Quantifying protein digestion kinetics of feed ingredients using a modified in vitro incubation assay

The kinetics of degradation of nutrients from diets or ingredients contributes to the nutritional value of feed ingredients. The aim of this study was to quantify the kinetics of nitrogen (N) solubilization and hydrolysis for feed ingredients varying in protein content and ileal protein digestibility by a modified 2-step enzymatic in vitro incubation assay. The amount of N subjected to in vitro incubation was standardized (approximately 0.48 g) among selected feed ingredients (rapeseed meal (RSM), fish meal, barley, peas, and zein). At the beginning of the stomach phase with pepsin, pH was set at 4 and then adjusted to 2 after 90 min, followed by a further 90 min of incubation. Subsequently, pH was adjusted to 6.8 and after 15 min, pancreatin and amyloglucosidase were added to simulate small intestinal digestion for 240 min. Total solubilized nitrogen (TSN) and low molecular weight nitrogen compounds (LMWN, <500 Da) were measured at several time points (0 to 435 min) during incubation. Flavourzyme (FL) or intestinal acetone powders from rat (IAPR) were added for 1 additional hour of incubation following the small intestine phase to simulate brush-border enzyme activity. Between 46% (RSM) and 73% (fish meal) of the TSN, and 39% (RSM) to 68% (fish meal) of the LMWN were seen during the stomach phase. Nitrogen solubilization in response to pH-change during the stomach phase was most pronounced in fish meal, having the highest rate of N solubilization during the first 10 min after adjusting pH from 4 to 2 (3.10%/min) and the highest N solubility (73%) and LMWN (68%) during the stomach phase. The kinetics of appearance of LMWN followed the pattern of N solubilization. During small intestinal phase, proteins in RSM and barley exhibited higher solubilization and hydrolysis yet achieved slightly lower solubility (76% for RSM and 77% for barley) and LMWN (73% for RSM and 70% for barley) compared with fish meal (86% for N solubility and 82% for LMWN) and peas (85% for N solubility and 72% for LMWN). The addition of FL or IAPR did not increase N solubilization or LMWN. Our modified in vitro assay allowed to quantify rate and extent of N solubilization and appearance of LMWN of feed ingredients varying in protein content and ileal digestibility, reflecting variation in protein characteristics, with potential effects on absorption kinetics in vivo.