Potential for Food-Grade Microbial Proteases in Meat Tenderisation and Bioactive Peptide Generation

Meat has been consumed by a wide range of cultures for thousands of years and is an important source of many essential dietary components. Research has suggested that consumers are more willing to consume meat if the cut is tender. Traditional methods of aging meat and allowing endogenous proteases to partially hydrolyse the meat protein structure contributes to increasing meat tenderness, however this has proven to be less effective with the toughest cuts of meat. More recently other methods of tenderisation have included the use of exogenous plant and microbial-derived proteases to partially hydrolyse some of the major protein components. In addition, the processing of meat for the consumer market leads to the generation of a range of waste materials rich in protein including blood, bones, meat trimmings and connective tissues and the production and disposal of these waste products can have significant environmental effects. Extensive hydrolysis of natural product proteins to produce short peptide sequences has been shown to generate a range of bioactivities including the inhibition of angiotensin I-converting enzyme and antioxidant properties. The recent availability of four fungal and one bacterial-derived protease preparations has led to the investigation reported in this thesis of their potential meat tenderising properties and their ability to produce bioactive peptides from meat myofibrillar and connective tissue protein extracts. Mass spectrometry analysis found that the commercial protease preparations each contained multiple protease components primarily originating from either Bacillus or Aspergillus species. Kinetic assays with a fluorescent casein substrate showed that the bacterial-derived HT proteolytic (HT) protease had a significantly faster reaction rate compared to that of the four fungal-derived proteases. However the acidic fungal protease (AFP) fungal-derived protease had the highest affinity for the substrate. Each protease preparation hydrolysed the meat myofibrillar and connective tissue extracts with different specificities and at different rates as assessed by time course hydrolysis experiments and analysis by 1D SDS PAGE. Western blotting was used to specifically investigate the hydrolysis of desmin and troponin that have been reported to be import contributors to meat tenderness. HT protease showed the least selectivity and most efficient hydrolysis for both the meat myofibrillar and connective tissue substrates. In general, the fungal proteases demonstrated slower and potentially more controllable degradation of the protein components as assessed by 1D-SDS-PAGE, however their overall effectiveness was reduced. HT protease was shown to produce the most complete hydrolysis pattern following extended incubation with both meat myofibrillar and connective tissue extracts as analysed on 1D-SDS-PAGE and confirmed with gel permeation chromatography. As peptide bioactivities are reported to be mainly associated with short peptide sequences it was elected to focus on HT alone for analysis of bioactive peptide production. HT protease produced peptide hydrolysates containing significant (angiotensin I-converting enzyme) ACE inhibitory and antioxidant activities from meat myofibrillar and connective tissue extracts. Following gel permeation chromatography it was found that in all cases these activities were associated with the small peptide fraction. Sub fractionation of the small peptide fraction was achieved with OFFGEL isoelectric focussing electrophoresis. The OFFGEL fractions displayed various levels of ACE inhibitory and antioxidant activities but preliminary separation of the peptides present in an OFFGEL fraction indicated a complex mixture. This indicated that further separation techniques would need to be employed to reduce the peptide complexity. Initial cytotoxicity assays indicated that the peptide-containing fractions had no significant cytotoxic effects towards Vero cells for any of the fractions tested