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. 2025 Jan 4:103:skaf190.
doi: 10.1093/jas/skaf190.

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

Shiyi Zhang  1   2 , Leon de Jonge  1 , Sonja de Vries  1 , Walter J J Gerrits  1
Affiliations

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

Shiyi Zhang et al. J Anim Sci. .

Abstract

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.

Keywords: amino acids; hydrolysis; peptides; solubilization.

Plain language summary

The nutritional value of pig feed is evaluated by nutrient digestion in the gastrointestinal tract. The kinetics (rate and extent) of amino acid absorption are important for assessing their availability for post-absorptive metabolism and are positively related to the kinetics of solubilization and hydrolysis. Soluble proteins flowing with the liquid phase of digesta move quickly from the stomach to the small intestine, allowing rapid hydrolysis and absorption. In vitro methods are commonly used to measure the kinetics of protein solubilization and hydrolysis, but they overlook the inconsistent pH in pig stomach and the brush border enzyme activity in the small intestine. We adapted an in vitro method to measure the kinetics of nitrogen (N) solubilization and hydrolysis and tested it in rapeseed meal, fish meal, peas, barley, and zein; sources that vary in protein content and digestibility. We found that the majority of N was solubilized during the stomach phase. The decrease of pH from 4 to 2 substantially increased N solubilization and hydrolysis, especially for fish meal. The appearance of low molecular weight N aligned closely with the N solubilization kinetics. The extended incubation, simulating hydrolysis of brush border enzymes using Flavourzyme or intestinal acetone powders from rats, did not enhance N hydrolysis in the selected ingredients.

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Figures

Figure 1.
Figure 1.
The effect of the addition of Flavourzyme or intestinal actone powders from rat on nitrogen (N) solubility (panel a) or appearance of low molecular weight nitrogen (LMWN) (panel b) during the subsequent 60 min incubation after a 3-step enzymatic in vitro incubation for rapeseed meal (RSM), fish meal, barley and peas. Error bars represent standard error of the mean, of 2 replicate observations, except for N-solubility of fish meal control where n = 1.
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