Digestion study of proteins from cooked meat using an enzymatic microreactor

Abstract

A semi automatic flow procedure with photometric detection was developed for the study of meat protein digestion. This system comprised two independent flow pathways, gathered by two compartments. The gastric compartment was simulated by an ultrafiltration cell fitted with a 10 KDa cut off membrane and the intestinal compartment was simulated by a 1 KDa cut off dialysis membrane. The pathways were filled with solutions simulating digestive conditions. The proposed system was employed in digestion studies of whole protein extracts from raw and cooked (100 °C) meat. A mathematical modelling for the determination of the digestive kinetic constants was established. The results show that meat cooking leads to an important decrease of protein digestibility by proteases of the digestive tract.

Introduction

Meat and meat products are a good source of proteins for humans. These proteins are well balanced in amino acids and contain all the essential amino acids that humans can not synthesize. Nevertheless, meat processes (storage, cooking, salting, irradiation) can affect the physicochemical state of proteins and thus the bioavailability of amino acids. Indeed, during these processes, proteins are the target of free radical attack. Oxidation of amino acids leads to protein aggregation through the formation of disulfide and dityrosine bridges. Such reactions have been detected during meat cooking (Santé-Lhoutellier, Astruc, Marinova, Grève, & Gatellier, 2008) or during exposure of meat proteins to a chemical oxidative system (Morzel, Gatellier, Sayd, Renerre, & Laville, 2006). Protein aggregation can also be generated by the interaction of free amino groups of lysine with aldehydic products of lipid peroxidation and such reactions have been detected during meat storage (Gatellier et al., 2007, Renerre et al., 1996). Recently, we have reported that the increase of protein hydrophobicity observed during meat cooking was linked to protein aggregation (Santé-Lhoutellier et al., 2008). Many authors have demonstrated that aggregation of meat proteins can affect their degradation by enzymes of the digestive tract (Kamin-Belsky et al., 1996, Liu and Xiong, 2000, Santé-Lhoutellier et al., 2007, Santé-Lhoutellier et al., 2008). Besides less efficient amino acid assimilation, a reduced protein digestion rate would have a negative impact on human health. It has been demonstrated that fermentation of non hydrolysed proteins by colonic flora increases the risk of colon cancer (Evenepoel et al., 1998, Geypens et al., 1997). Thus estimation of protein digestion rate is of great interest in the evaluation of the nutritional and healthy qualities of meat products and that sensitive tools for the study of this process are required.

In digestion studies, animal models such as rats or cannulated mini pigs provide realistic information about the degradation and assimilation of proteins which is closely related to human digestion. Nevertheless these experiments are difficult and need procedures that few laboratories have at their disposal and they are inappropriate for large tests. Therefore it is not surprising that this approach is not commonly used and that in vitro models simulating the conditions existing in vivo have been proposed. Up till now, two in vitro approaches have been exploited for protein digestion rate analysis.

Some authors have reported the hydrolysis of proteins, in test tubes, using proteases of the digestive tract (pepsin, trypsin and chymotrypsin) in conditions of pH and temperature which mimic the digestive system (Kamin-Belsky et al., 1996, Liu and Xiong, 2000). Recently we monitored myofibrillar protein digestibility by such a method (Santé-Lhoutellier et al., 2007, Santé-Lhoutellier et al., 2008). These experiments in test tubes have many advantages. They are usually not expensive or time consuming as they only need basic equipment available in most laboratories. Nevertheless, these tests have a number of drawbacks and merit improvement to mimic the real digestive process. In vivo there is a continuity of protein cut by proteases while this in vitro approach always involves separate use of the gastric and pancreatic proteases. There is no doubt that treatment by pepsin prior to trypsin/chymotrypsin treatment will affect the nature of the latter enzyme products. Moreover, to decrease analysis time and increase sensitivity, high concentrations of enzyme are generally used which tends to produce undesirable auto digestion of the enzyme itself. Finally, isolation of hydrolysed amino acids and peptides from proteins always requires trichloro-acetic acid treatment and the cut off of this chemical treatment corresponds to compounds of approximately 20 KDa molecular weight which cannot migrate through the intestinal wall. In fact, these kinds of measurements, are more accurately described as in vitro enzymatic hydrolysis of protein than in vitro digestibility of protein.

There are computer-controlled systems that allow closer simulation of in vivo dynamic physiological processes occurring within the stomach and small intestine of humans. For example the gastric-small intestine system TIM-1 (TNO, Zeist, The Netherlands) reproduces with high reliability the main parameters of digestion, such as pH, temperature, gastric, biliary and pancreatic secretion, peristaltic mixing and passive absorption of water and small molecules. This system has been extensively used in pharmaceutical studies of drugs sensitive to digestive conditions (Blanquet et al., 2004, Blanquet et al., 2005). Even though this system might be closest to an in vivo situation, until now, it has not been applied to meat protein digestibility, perhaps because of the high cost and the difficulty of carrying out such experiments routinely.

This report describes efforts to develop an artificial digestive system more closely related to the in vivo process than the test tube method and less expensive than the gastric-small intestine system, involving the establishment of a semi automatic flow procedure that enables both the continuous monitoring of protein hydrolysis by the proteases of the digestive tract and the passive diffusion of amino acids and small peptides through a dialysis membrane. A mathematical model for the determination of the digestive kinetic constants was established and feasibility was ascertained by performing protein digestion assays with raw and cooked meat.