Thermal processing is central to the production of many feed ingredients. The temperature and chemicals during thermal processing and drying may result in significant changes to proteins, and amino acids that compose them, through a variety of chemical reactions. This “heat damage” can have deleterious effects on the nutritional quality of ingredients. Processing of proteins at high temperatures can result in protein cross-linking, the formation of covalent bonds between polypeptide chains within a protein or between proteins. There is emerging evidence that the presence of crosslinks in the structure of proteins may make parts of these proteins more resistant to the action of proteases. This study aimed to examine protein cross-linking associated with thermal processing and the impacts of heat damage on the digestibility of model animal protein.
Freeze-dried chicken breast meat (CM) samples were subjected to processing at different temperatures (70, 80, 95, 110, 120, and 130°C) for 45 min in an autoclave in absence (Control) or presence of chemical agents. L-serine and ferrous sulfate were used to stimulate amino acid cross-linking (Cross-Linking) and protein oxidation (ProtOx), respectively. Amino acids and cross-linked amino acids, namely lysinoalanine (LAL), lanthionine (LAN) and β-aminoalanine (BAL), were quantified using a standard amino acid analysis technique by UPLC. The content of sulfhydryl (S-H) and disulphide (S-S) bonds in CM was monitored using FT-Raman spectroscopy. The pepsin digestibility of heat-treated CMs was determined at two pepsin concentrations (i.e. 0.2% and 0.002%).
Exposing CM samples to increasing temperatures in the absence or presence of L-serine and ferrous sulfate resulted in significant decreases in several essential amino acids (lysine, arginine, histidine, threonine, and methionine) and significant increases in the formation of cross-linked amino acids, notably LAL and LAN. Higher concentrations of cross-linked amino acids were observed in Cross-Linking and ProtOx treatments compared to Control groups. Processing temperatures exceeding 110°C were associated with increased concentrations of LAL and LAN. S-H and S-S bonds contents of CM decreased quadratically in the Control and ProtOx treatment groups with increasing temperature, indicating that the formation of disulphide bridge did not appear to be significant under the conditions examined. In vitro pepsin digestibility suggests that thermal processing could significantly affect the protein digestibility of heat-treated CMs at either 0.2% or 0.002% pepsin concentrations. Negative and significant correlations were found between in vitro protein digestibility and cross-linked amino acids formation when using a lower concentration of pepsin. These covalent cross-links can promote the formation of aggregates in proteins, lowering the bio-availability of amino acids surrounding the cross-linked amino acids and decreasing the nutritional value of heat-damaged protein. This study illustrated the importance of protein cross-linking in heat damage and suggests that cross-linked amino acids can be practical indicators of heat damage for protein ingredients.