digestive tract, and their products may exhibit physiological activities. In vitro protease digestion mimics digestion of food protein in the body. When crude herbal drugs used in Japanese Kampo medicines are subjected to in vitro protease digestion, which mimics in vivo processes, decarboxylated amino acids (biogenic amines) are detected in the digest. The protease-resistant dipeptide, pyroglutamyl leucine (pyroGlu-Leu), is present in fermented foods. In vitro protease digestion of functional foods and ingredients has not yet been studied. Objective: To identify protein-derived compounds by in vitro protease digestion of three functional ingredients that possess anti-inflammatory effects: a standardized extract of Asparagus officinalis stem (EAS), a standardized extract of cultured Lentinula edodes mycelia (ECLM), and a standardized oligomerized-polyphenol from Litchi chinensis fruit extract (OPLFE). Methods: Functional ingredients were treated with endoproteinases and exopeptidases. To detect decarboxylated amino acids, amino groups were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), and a precursor ion scan was performed using liquid chromatography separation–tandem mass spectrometry (LC–MS/MS) to target the product ions. AQC-derivatized amines were identified, and decarboxylated amino acids and pyroGlu-Leu in the digests were analyzed and quantified using LC–MS/MS in the multiple reaction monitoring (MRM) mode. Undigested functional ingredients were analyzed by LC–MS/MS and used as the negative control. Results: Decarboxylated amino acids with anti-inflammatory activity were detected in both undigested functional ingredients and their protease digestion. Tyramine was abundant in OPLFE, but its content was significantly decreased after in vitro protease digestion. 2-Methylbutylamine and isoamylamine were present in both undigested ECLM and its digest, whereas trace amounts of isobutylamine were also detected. In addition, pyroGlu-Leu, which also exhibits anti-inflammatory effects, was highly abundant in undigested ECLM, and its content was significantly increased after in vitro protease digestion. Conclusion: We first reported that decarboxylated amino acids and pyroGlu-Leu are present in undigested functional ingredients and/or formed from their proteins by in vitro protease digestion. When functional ingredients are ingested, these protein-derived compounds may exert anti-inflammatory and other beneficial effects. This methodology may be applied to other functional foods and ingredients, leading to understanding of their effects in the body. Keywords: Functional food, bioactive compound, decarboxylated amino acid, trace amine, in vitro protease digestion, LC–MS/MS.