In pigs, high protein diets have been related to post-weaning diarrhoea, which may be due to the production of protein fermentation metabolites that were shown to have harmful effects on the intestinal epithelium in vitro. In this review, we discussed in vivo effects of protein fermentation on the microbial composition and their protein catabolic activity as well as gut and overall health. The reviewed studies applied different dietary protein levels, which was assumed to result in contrasting fermentable protein levels. A general shift to N-utilisation microbial community including potential pathogens was observed, although microbial richness and diversity were not altered in the majority of the studies. Increasing dietary protein levels resulted in higher protein catabolic activity as evidenced by increased concentration of several protein fermentation metabolites like biogenic amines in the digesta of pigs. Moreover, changes in intestinal morphology, permeability and pro-inflammatory cytokine concentrations were observed and diarrhoea incidence was increased. Nevertheless, higher body weight and average daily gain were observed upon increasing dietary protein level. In conclusion, increasing dietary protein resulted in higher proteolytic fermentation, altered microbial community and intestinal physiology. Supplementing diets with fermentable carbohydrates could be a promising strategy to counteract these effects and should be further investigated.

We present a dynamic, semi-mechanistic, compartmental protein digestion model to study the kinetics of protein digestion. The digestive system is described as a series of eight compartments: one for the stomach, one for the duodenum, two for the jejunum and four for the ileum. The digestive processes are described by a set of zero or first order differential equations. The model considers ingestion of a meal, secretion of gastric and pancreatic juices, protein hydrolysis, grinding, transit and amino acid absorption. The model was used to simulate protein digestion of a meal composed of a solid and a liquid phase or one where both phases are blended into a homogeneous phase. Luminal volumes and pH of gastric and duodenal contents were estimated for both meals. Further, gastric emptying is described as a function of the energy density of the bolus, instead of the more common mass action approach.

Assessment of amino acid bioavailability is of key importance for the evaluation of protein quality; however, measuring ileal digestibility of dietary proteins in humans is challenging. Therefore, a less-invasive dual stable isotope tracer approach was developed. We aimed to test the assumption that the 15N:13C enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of 15N test protein. In a crossover design, 10 healthy adults were given a semi-liquid mixed meal containing 25 g (low protein) or 50 g (high protein) of 15N-labeled milk protein concentrate simultaneous with 0.4 g of highly 13C–enriched spirulina. The meal was distributed over multiple small portions, frequently provided every 20 min during a period of 160 min. For several amino acids, the blood 15N- related to 13C-isotopic enrichment ratio was determined at t = 0, 30, 60, 90, 120, 180, 240, 300, and 360 min and differences between the 2 meals were compared using paired analyses. No differences in 13C AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas 15N AUC of amino acids was ∼2 times larger on the high-protein meal (P < 0.001). Doubling the intake of 15N-labeled amino acids increased the 15N:13C ratio by a factor of 2.04 ± 0.445 for lysine and a factor between 1.8 and 2.2 for other analyzed amino acids, with only phenylalanine (2.26), methionine (2.48), and tryptophan (3.02) outside this range. The amino acid 15N:13C enrichment ratio in the peripheral circulation increased proportionally to the quantity of 15N-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using 15N-labeled protein, correction for, e.g., α-carbon 15N atom transamination is advised for determination of bioavailability of individual amino acids. This trial was registered at www.clinicaltrials.gov as NCT02966704.

Fermentation of dietary and endogenous protein in the hindgut is generally considered detrimental to the health of pigs. We investigated the in vitro fermentation potential of porcine endogenous protein in ileal digesta and colonic mucus, using a N-free buffer with an excess of fermentable carbohydrates. Urea, whey protein isolate (WPI, positive control), WPI hydrolysate (WPIH), and combinations of the latter two were used to validate the assay. A new biphasic model, including a linear end simulation, fitted to the gas production data over a 48-h period identified the time point when substrate fermentation ended. A higher degree of hydrolysis of WPI resulted in a higher maximum gas production rate (R_max, P < 0.01). Differences in R_max and the time required to reach R_max were observed among ileal digesta samples, with R_max increasing with the insoluble protein content, and the highest R_max occurring with colonic mucus samples (P < 0.05). The endogenous proteins entering the large intestine of pigs can ferment more rapidly compared to highly soluble and digestible protein sources, with R_max positively correlated with decreasing solubility of endogenous nitrogenous components. Protein fermentation in the hindgut of pigs can impact their health, affecting factors like growth rates and feed efficiency. Besides dietary protein, up to 50% of the protein entering the large intestine of growing pigs may be of endogenous origin. Therefore, we explored the fermentation potential of endogenous proteins compared to a well-known protein source, whey protein isolate (WPI). In developing and validating an in vitro gas production technique, we employed urea, WPI, WPI hydrolysate, and various combinations as substrates. The study introduces a new biphasic model for in vitro gas production, offering a detailed analysis of the fermentation process over a 48-h period. Our results revealed that porcine endogenous proteins can undergo rapid fermentation because the maximum gas production rate was higher compared to WPI. This insight is crucial for understanding the dynamics of protein fermentation in pigs. Additionally, we explored the solubility and molecular size of proteins, providing a comprehensive understanding of their fermentation characteristics. We found that endogenous proteins were less soluble compared to WPI but contained more smaller peptides. Unraveling the complexities of protein fermentation in pigs contributes to improvement of feed formulation for optimal gut health.

Understanding protein fermentation in the hindgut of pigs is essential due to its implications for health, and ileal digesta is commonly used to study this process in vitro. This study aimed to assess the feasibility of utilising in vitro digested residues as a replacement for ileal digesta in evaluating the protein fermentation potential. In vitro residues from cottonseed meal, maize germ meal, peanut meal, rapeseed cake, rapeseed meal, soyabean meal and sunflower meal were analysed using a modified gas production (GP) technique and curve fitting model to determine their fermentation dynamics and compare with the use of ileal digesta. Significant variations were observed in GP parameters between in vitro digested residues, indicating differences in nitrogen utilisation by fecal microbiota. Soyabean meal and sunflower meal exhibited the highest maximum GP rates (R_max), with values of 29·5 ± 0·6 and 28·0 ± 1·2 ml/h, respectively, while maize germ meal showed slowest protein utilisation (17·3 ± 0·2 ml/h). A positive relationship was found between the R_max of in vitro residues and ileal digesta (R² = 0·85, P < 0·01). However, GP potential (GP_s) showed a tendency for a negative relationship (R² = 0·39, P < 0·1), likely due to narrow observed GP_s values and the presence of varied endogenous proteins in ileal digesta. Our results demonstrate the potential of using in vitro digested residues as a substitute for ileal digesta in assessing the fermentation potential of protein ingredients, particularly regarding the rate of protein fermentation.

The dual isotope tracer approach was developed as a less invasive alternative for the measurement of ileal amino acid (AA) digestibility. This study aimed to validate the dual isotope tracer approach with the standardized oro-ileal balance method in growing pigs. Eight pigs were fitted with jugular catheters and ileal T-cannulas. At the test day, feed, containing intrinsically labeled ¹⁵N-milk protein and ¹³C-spirulina, was provided every half hour, during 240 minutes. Ileal digesta and ten jugular blood samples were collected between 0-540 minutes. Digesta samples were analyzed for isotopic enrichment, amino acid, and titanium concentrations for balance method calculations. Serum AA isotopic enrichment was measured for dual stable isotope tracer calculations. Using the oro-ileal balance method the average ileal AA digestibility of milk protein concentrate was 97.8±0.59% and of spirulina 81.5±2.44% (mean ± SD). Lysine digestibility was specifically evaluated, as it does not transaminate. Lysine digestibility of milk protein concentrate calculated according to the dual isotope tracer approach was 88.9 ± 8.35%, 9 % point lower than the value obtained with the oro-ileal method (98.1 ± 0.36, p=0.04). Moreover, Bland-Altman analysis showed the difference between methods was higher with lower mean lysine digestibility. This study observed differences between the dual isotope tracer approach and oro-ileal balance method for estimating lysine digestibility under the current experimental conditions with six pigs. This result may be due to methodological issues. Considering the use of ¹⁵N protein, conclusions on other AA that do not transaminate could not be drawn.