To meet the protein and amino acid requirements of individuals and of populations requires information not only about their requirements but also about the capacity of available foods to meet those requirements. Most of our current knowledge of the digestibility of food proteins and the methods to estimate it has been derived from work with animals. Because the microbiota of the large intestine alter the amino acid composition of the digesta, and because only trivial quantities of amino acids are absorbed intact from the large intestine, the current method of choice for assessing amino acid digestibility is ileal digestibility corrected for basal endogenous losses, that is, standardized ileal digestibility. For protein as a whole, however, because nitrogen absorbed in forms other than as amino acids can contribute to the nitrogen economy, the absorption of nitrogen over the whole digestive tract is the more appropriate measure. Most of the methods developed for estimating ileal amino acid outflow in animals are not directly applicable to man: the exception is the use of volunteers with an ileostomy. The flow and composition of ileal digesta in human subjects can also be measured by the infusion of a marker and withdrawal of samples through a naso-intestinal tube. However, this method is too demanding for routine use and is likely to be restricted to validating the application to humans of digestibility data obtained either from animals, of which the pig seems most suitable, or in vitro methods. Microbial activity in the gastrointestinal (GI) tract is not confined to the large intestine: the numbers and metabolic activity of the upper GI microbiota lead to substantial amounts of microbial protein leaving the ileum. It appears however that a large proportion of the amino acids used by the upper GI microbiota are preformed - from the diet or from endogenous materials - rather than from de novo synthesis. Although there are still uncertainties about the impact of microbial activity in the upper GI tract, the amino acid composition of ileal digesta provides the best available basis for estimating the proportion of dietary amino acids available for metabolism.

A comparative non-ruminant species view of the contribution of the large intestinal metabolism to inaccuracies in nitrogen and amino acid absorption measurements is provided to assess potential implications for the determination of crude protein/amino acid digestibility in adult humans consuming lower digestible protein sources. Most of the amino acids in the hindgut are constituents of the microorganisms and significant microbial metabolism of dietary and endogenous amino acids occurs. Bacterial metabolism of nitrogen-containing compounds leads to a significant disappearance of nitrogen in the large intestine. Literature data show that some 79 % of the nitrogen entering the large intestine of the horse is absorbed. For dogs, sows, and growing pigs these estimates are 49, 34 and 16 %, respectively. The coefficient of gut differentiation of humans compares closely to that of dogs while the coefficient of fermentation in humans is the lowest of all non-ruminant species and closest to that of cats and dogs. Large intestinal digesta transit times of humans compare closest to adult dogs. Significant amino acid metabolism has been shown to occur in the large intestine of the adult dog. Use of the growing pig as an animal model is likely to underestimate the fermentation of amino acids in the human large intestine. Based on the significant degree of fermentation of nitrogen-containing components in the large intestine of several non-ruminant species, it can be expected that determination of amino acid digestibility at a faecal level in humans consuming low quality proteins would not provide accurate estimates of the amino acids absorbed by the intestine.

The true ileal digestibility assay provides the most informative measure of digestibility to assess bioavailability of amino acids in foods for humans. To determine ‘true’ estimates of ileal amino acid digestibility, requires that endogenous amino acids present in digesta at the terminal ileum be quantified. The amounts of endogenous amino acids in ileal digesta can be determined after feeding an animal or human a protein-free diet (traditional approach) or by various methods after giving a protein-containing diet. When the protein-free method has been applied with adult human subjects an overall mean value (three separate studies) for endogenous ileal nitrogen flow of 800 mg N/d has been reported. This value is considerably lower than a comparable value obtained after feeding protein of 1852 mg N/d (mean of four separate studies), and thus endogenous ileal N and amino acids should be measured under conditions of protein alimentation. There is some confusion concerning the terminology used to define digestibility, with the term “true” digestibility having different adopted meanings. Here, true amino acid digestibility is defined as apparent amino acid digestibility corrected for the basal amino acid losses determined after giving either a protein-free or a protein-containing diet. Basal losses should be determined at a defined dry-matter and protein intake. The protein-free diet approach to determining endogenous amino acids is considered unphysiological and basal losses refer to ileal endogenous amino acid flows associated with digesta dry-matter flow, and not including “specific” effects of dietary factors such as non starch polysaccharides and anti nutritional factors. Arguments are advanced that the enzyme hydrolysed protein/ultra filtration method may be suitable for routine application with a cannulated pig model, to obtain physiologically-valid basal estimates of ileal endogenous amino acids to allow calculation of true ileal amino acid digestibility in the pig, and then prediction (via statistical relationships) of true coefficients of amino acid digestibility in humans.

Measures of amino acid (AA) digestibility are used widely to estimate bioavailability of AA in feed and food ingredients for monogastric animals. In principle, the digestibility assay is simpler than in vivo assessments of AA bioavailability and allows for simultaneous estimation of the bioavailability of all AA in an experimental diet. It is generally assumed that absorption of intact AA in the hindgut of monogastrics is minimal, even though colonocytes do contain AA transporters and have been shown to absorb AA. This assumption is supported by the observation that infusion of AA into the hindgut does not improve nitrogen balance in monogastrics. In addition, growth performance of monogastrics is more highly correlated with ileal than faecal AA digestibility. Therefore, ileal digestibility coefficients provide better estimates of AA bioavailability than faecal digestibility coefficients. Measures of apparent ileal digestibility (AID) of AA are confounded with endogenous gut AA losses (EAAL). The curvilinear increase in AID of AA with increasing dietary AA level has been attributed to the relatively large contribution of EAAL to total ileal AA flows at low dietary AA levels. Subtracting basal EAAL from total ileal AA flows yields standardized ileal digestibility (SID) coefficients that appear to be more additive than AID coefficients in mixtures of feed ingredients. An implicit assumption when using SID AA coefficients in diet formulation is that the post-absorptive utilization of AA is not influenced by the dietary protein source. This assumption appears inappropriate when using feed or food ingredients that have been over-heated, induce large amounts of EAAL, or contain substantial amounts of fermentable fibre. Improved understanding of processes that contribute to the discrepancy between bioavailability and ileal digestibility will allow a more effective use of AA digestibility coefficients in diet formulation.

Animal models have been commonly used for determining amino acid digestibility in humans. This allows digestibility assays to be undertaken more efficiently than those undertaken using humans directly. The laboratory rat, usually considered as a suitable animal model, has been widely used, especially as the rat is easy to raise and relatively inexpensive to house. Although more technically demanding, the pig has also been promoted as a useful model for human nutrition studies. It may be a better model than the rat, as it is a meal eater, its upper digestive tract is anatomically and physiologically closer to that of humans and it eats most foods consumed by humans. Amino acid digestibility may be determined either at the faecal or the ileal level, the latter being considered the most accurate. This contribution evaluates the suitability of the rat and pig as animal models for assessing ileal and faecal amino acid digestibility in humans. The drawbacks and advantages of using these animal models are discussed. The review is based mainly on results from controlled studies comparing both species; however, as the number of these studies is limited, data from indirect comparisons also provide insight.

The development, refinement and validation of in vitro digestibility assays for dietary protein and amino acids for single stomached mammals are reviewed. The general principles of in vitro digestibility assays and their limitations are discussed. In vitro protein digestibility assays must be accurate, rapid, cheap, simple, robust, adaptable and relevant to the processes of digestion, absorption, and metabolism. Simple in vitro methods have the potential to give useful measures of in vivo amino acid and protein digestibility for humans. In vitro methods, including the complex multi-component models of digestion simulating the various physical and chemical processes, require independent validation with in vivo data from the target species or an acceptable animal model using the most appropriate in vivo measure of digestibility. For protein sources devoid of anti-nutritional factors or plant fibre, true ileal digestibility is the recommended in vivo baseline, while for plant proteins the recommended in vivo assay is real ileal digestibility. More published comparative studies are required to adequately validate in vitro digestibility assays.

In this paper, we give an overview of our research exploring the impact of physical and chemical processing on food proteins. There are three themes, applied to the proteins of wheat, soya, egg and dairy foods. Firstly, the impact of the Maillard reaction on food proteins is discussed, with a particular focus on how the reactions might be harnessed to manipulate food texture. Secondly, the potential of enzymatic protein-protein crosslinking is considered, especially the enzyme transglutaminase. Thirdly, the broader question of how the aggregation of proteins within a food is altered by chemical and physical modification and how, in turn, this might impact on the overall nutritional quality of the food is considered.

Available amino acids are those absorbed from the gastrointestinal tract in a form suitable for body protein synthesis. True ileal digestible amino acids are determined based on the difference between dietary amino acid intake and unabsorbed dietary amino acids at the terminal ileum. The accuracy of ileal digestible amino acid estimates for predicting available amino acid content depends on several factors, including the accuracy of the amino acid analysis procedure. In heat processed foods, lysine can react with compounds to form nutritionally unavailable derivatives that are unstable during the hydrochloric acid hydrolysis step of amino acid analysis and can revert back to lysine causing an overestimate of available lysine. Recently, the true ileal digestible reactive (available) lysine assay based on guanidination has provided a means of accurately determining available lysine in processed foods. Methionine can be oxidised during processing to form methionine sulphoxide and methionine sulphone and cysteine oxidised to cysteic acid. Methionine sulphoxide, but not methionine sulphone or cysteic acid, is partially nutritionally available in some species of animal. Currently, methionine and cysteine are determined as methionine sulphone and cysteic acid respectively after quantitative oxidation prior to acid hydrolysis. Consequently, methionine and cysteine are overestimated if methionine sulphone or cysteic acid are present in the original material. Overall, given the problems associated with the analysis of some amino acids in processed foodstuffs, the available amino acid content may not always be accurately predicted by true ileal amino acid digestibility estimates. For such amino acids specific analytical strategies may be required.

The nutritive value of food protein sources is dependent on the amino acid composition and the bioavailability of the nutritionally indispensable amino acids. Traditionally the methods developed to determine amino acid bioavailability have focused on intestinal absorption or digestibility, which is calculated as the percent of amino acid intake that does not appear in digesta or faeces. Traditional digestibility based methods do not always account for gut endogenous amino acid losses or absorbed amino acids which are unavailable due to the effect of heat processing and the presence of anti-nutritional factors, though methods have been developed to address these issues. Furthermore, digestibility based methods require the use of animal models, thus there is a need to develop in vivo methods that can be applied directly in human subjects to identify the proportion of dietary amino acids which is bioavailable, or metabolically available to the body for protein synthesis following digestion and absorption. The indicator amino acid oxidation (IAAO) method developed in our laboratory for humans has been systematically applied to determine almost all indispensable amino acid requirements in adult humans. Oxidation of the indicator amino acid is inversely proportional to whole body protein synthesis and responds rapidly to changes in the bioavailability of amino acids for metabolic processes. Using the IAAO concept, we developed a new in vivo method in growing pigs, pregnant sows and adult humans to identify the metabolic availability of amino acids in foods. The stable isotope based metabolic availability method is suitable for rapid and routine analysis in humans, and can be used to integrate amino acid requirement data with dietary amino acid availability of foods.