**2.1 An overview of the protein digestion in the digestive tract**

First of all, is should be noted, that excellent reviews and textbooks are readily available on the topic of digestive tract physiology and biochemistry (Freeman & Kim 1978; Widmaier et al. 2011), and we refer a curious reader them for more details; here only a short overview of this process is presented.

The digestive tract consists of several interconnected compartments: mouth (grinding and moisturizing of food), esophagus (connection and separation), stomach, small intestine (food digestion and absorption), and large intestine (food remnants digestion by symbiotic microbiota and undigested material excretion). Protein digestion is initiated in lumen of stomach, then proceeds in the intestinal lumen and is finalized at the surface and in the cytoplasm of intestinal mucosa cells. Additional digestion events occur in large intestine, where symbiotic microorganisms either digest protein remnants to amino acids, or convert them to other compounds of nutritional value.

Stomach is the first compartment, which is chemically active towards proteins. Its acidic pH serves three major functions: it sterilizes the ingested material, denaturates food protein and activates local proteolytic enzymes. Only one protease, pepsin, is secreted in stomach. Pepsin does not destruct proteins to amino acids, rather it splits them to smaller parts to increase its accessibility for successive digestion steps (Widmaier et al. 2011).

The core function of stomach is the control of graduated food delivery into small intestine, and so it is rather dispensable for the overall protein assimilation. The experimental data on animals and patients with total gastrectomy show that the organism even under this condition retains the ability to assimilate dietary protein adequately and remains in positive nitrogen balance (Freeman & Kim 1978).

The next digestion phase, pancreatic, occurs in small intestine. It is thought to be more critical than the gastric phase. Here the pH changes to more basic and several different sources of proteases come into play. The most abundant ones: trypsin, chymotrypsin, elastase and several carboxypeptidases, are secreted by pancreatic acinar cells in a form of precursors, which are activated by proteolytic cleavage (Widmaier et al. 2011). The first three enzymes are endopeptidases; they cleave protein or polypeptide substrates at different specific peptide linkages within their secondary structures. Carboxypeptidases are exopeptidases and act on terminal peptide bonds at the carboxyl terminus of protein. The products of the combined action of the aforementioned enzymes are small peptides and amino acids. These small peptides consist of 2-6 amino acid residues and account for over 60% of luminal amino nitrogen; the remaining 40% is associated with amino acids (Freeman & Kim 1978).

The results of Savoie *et al.* indicate that the amount and size of the resultant peptides and their stability and resistance to exhaustive hydrolysis is strongly influenced by the nature of the source protein. For example, while casein and gluten have the highest proportion of the 1-10 kDa peptide fraction in the first 2 h of digestion, the peptides from gluten are evidently less degraded in the subsequent 4 h. The folding and spatial orientation of the protein chains could explain this observation; the high number of Pro and Glu residues is expected to have contributed heavily in the low accessibility and efficacy of the hydrolytic action of pancreatic enzymes on Pro- and Glu-reach proteins (Savoie et al. 2005).

First of all, is should be noted, that excellent reviews and textbooks are readily available on the topic of digestive tract physiology and biochemistry (Freeman & Kim 1978; Widmaier et al. 2011), and we refer a curious reader them for more details; here only a short overview of

The digestive tract consists of several interconnected compartments: mouth (grinding and moisturizing of food), esophagus (connection and separation), stomach, small intestine (food digestion and absorption), and large intestine (food remnants digestion by symbiotic microbiota and undigested material excretion). Protein digestion is initiated in lumen of stomach, then proceeds in the intestinal lumen and is finalized at the surface and in the cytoplasm of intestinal mucosa cells. Additional digestion events occur in large intestine, where symbiotic microorganisms either digest protein remnants to amino acids, or convert

Stomach is the first compartment, which is chemically active towards proteins. Its acidic pH serves three major functions: it sterilizes the ingested material, denaturates food protein and activates local proteolytic enzymes. Only one protease, pepsin, is secreted in stomach. Pepsin does not destruct proteins to amino acids, rather it splits them to smaller parts to

The core function of stomach is the control of graduated food delivery into small intestine, and so it is rather dispensable for the overall protein assimilation. The experimental data on animals and patients with total gastrectomy show that the organism even under this condition retains the ability to assimilate dietary protein adequately and remains in positive

The next digestion phase, pancreatic, occurs in small intestine. It is thought to be more critical than the gastric phase. Here the pH changes to more basic and several different sources of proteases come into play. The most abundant ones: trypsin, chymotrypsin, elastase and several carboxypeptidases, are secreted by pancreatic acinar cells in a form of precursors, which are activated by proteolytic cleavage (Widmaier et al. 2011). The first three enzymes are endopeptidases; they cleave protein or polypeptide substrates at different specific peptide linkages within their secondary structures. Carboxypeptidases are exopeptidases and act on terminal peptide bonds at the carboxyl terminus of protein. The products of the combined action of the aforementioned enzymes are small peptides and amino acids. These small peptides consist of 2-6 amino acid residues and account for over 60% of luminal amino nitrogen; the remaining 40% is associated with amino acids (Freeman

The results of Savoie *et al.* indicate that the amount and size of the resultant peptides and their stability and resistance to exhaustive hydrolysis is strongly influenced by the nature of the source protein. For example, while casein and gluten have the highest proportion of the 1-10 kDa peptide fraction in the first 2 h of digestion, the peptides from gluten are evidently less degraded in the subsequent 4 h. The folding and spatial orientation of the protein chains could explain this observation; the high number of Pro and Glu residues is expected to have contributed heavily in the low accessibility and efficacy of the hydrolytic action of

pancreatic enzymes on Pro- and Glu-reach proteins (Savoie et al. 2005).

increase its accessibility for successive digestion steps (Widmaier et al. 2011).

**2.1 An overview of the protein digestion in the digestive tract** 

this process is presented.

them to other compounds of nutritional value.

nitrogen balance (Freeman & Kim 1978).

& Kim 1978).

The core properties of the luminal proteases are summarized in table 1, to give the reader an understanding of the processes, which take place at different stages of digestion. It should be noted that most digestive enzymes are present not as a single entity, but rather as a group of slightly different molecules with quite similar substrate specificity and kinetic properties. Some authors suggest it to be a sign of evolutionary importance of these enzymes (Silk et al. 1976). A detailed discussion of these enzymes is, however, beyond the scope of this chapter, and we refer a curious reader to the excellent reviews available (Freeman & Kim 1978; Turner 1968; Whitcomb & Lowe 2007).

Besides the common luminal enzymes, which are secreted by specialized organ – pancreas, a significant amount of intestinal wall proteases is also present in the lumen. The sources of these enzymes are desquamated mucosal cells resulting from active replacement of the lining epithelium. An approximate quantity of these enzymes is about 30 g per day (Freeman & Kim 1978; Gropper et al. 2009).

Chemical assay data, thermostability studies, and examination of electrophoretic mobilities of luminal peptide hydrolases indicate that aforementioned jejunal enzymes originate predominantly from the cytoplasm of intestinal mucosal cells, whereas the brush border of mucosal cells is a major source of the enzymes in the ileum. All of these enzymes recognize primarily short peptides and are aminopeptidases; they are discussed in more detail in the next section.

The products of luminal proteolysis are free amino acids and small peptides having a chain length of two to six amino acid residues. Analysis of post-prandial intestinal contents aspirated from human jejunum reveals that approximately only one-third of the total amino acid content exists in the free form (Silk et al. 1985). Some of the short peptides are directly absorbed by specific transport systems. However, to increase the overall efficiency of the assimilation process the mucosal cells of intestinal wall provide an additional source of proteolytic enzymes. As these cells line the intestinal wall and are intensively shed into lumen, they contribute both to luminal and parietal digestion. A profound characteristic of mucosal cells is high aminopeptidase activity. This peptidase activity is located in two main subcellular fractions: the cytoplasm and the brush border membrane. The brush border membrane fraction is associated with less than 10% of the total hydrolytic cellular activity for dipeptides but as much as 60% for tripeptides (Freeman & Kim 1978; Sterchi & Woodley 1980a).

The whole digestion process is quite rapid in man, as well as in rat. So, according to the data of Curtis *et al.*, 44.9 % of the meal had left the stomach of a rat in 30 min, and 84.7 % of the meal had left the stomach in 60 min. At 30 min and thereafter, negligible amounts of 14Clabeled protein, which was included in the meal, were detected in intestinal contents of proximal third of small intestine. At 30 min and 1 hr, 14C accumulated in medial and distal thirds of small intestine. At 2, 3 and 4 hr small quantities of 14C were detected in intestinal contents of distal third of small intestine and the colon. The absorption of ingested protein was virtually complete 2 hr after administration of the meal (Curtis et al. 1978). In human, the situation is quite the same: digestion and absorption of dietary protein are completed in the jejunum, the whole process of digestion and absorption reaches its maximum at about 45 min or possibly earlier, and absorption may be largely complete within 75 to 90 min. However, the data of some studies show that the remnants of dietary protein can be recovered from the ileum as late as 4 hr after a meal (Adibi 1976). Nevertheless, it appears

Methods of Protein Digestive Stability Assay – State of the Art 215

reasonable to assume that under normal conditions the greatest portion of dietary proteins is digested and absorbed in the upper small intestine (Adibi 1976; Silk 1980; Silk et al. 1985).

In addition to the luminal proteases, the mucosal cells that line the intestinal wall also possess two kinds of proteolytic enzymes: membrane-bound, which are primarily localized in the brush border area, and cytoplasmic. The core difference of these enzymes from the luminal ones is that they are oligopeptidases, that is, recognize only short peptides of 2-6 amino acids in length. The profound characteristics of these enzymes are that they are aminopeptidases, so the N-terminal amino acid residue of peptides is the determinant of their rate and substrate specificity, and all have pH optima in alkaline (7.5-8.5) range (Adibi

To date, in brush border membranes in human and rat were identified at least 6 aminopeptidases (aminopeptidase 1, aminopeptidase 2, dipeptidylpeptidase IV, gammaglutamyltranspeptidase, aminopeptidase A and aminopeptidase M) (Kim et al. 1972; Sterchi & Woodley 1980b), and in the cytosol of the mucosal epithelial cells at least 4 enzymes were detected (Kim et al. 1972; Schiller et al. 1977). The specificity towards peptide length is distributed unequally between the subcellular fractions: the majority of dipetides (80 to 95%) are hydrolyzed by cytoplasmic enzymes (Adibi 1976; Krzysik & Adibi 1977), 10-60% of the tripeptidase activity is also in the brush border membrane (Sterchi & Woodley 1980a), and activity against tetrapeptides is located exclusively in the brush border membrane (Sterchi & Woodley 1980a). The ability of brush border enzymes to hydrolyze penta- and hexapeptides has been reported (Adibi 1976), however, little details are still known about

Some, but not all, cytosolic and brush border membrane peptidases also display some preference to the amino acid composition of substrate. Thus, aminopeptidase 1 from brush border is most active towards aliphatic tripeptides (Sterchi & Woodley 1980b), although most activity against aliphatic peptides is located in cytosol; dipeptidases of brush border prefer substrates with aromatic amino acid at the N-terminus (Sterchi & Woodley 1980a). An interesting exception are proline-containing peptides, which are hydrolyzed by a special hydrolase in the cytosolic fraction (Freeman & Kim 1978; Smith & Bergmann 1944). In normal conditions, 80-90% of hydrolase activity in rat and human is localized within the cytosolic fraction (Kim et al. 1972), starvation increases the peptide hydrolase activity of the

The distribution of peptide hydrolase activity between brush border membrane and cytosol varies considerably with species (Sterchi & Woodley 1980a), the enzymes from different species have different electrophoretic mobility, but, at the same time, by the activity and substrate preference they often could be grouped in a strikingly similar manner (Kim et al.

The absorption is the last and the most controversial phase of protein digestion. Theoretically, an organism requires only amino acids to produce energy and rebuild lost proteins. The foreign proteins and peptides should be avoided, as they could transmit

cytosol fraction, while that of the brush border fraction is decreased (Adibi 1976).

**2.3 Peptide and protein transport across the intestinal wall** 

**2.2 Enzymes of the intestinal wall** 

1976).

this activity.

1972).


Table 1. Principal properties of protein hydrolases

reasonable to assume that under normal conditions the greatest portion of dietary proteins is digested and absorbed in the upper small intestine (Adibi 1976; Silk 1980; Silk et al. 1985).

### **2.2 Enzymes of the intestinal wall**

214 New Advances in the Basic and Clinical Gastroenterology

Stomach lumen Endopeptidase,

Ileum lumen Endopeptidase;

Ileum lumen Endopeptidase,

and localization Properties References

cleaves at Phe, Tyr, Leu, and Val

cleaves peptide bond at Lys and Arg

cleaves bonds at aliphatic amino acid

cleaves at Ala, Gly, Ser

aromatic amino acids from carboxyl terminus of substrate

Arg or Lys from Cterminus of substrate

Hydrolyze amino acid residue at the Nterminus of hexa- to

Hydrolyze amino acid residue at the Nterminus of tri- and

tripeptides

dipeptides

residues

Ileum lumen Exopeptidase, cleaves

Ileum lumen Exopeptidase, cleaves

(Freeman & Kim 1978; Szecsi 1992; Turner 1968; Whitecross et al.

(Feinstein et al. 1974; Freeman & Kim 1978; Savoie et al. 2005; Whitcomb & Lowe 2007)

(Bender & Killheffer 1973; Savoie et al. 2005; Whitcomb & Lowe

(Whitcomb & Lowe 2007)

(Whitcomb & Lowe 2007)

(Whitcomb & Lowe 2007)

(Kim et al. 1972; Sterchi & Woodley

(Kim et al. 1972; Schiller et al. 1977)

1980b)

2007)

1973)

Enzymes Compartment

Elastases: Two isoforms Ileum lumen Endopeptidase,

Small intestine epithelium brush

Small intestine epithelium cytoplasm

Table 1. Principal properties of protein hydrolases

border

Stomach proteases

Pancreatic proteases

Chimotrypsins: Two isoforms + chymotrypsin-

Carboxypeptidases A: A1,

Carboxypeptidases B: B1, B2,

Intestinal wall proteases

aminopeptidases (distinct enzymes): aminopeptidase 1,

glutamyltranspeptidase, aminopeptidase A, aminopeptidase M

aminopeptidases: four distinct enzymes

pancreasin

like protease

A2, A3

N, M, E, AEBP1

Brush border

gamma-

Cytoplasmic

aminopeptidase 2, dipeptidylpeptidase IV,

Trypsins: Cationic trypsin, anionic trypsin, mesotrypsin,

Pepsins: Pepsin, pepsin B, gastricsin, chymosin A, renin

In addition to the luminal proteases, the mucosal cells that line the intestinal wall also possess two kinds of proteolytic enzymes: membrane-bound, which are primarily localized in the brush border area, and cytoplasmic. The core difference of these enzymes from the luminal ones is that they are oligopeptidases, that is, recognize only short peptides of 2-6 amino acids in length. The profound characteristics of these enzymes are that they are aminopeptidases, so the N-terminal amino acid residue of peptides is the determinant of their rate and substrate specificity, and all have pH optima in alkaline (7.5-8.5) range (Adibi 1976).

To date, in brush border membranes in human and rat were identified at least 6 aminopeptidases (aminopeptidase 1, aminopeptidase 2, dipeptidylpeptidase IV, gammaglutamyltranspeptidase, aminopeptidase A and aminopeptidase M) (Kim et al. 1972; Sterchi & Woodley 1980b), and in the cytosol of the mucosal epithelial cells at least 4 enzymes were detected (Kim et al. 1972; Schiller et al. 1977). The specificity towards peptide length is distributed unequally between the subcellular fractions: the majority of dipetides (80 to 95%) are hydrolyzed by cytoplasmic enzymes (Adibi 1976; Krzysik & Adibi 1977), 10-60% of the tripeptidase activity is also in the brush border membrane (Sterchi & Woodley 1980a), and activity against tetrapeptides is located exclusively in the brush border membrane (Sterchi & Woodley 1980a). The ability of brush border enzymes to hydrolyze penta- and hexapeptides has been reported (Adibi 1976), however, little details are still known about this activity.

Some, but not all, cytosolic and brush border membrane peptidases also display some preference to the amino acid composition of substrate. Thus, aminopeptidase 1 from brush border is most active towards aliphatic tripeptides (Sterchi & Woodley 1980b), although most activity against aliphatic peptides is located in cytosol; dipeptidases of brush border prefer substrates with aromatic amino acid at the N-terminus (Sterchi & Woodley 1980a). An interesting exception are proline-containing peptides, which are hydrolyzed by a special hydrolase in the cytosolic fraction (Freeman & Kim 1978; Smith & Bergmann 1944). In normal conditions, 80-90% of hydrolase activity in rat and human is localized within the cytosolic fraction (Kim et al. 1972), starvation increases the peptide hydrolase activity of the cytosol fraction, while that of the brush border fraction is decreased (Adibi 1976).

The distribution of peptide hydrolase activity between brush border membrane and cytosol varies considerably with species (Sterchi & Woodley 1980a), the enzymes from different species have different electrophoretic mobility, but, at the same time, by the activity and substrate preference they often could be grouped in a strikingly similar manner (Kim et al. 1972).

#### **2.3 Peptide and protein transport across the intestinal wall**

The absorption is the last and the most controversial phase of protein digestion. Theoretically, an organism requires only amino acids to produce energy and rebuild lost proteins. The foreign proteins and peptides should be avoided, as they could transmit

Methods of Protein Digestive Stability Assay – State of the Art 217

towards possible immune response. Paracellular transport across the epithelial barrier is highly regulated and is able to leave access to small molecules (44 angstroms), but also allows the passage of larger molecules including small peptides and bacterial lipopolysaccharides (500-1500 Da). A particular subset of dendritic cells (CX3CR11) is known to extend their dendrites in the lumen to collect bacteria under steady-state conditions, a process further accentuated upon inflammation. It is not yet clear whether this process can also take place for food antigens and whether this could contribute to the selective passage of antigens across the intestinal barrier and further presentation to the

Normally, large molecules are restricted from access to the intestinal wall by the mucus layer, which blocks passage of molecules of over 17 kDa (Jin et al. 2006). The intestinal cell layer permeability towards peptides and large molecules, on the other hand, is tightly bound to the external conditions. Thus, during fasting the efficiency of small peptide absorption increases (Silk et al. 1985). Then, a specific consequence of allergic reactions is the increased permeability towards large molecules via paracellular pathway, which can persist for a significant time after acute immune response is over (Perrier & Corthésy 2011). *In vitro* studies on confluent cultures of the enterocyte-like cell line Caco-2 have shown, that a model protein beta-lactoglobulin is taken by endocytosis, and whilst the majority of the protein was degraded intracellularly, around a third was transported across the cells (Wickham et

The functional importance of different stages of protein digestion process is unequal, albeit the exact significance of each of them varies depending on whether food toxicity/allergenicity or nutritive value is considered. As one could expect, the nutritive value of a protein is the most stable characteristic in context of the involvement of different digestive tract compartments. Thus, stomach and mucosal peptidases are dispensable for this function, and individual amino acid transport is also not very important if peptide transport is alive. The core functionality appears to be the one of pancreatic endopeptidases, which should split food protein into peptides of 3-2 amino acids; this activity is logically

In addition to this, it should be noted, that peptide transport role is more impotant than the amino acid one. In evaluations involving both highly digestible proteins such as egg and less digestible protein sources such as corn as well as free amino acid mixtures, the food source of protein routinely gave better nitrogen balance test results than did the counterpart amino acid diets. Explanations for these results included the following: (a) that the immediate availability of purified amino acids might lead to overall overtaxing of absorption mechanism systems or competition among particular amino acids might lead to selected competitive inhibition of absorption of particular amino acids and hence to reduced efficiency of their utilization; (b) that rapid absorption of amino acids over a short period of time rather than over a longer period of time might overtax the body's ability to efficiently use amino acids for protein synthesis purposes, thus leading to increased deamination of amino acids and utilization for energy purposes; (c) that experimental diets containing purified amino acids were not truly matched to those containing food proteins in contents of

mucosal immune system (Perrier & Corthésy 2011).

**2.4 Comparison of relative roles of protein digestion stages** 

protected by the existence of multiple enzyme isoforms.

all trace nutrients and results reflected these deficits (Kies 1981).

al. 2009).

unwanted signals or even be toxic or mutagenic. The reality, however, is much more complicated. Although amino nitrogen in postprandial blood is almost entirely in the form of free amino acids, few small peptides do appear in portal blood. These include short proline- and hydroxyproline-containing peptides after gelatin ingestion, and both carnosine and anserine after ingestion of certain meats, specifically chicken breast. Significant amounts of whole protein and other large macromolecules are absorbed intact by fetal and neonatal small intestine (Freeman & Kim 1978).

The transport of amino acids is active and depends on a gradient of sodium ions across the brush border membrane of intestinal epithelial cell. Three major group-specific active transport systems exist for: a) monoamino monocarboxylic (neutral) amino acids, b) dibasic amino acids and cysteine, and c) dicarboxylic (acidic) amino acids (Silk et al. 1985). Methionine and the branched chain amino acids (leucine, isoleucine, and valine) are absorbed most rapidly from amino acid mixture. Among the 18 common dietary amino acids, aspartate and glutamate are most slowly absorbed, as are tryptophan and threonine among the eight essential amino acids. As a group, essential amino acids are better absorbed than nonessential amino acids (Adibi 1976).

Small peptides are also actively absorbed, but their transporters are different from those for amino acids. As such, dipeptides and tripeptides are actively transported against a concentration gradient, and they share a common transport mechanism. There are at least two general strategies for transport of small peptides. First, small peptides resistant to brush border membrane peptidases appear to be transported intact across the brush border membrane and are later hydrolyzed by the cytoplasmic peptidases. Second, small peptides, having a higher affinity for brush border membrane peptidases are likely hydrolyzed primarily at the brush border membrane. The hydrolysis products, including free amino acids and dipeptides or tripeptides, are then absorbed by their respective transport systems (Freeman & Kim 1978; Sterchi & Woodley 1980a). There are little data on the active transport of tetrapeptides, and these data do not support the hypothesis on existence of such process (Silk et al. 1985).

One of important differences between amino acid and small peptide transport is that the capacity of free amino acid absorption is far greater in the jejunum than in the ileum, while that for peptide transport is evenly distributed. High-affinity amino acids competitively inhibit the absorption of low-affinity amino acids. However, absorption rates of amino acids by the intestinal epithelial cells are significantly greater in the form of low molecular weight peptides than the corresponding free amino acids, with the competition between amino acids being abolished. These spatio-temporal events directly influence amino acid concentration in blood flow, which in turn affects many physiological and metabolic phenomena as well as influencing the overall rate of protein synthesis (Savoie et al. 2005).

As to the peptide and protein molecules that are larger than 6 amino acids, so they pass through the intestinal wall differently. There are several routes, which allow the more or less non-specific passage of molecules: specialized microfold cells of Peyer's patches, isolated lymphoid follicles, through the epithelial cells (transcellular pathway) and between these cells (paracellular pathway) (Perrier & Corthésy 2011). The mucosa-associated lymphoid tissue, comprising Peyer's patches and isolated lymphoid follicles, is covered by the follicleassociated epithelium containing microfold cells. These cells have the capacity to engulf particulate antigens, and transport them to underlying dendritic cells thus contribute

unwanted signals or even be toxic or mutagenic. The reality, however, is much more complicated. Although amino nitrogen in postprandial blood is almost entirely in the form of free amino acids, few small peptides do appear in portal blood. These include short proline- and hydroxyproline-containing peptides after gelatin ingestion, and both carnosine and anserine after ingestion of certain meats, specifically chicken breast. Significant amounts of whole protein and other large macromolecules are absorbed intact by fetal and neonatal

The transport of amino acids is active and depends on a gradient of sodium ions across the brush border membrane of intestinal epithelial cell. Three major group-specific active transport systems exist for: a) monoamino monocarboxylic (neutral) amino acids, b) dibasic amino acids and cysteine, and c) dicarboxylic (acidic) amino acids (Silk et al. 1985). Methionine and the branched chain amino acids (leucine, isoleucine, and valine) are absorbed most rapidly from amino acid mixture. Among the 18 common dietary amino acids, aspartate and glutamate are most slowly absorbed, as are tryptophan and threonine among the eight essential amino acids. As a group, essential amino acids are better absorbed

Small peptides are also actively absorbed, but their transporters are different from those for amino acids. As such, dipeptides and tripeptides are actively transported against a concentration gradient, and they share a common transport mechanism. There are at least two general strategies for transport of small peptides. First, small peptides resistant to brush border membrane peptidases appear to be transported intact across the brush border membrane and are later hydrolyzed by the cytoplasmic peptidases. Second, small peptides, having a higher affinity for brush border membrane peptidases are likely hydrolyzed primarily at the brush border membrane. The hydrolysis products, including free amino acids and dipeptides or tripeptides, are then absorbed by their respective transport systems (Freeman & Kim 1978; Sterchi & Woodley 1980a). There are little data on the active transport of tetrapeptides, and these data do not support the hypothesis on existence of such process

One of important differences between amino acid and small peptide transport is that the capacity of free amino acid absorption is far greater in the jejunum than in the ileum, while that for peptide transport is evenly distributed. High-affinity amino acids competitively inhibit the absorption of low-affinity amino acids. However, absorption rates of amino acids by the intestinal epithelial cells are significantly greater in the form of low molecular weight peptides than the corresponding free amino acids, with the competition between amino acids being abolished. These spatio-temporal events directly influence amino acid concentration in blood flow, which in turn affects many physiological and metabolic phenomena as well as influencing the overall rate of protein synthesis (Savoie et al. 2005). As to the peptide and protein molecules that are larger than 6 amino acids, so they pass through the intestinal wall differently. There are several routes, which allow the more or less non-specific passage of molecules: specialized microfold cells of Peyer's patches, isolated lymphoid follicles, through the epithelial cells (transcellular pathway) and between these cells (paracellular pathway) (Perrier & Corthésy 2011). The mucosa-associated lymphoid tissue, comprising Peyer's patches and isolated lymphoid follicles, is covered by the follicleassociated epithelium containing microfold cells. These cells have the capacity to engulf particulate antigens, and transport them to underlying dendritic cells thus contribute

small intestine (Freeman & Kim 1978).

than nonessential amino acids (Adibi 1976).

(Silk et al. 1985).

towards possible immune response. Paracellular transport across the epithelial barrier is highly regulated and is able to leave access to small molecules (44 angstroms), but also allows the passage of larger molecules including small peptides and bacterial lipopolysaccharides (500-1500 Da). A particular subset of dendritic cells (CX3CR11) is known to extend their dendrites in the lumen to collect bacteria under steady-state conditions, a process further accentuated upon inflammation. It is not yet clear whether this process can also take place for food antigens and whether this could contribute to the selective passage of antigens across the intestinal barrier and further presentation to the mucosal immune system (Perrier & Corthésy 2011).

Normally, large molecules are restricted from access to the intestinal wall by the mucus layer, which blocks passage of molecules of over 17 kDa (Jin et al. 2006). The intestinal cell layer permeability towards peptides and large molecules, on the other hand, is tightly bound to the external conditions. Thus, during fasting the efficiency of small peptide absorption increases (Silk et al. 1985). Then, a specific consequence of allergic reactions is the increased permeability towards large molecules via paracellular pathway, which can persist for a significant time after acute immune response is over (Perrier & Corthésy 2011). *In vitro* studies on confluent cultures of the enterocyte-like cell line Caco-2 have shown, that a model protein beta-lactoglobulin is taken by endocytosis, and whilst the majority of the protein was degraded intracellularly, around a third was transported across the cells (Wickham et al. 2009).

#### **2.4 Comparison of relative roles of protein digestion stages**

The functional importance of different stages of protein digestion process is unequal, albeit the exact significance of each of them varies depending on whether food toxicity/allergenicity or nutritive value is considered. As one could expect, the nutritive value of a protein is the most stable characteristic in context of the involvement of different digestive tract compartments. Thus, stomach and mucosal peptidases are dispensable for this function, and individual amino acid transport is also not very important if peptide transport is alive. The core functionality appears to be the one of pancreatic endopeptidases, which should split food protein into peptides of 3-2 amino acids; this activity is logically protected by the existence of multiple enzyme isoforms.

In addition to this, it should be noted, that peptide transport role is more impotant than the amino acid one. In evaluations involving both highly digestible proteins such as egg and less digestible protein sources such as corn as well as free amino acid mixtures, the food source of protein routinely gave better nitrogen balance test results than did the counterpart amino acid diets. Explanations for these results included the following: (a) that the immediate availability of purified amino acids might lead to overall overtaxing of absorption mechanism systems or competition among particular amino acids might lead to selected competitive inhibition of absorption of particular amino acids and hence to reduced efficiency of their utilization; (b) that rapid absorption of amino acids over a short period of time rather than over a longer period of time might overtax the body's ability to efficiently use amino acids for protein synthesis purposes, thus leading to increased deamination of amino acids and utilization for energy purposes; (c) that experimental diets containing purified amino acids were not truly matched to those containing food proteins in contents of all trace nutrients and results reflected these deficits (Kies 1981).

Methods of Protein Digestive Stability Assay – State of the Art 219

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

An intermediate between *in vivo* and *in vitro* protein digestibility assays is the utilization of isolated digestive tract compartments (Polovic et al. 2010; Wickham et al. 2009). The experimental procedure requires an animal to be sacrificed immediately before the measurement. After that, the required compartment is surgically removed and either placed into a vial with a simulated fluid of this compartment, or sealed both sides with the salt mixture with substrate within. A variation of this technique implies the inversion of a digestive tract compartment inside out before sealing, so the resultant bag is put into a beaker with the substrate-containing solution, and the digestion products diffuse to the internal volume of such bag. Some researchers add glucose to the incubation medium to

The strong point of this approach is that the digestion and absorption systems of the intestine remain intact, and so the biological significance of the obtained results is very high, and the question on whether the digestion products really pass through the intestinal wall is immediately answered. The isolation of reaction products is also rather simple, as they are automatically separated from the source protein and mucosal proteins (if a sealed compartment bag is employed). The downsides of this method are, of course, high animal

*In vivo* methods, albeit the most complex ones, are at the same time the most informative ones. The two possible strategies within this group are based on the evaluation of either the remnants of the substrate within or at the end of digestive tract, or of the assimilation of the

The methods based on protein nitrogen utilization by an organism are subject of several great reviews, and we refer a curious reader to them for more details (Bender 1958; Darragh & Hodgkinson 2000; von der Decken 1983). Below, a short description of method families

The first group of methods (protein efficiency ratio, net protein retention, rat-repletion method) use direct determination of the weight of animals, fed with a test protein. The

of isolated allergenic proteins» (Wickham et al. 2009).

requirements and low suitability for high-throughput screening.

**3.2 Isolated digestive tract compartments** 

provide the tissue with energy.

**3.3** *In vivo* **methods** 

will be given.

digestion products by the organism.

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 chunks, thus destroying potential immunological epitopes.
