**5. Conclusion**

Significant progress has been made in the modeling of protein digestion and absorption in gastrointestinal tract since the first works on this topic in the beginning of the XX century. A multitude of methods of various complexity, biological significance, animal requirements and human applicability now exist to answer the question on digestibility of proteins and peptides. The degree of biological processes imitation ranges from single luminal enzymes in simulated gastric or intestinal fluid model to sophisticated devices, which take in account grinding, mixing and turbulence forces and transition times, as well as isolated animal organs.

There is, however, no single method, which will suit every possible research goal. The problem is, that as the biological significance increases, time and labor requirements also rise tremendously, and methods became progressively less suitable for high throughput screening, which is often required. In addition to this, there is no experimental model, which could imitate every last component of the digestion process. The enzyme-only models and organ fragments ignore mechanical forces, digestive devices lack intracellular hydrolases and transport steps, isolated organs usually represent only one compartment of the whole system, and they fail to exert mechanical forces as well. As there is no efficient methodology of sampling of the digested products after they are absorbed, but before they enter blood or lymph, whole animal models are also limited – they can only answer questions, whether a protein was absorbed and if it was suitable for the sustaining normal growth of an organism. A very promising approach has appeared with the development of the highperformance computation methods, that is, molecular modeling. Theoretically, mathematical modeling is able to join data on the digestion patterns of various proteins from *in vitro* experiments with the results of *in vivo* tests of the ability of an organism to

one picture (*cf.,* for example, the discussion in the work by Sterchi et al. (Sterchi & Woodley

The aim of a substantial part of pharmacological studies of peptide- and protein-based therapeutics is often the evaluation of the disintegration, dissolution and drug release profiles of oral drug formulations (Vardakou et al. 2011). Thus another aspect of food digestion becomes important – the mechanical grinding and mixing forces, exerted on a pharmaceutical formulation by various digestive tract compartments. To simulate such conditions devices of various complexity are employed (Schasteen et al. 2002; Vardakou et al. 2011). The applicability of such devices for the drug form disintegration testing is unquestionable, however, usually they reproduce only the luminal phase of food digestion. Thus the evaluation of the ability of released protein or peptide to get to the blood flow

A short reference of available method types is provided in table 2. It should be noted, that some *in vitro* methods could be combined and expanded to provide more relevant data. For example, single-enzyme digestions by stomach and small intestine could be performed sequentially, the Caco-2 cell culture is often overlaid by a simulated gastric fluid and possibly mucin (to protect cells and imitate the similar layer present *in vivo*). The computational methods are the most adaptive and powerful ones, however, to date their usability is limited to large screening studies due to huge preparatory experimental work.

Significant progress has been made in the modeling of protein digestion and absorption in gastrointestinal tract since the first works on this topic in the beginning of the XX century. A multitude of methods of various complexity, biological significance, animal requirements and human applicability now exist to answer the question on digestibility of proteins and peptides. The degree of biological processes imitation ranges from single luminal enzymes in simulated gastric or intestinal fluid model to sophisticated devices, which take in account grinding, mixing and turbulence forces and transition times, as well as isolated animal

There is, however, no single method, which will suit every possible research goal. The problem is, that as the biological significance increases, time and labor requirements also rise tremendously, and methods became progressively less suitable for high throughput screening, which is often required. In addition to this, there is no experimental model, which could imitate every last component of the digestion process. The enzyme-only models and organ fragments ignore mechanical forces, digestive devices lack intracellular hydrolases and transport steps, isolated organs usually represent only one compartment of the whole system, and they fail to exert mechanical forces as well. As there is no efficient methodology of sampling of the digested products after they are absorbed, but before they enter blood or lymph, whole animal models are also limited – they can only answer questions, whether a protein was absorbed and if it was suitable for the sustaining normal growth of an organism. A very promising approach has appeared with the development of the highperformance computation methods, that is, molecular modeling. Theoretically, mathematical modeling is able to join data on the digestion patterns of various proteins from *in vitro* experiments with the results of *in vivo* tests of the ability of an organism to

1980a)).

intact requires additional methods.

**5. Conclusion** 

organs.

utilize these proteins. However, a vast array of preliminary data is required for optimization and validation of such models, and so they are relatively rare.

Thus, when considering various methods of protein digestibility assay, one should first of all define the limitations of the research at hand. If a high throughput screening is required? If there is a need for any data on digestion products? If absorption efficiency could vary and is essential? If a protein should serve as food or not? A possible decision tree for method optimization is presented on figure 1, albeit it should be considered more as a reference than a rule.

Fig. 1. A decision tree for protein or peptide digestibility assay choice
