**3. The evaluation of protein and peptide digestibility**

There are several approaches to the evaluation of protein digestibility. The most common of these include single-enzyme systems, based on pepsin and/or trypsin; whole-organ systems; cannula incision; and immobilized multiple digestive enzymes system. The latter model provides the most relevant data, while pepsin hydrolysis is the simplest. Several more rare models will be also described (for example, the use of cell fractions of small intestine, culture of Caco-2 cells, yeast surface enzymes, tissue fragments).

#### **3.1 Isolated enzymes**

One of the simplest models of protein digestion is the use of the purified major enzymes of stomach (pepsin) and pancreas (trypsin and chymotrypsin) (Horii et al. 2009; Huang et al. 2010; Kamata et al. 1982; Nielsen et al. 1988; Stanic et al. 2010; Yagami et al. 2000), implemented either separately of subsequently. The products of the digestion are usually analyzed using SDS-PAGE with the separation range from 3 to 20-30 kDa (Stanic et al. 2010; Yu et al. 2011) or reverse-phase HPLC (Bublin et al. 2008). In rare cases, mass spectrometry is also employed to characterize the digestion products (Dupont et al. 2010).

The procedure of pepsin and trypsin/chymotrypsin digestion is defined in the US and EU Pharmacopoeias (Council of Europe 2004; United States Pharmacopeial Convention 2006), and prescribes the exact composition of the incubation medium, enzyme to protein ratio and incubation time.

Several optimizations of enzyme digestion assay exist. First of all, the inclusion of non-protein components of gastric and intestinal juice, such as phosphatidyl choline and bile, and some potential food components (pectin) could significantly change the analysis result (Bublin et al. 2008; Dupont et al. 2010; Mandalari et al. 2009; Polovic et al. 2010). Not all proteins respond to such changes, but sometimes partially unfolded form of the protein is able to penetrate into the phosphatidyl choline vesicles. These interactions are probably responsible for slowing of gastric digestion by reducing the accessibility of the protein to pepsin (Moreno et al. 2005). Another option is the use not only of the major luminal proteases, but also of other enzymes: aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y (Dizdaroglu et al. 1984; Tonglet et al. 2001), or modeling of the subsequent interaction of food protein with saliva, gastric juice and duodenal juice (Versantvoort et al. 2005), and heating during food processing (Nielsen et al. 1988), a group of approaches which should be definitively considered if some protein seems to be stable. Horii *et al.* implemented another interesting modification of enzyme digestion procedure. They introduced test protein into the incubation medium not as a solution of individual molecules, but as a component, expressed

The situation changes significantly, as one tries to evaluate food protein toxicity and allergenicity. In this case stomach phase value greatly increases, as the low pH in this compartment often provides denaturation and thus inactivation and detoxification of ingested proteins. The oligopeptidases of the intestinal mucosa are not very important for the toxicity prevention, albeit they play a role as protectors against exogenous signals, as many proteins could possess hormonal properties (Widmaier et al. 2011). The pancreatic enzymes are still important for the defensive function, as they split large proteins into small

There are several approaches to the evaluation of protein digestibility. The most common of these include single-enzyme systems, based on pepsin and/or trypsin; whole-organ systems; cannula incision; and immobilized multiple digestive enzymes system. The latter model provides the most relevant data, while pepsin hydrolysis is the simplest. Several more rare models will be also described (for example, the use of cell fractions of small

One of the simplest models of protein digestion is the use of the purified major enzymes of stomach (pepsin) and pancreas (trypsin and chymotrypsin) (Horii et al. 2009; Huang et al. 2010; Kamata et al. 1982; Nielsen et al. 1988; Stanic et al. 2010; Yagami et al. 2000), implemented either separately of subsequently. The products of the digestion are usually analyzed using SDS-PAGE with the separation range from 3 to 20-30 kDa (Stanic et al. 2010; Yu et al. 2011) or reverse-phase HPLC (Bublin et al. 2008). In rare cases, mass spectrometry

The procedure of pepsin and trypsin/chymotrypsin digestion is defined in the US and EU Pharmacopoeias (Council of Europe 2004; United States Pharmacopeial Convention 2006), and prescribes the exact composition of the incubation medium, enzyme to protein ratio and

Several optimizations of enzyme digestion assay exist. First of all, the inclusion of non-protein components of gastric and intestinal juice, such as phosphatidyl choline and bile, and some potential food components (pectin) could significantly change the analysis result (Bublin et al. 2008; Dupont et al. 2010; Mandalari et al. 2009; Polovic et al. 2010). Not all proteins respond to such changes, but sometimes partially unfolded form of the protein is able to penetrate into the phosphatidyl choline vesicles. These interactions are probably responsible for slowing of gastric digestion by reducing the accessibility of the protein to pepsin (Moreno et al. 2005). Another option is the use not only of the major luminal proteases, but also of other enzymes: aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y (Dizdaroglu et al. 1984; Tonglet et al. 2001), or modeling of the subsequent interaction of food protein with saliva, gastric juice and duodenal juice (Versantvoort et al. 2005), and heating during food processing (Nielsen et al. 1988), a group of approaches which should be definitively considered if some protein seems to be stable. Horii *et al.* implemented another interesting modification of enzyme digestion procedure. They introduced test protein into the incubation medium not as a solution of individual molecules, but as a component, expressed

chunks, thus destroying potential immunological epitopes.

**3.1 Isolated enzymes**

incubation time.

**3. The evaluation of protein and peptide digestibility**

intestine, culture of Caco-2 cells, yeast surface enzymes, tissue fragments).

is also employed to characterize the digestion products (Dupont et al. 2010).

on the surface of a whole yeast cell. The aim of this study was to check the stability of expressed oral vaccine, and it has shown that indeed, a protein displayed on the yeast cell surface had an increased resistance to pepsin, possibly due to pepsin underwent steric hindrance and only digested the outermost side of the yeast cell wall protein (Horii et al. 2009).

An unquestionable advantage of individual enzyme digestion method is its simplicity and speed, as the whole analysis takes only about 2 to 3 hours to complete (including the products separation) and does not require any specific instrumentation or animal procedures. Wickham *et al.*, however, notices, that such models «are particularly useful where there is limited digestion, e.g. stomach, but are less applicable for total digestion studies. These types of models are predominately used for digestion studies on simple foods and isolated or purified nutrients, and are therefore ideal for assessments of the digestibility of isolated allergenic proteins» (Wickham et al. 2009).