E.E. O’Neill

Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate.

Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37°C for 2h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a(w)) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe(2+) chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use.

The effects of different cooking regimes on the cook yield and tenderness of non-injected and injection enhanced forequarter beef muscles.

The aim was to investigate the effect of different cooking regimes on the cook yield and tenderness of non-injected and brine injected (0.5% residual NaCl) bovine M. triceps brachii caput longum (TB), M. supraspinatus (SP) and M. pectoralis profundus (PP). Injected and non-injected TB, SP and PP muscle sections (400 g) were (a) conventionally oven cooked to 72 degrees C or cooked slowly (using a Delta10 programme) to 72 degrees C or (b) cooked in a water bath to 72 degrees C or cooked in a water bath to 55 degrees C and held at this temperature for 2 h before heating to 72 degrees C. In addition, injected PP muscle sections were oven cooked to 69 degrees C and held at this temperature for up to 12 h. Slow cooking using a Delta10 programme had no significant (P<0.05) effect on WBSF values of injected or non-injected SP and TB muscles but significantly (P<0.05) decreased the WBSF values of injected and non-injected PP muscles when compared to conventional cooking. Slow cooking significantly (P<0.05) increased % cook yield of injected PP, SP and TB muscles. Staged cooking significantly (P<0.05) decreased the WBSF values and had no significant effect on sensory tenderness ratings of non-injected TB, SP and PP muscles and injected PP muscles. Staged cooked injected or non-injected PP, SP and TB muscles had lower % cook yield values than those cooked straight to 72 degrees C. Increasing the cooking time of injected PP muscles at 69 degrees C to 8 and 12 h decreased % cook yield, decreased WBSF values and increased sensory tenderness ratings. It also alleviated the problem of residual chewiness which was evident in PP muscles cooked using the other regimes.

Manipulation of the pre-rigor phase to investigate the significance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi.

The objective of this study was to evaluate the importance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi (LD) muscle over a 21-day period. This was done by altering the pre-rigor glycolytic behaviour of hot-boned LD muscles using different early post-mortem temperature regimes. Hot-boned LD muscles (n=8) were cut in two, randomised, placed in polythene bags and submerged in a water bath set at 5°C (rapidly chilled) and 15°C (slowly chilled) for 8h post-mortem. All muscles were then stored at 2°C for up to 21 days post-mortem. The temperature regimes altered the glycolytic behaviour of the muscles in the pre-rigor period. The slowly chilled muscles exhibited a faster (P<0.01) pH fall than rapidly chilled muscles. Cold shortening was induced in rapidly chilled muscles as they had shorter (P<0.01) sarcomere lengths than slowly chilled muscles up to day 21 post-mortem. Warner Bratzler shear force values (WBSF) deemed cold-shortened muscles as tougher than noncold shortened up to day 14 post-mortem. Both cold-shortened and noncold-shortened muscles tenderised over time to an extent where there was no significant difference in WBSF values by day 21 post-mortem. SDS-PAGE protein profiles indicated that the rate of proteolysis was faster in slowly chilled muscles when compared to rapidly chilled muscles. However by day 21 post-mortem it appeared that rapidly and slowly chilled muscles underwent proteolysis to the same extent.

Manipulation of the pre-rigor glycolytic behaviour of bovine M. longissimus dorsi in order to identify causes of inconsistencies in tenderness

The aim of this study was to monitor the effects of the alteration of the pre-rigor environment of the bovine LD muscle using controlled temperature regimes in order to gain an insight into the early post-mortem pH/temperature/time interactions which are important from the point of view of tenderness and to identify possible reasons for inconsistencies in beef tenderness. LD muscles (n=12) were hot-boned within 90min post-slaughter, cut into three pieces which were randomly placed in polyethylene bags and submerged in water baths pre-set at the following temperatures; 0, 5, 10, 15, 20 and 25°C for 8 h post-mortem then stored at 2°C for up to 14-days post-mortem. The rate of pH decline increased with increasing temperature. Muscles incubated at 0 and 5°C were cold shortened however not all of these muscles were tough as indicated by Warner Bratzler shear force values (WBSF). A pH range of 5.9-6.2 at 3 h post-mortem (pH(3)) produced consistently tender beef where cold-shortening was avoided. Cold shortened muscles showed the greatest variation in tenderness at 14 days post-mortem and underwent the greatest amount of tenderisation (ΔWBSF) and proteolysis between days 2 and 14 post-mortem. Proteolysis of cold shortened muscle may induce variation in tenderness in these muscles.

The effect of sodium lactate, potassium lactate, carrageenan, whey protein concentrate, yeast extract and fungal proteinases on the cook yield and tenderness of bovine chuck muscles

This study evaluated the efficacy of injection with enhancement solutions containing sodium lactate (NaLac), potassium lactate (KLac), carrageenan, whey protein concentrate (WPC), yeast extract or fungal proteinases alone or in combination with NaCl at increasing the tenderness and cook yield of bovine M.supraspinatus and M. tricepsbrachii caput longum. Muscle sections (400g) were injected with enhancement solutions at an injection rate of 110% to give specific residual concentrations as follows: 0.5% NaCl; 2% NaLac; 2% KLac; 0.5% NaCl+1 or 2% NaLac; 0.5% NaCl+1% KLac; 0.5% NaCl+0.4% sodium tripolyphosphate (STPP)+2% NaLac; 0.5% NaCl+0.4% STPP, 1.5% WPC+/-0.5% NaCl, 1.0% carrageenan+/-0.5% NaCl, 0.5% yeast extract or 0.0005% and 0.001% fungal enzymes. Injection with solutions containing sodium or potassium lactates (alone or in combination with NaCl and STPP), WPC, carrageenan and yeast extract significantly (P<0.05) decreased WBSF values and increased sensory tenderness ratings, compared to non-injected controls. All of these ingredients in water also significantly (P<0.05) increased cook yield with the exception of yeast extract which had no significant effect. The fungal enzymes in a water carrier acted as effective tenderising agents in both muscles but when used at the higher level resulted in a significant decrease in percentage cook yield. Injection of the chuck muscles with these ingredients and water represents an effective method of improving their tenderness and in most cases also increasing percentage cook yield.

Proteolytic specificity of cathepsin D on bovine F-actin

Proteolysis of bovine F-actin by cathepsin D (E.C. 3.4.23.5) in 50 mM Na acetate buffer, pH 5.5, at 37°C was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high performance liquid chromatography (RP-HPLC). Actin was hydrolyzed by cathepsin D during incubation to peptides detectable by RP-HPLC, although no degradation products were detected by SDS-PAGE. Peptides (2% trichloroacetic acid-soluble) from the hydrolyzate were isolated by RP-HPLC on a C(18) column using an acetonitrile/water gradient and identified from their N-terminal sequence and mass. Cathepsin D cleavage sites were identified at Cys(12)-Asp(13), Gly(22)-Phe(23), Arg(30)-Ala(31), Thr(79)-Asn(80), Ile(87)-Trp(88), Thr(91)-Phe(92), Phe(92)-Tyr(93), Arg(97)-Val(98), His(103)-Pro(104), Leu(107)-Thr(108), Thr(108)-Glu(109), Lys(120)-Met(121), Leu(144)-Tyr(145), Ile(153)-Val(154), Leu(155)-Asp(156), Ile(167)-Tyr(168), Leu(180)-Asp(181), Met(192)-Lys(193), Leu(195)-Thr(196), Arg(208)-Glu(209), Arg(212)-Asp(213), Leu(223)-Asp(224), Lys(240)-Ser(241), Thr(262)-Leu(263), Trp(342)-Ile(343), Arg(349)-Ser(350), Trp(358)-Ile(359), and Lys(375)-Cys(376). In general, cathepsin D preferentially cleaved bonds containing at least one hydrophobic amino acid residue. The results of this study showed that actin was degraded extensively by cathepsin D with peptides released from numerous locations in the protein molecule.

Effects of environmental conditions on microbial proteolysis in a pork myofibril model system

P.M. KENNEALLY, N.G. FRANSEN, H. GRAU, E.E. O’NEILL & E.K. ARENDT.1999.A number of bacterial strains used for meat fermentations were screened for proteolytic activity. A strain of Micrococcus which was found to be proteolytic was evaluated for the effects of environmental conditions on its proteolytic activity against pork myofibrillar proteins using response surface methodology. Three strains of micrococci were also tested for the ability to produce free amino acids from pork myofibrils. Analysis of the effects of environmental conditions showed that proteolytic activity would be minimal under conditions normally found in fermented sausages, thereby suggesting that proteolysis in these products is largely due to endogenous meat enzymes. The three strains of micrococci were shown to produce free amino acids from pork myofibrils, thereby demonstrating the presence of peptidase activity in these strains.

Effects of enhancement procedures on whole and re-formed beef forequarter muscles.

Beef Supraspinatus and Triceps brachii muscles were subjected to three enhancement and/or re-forming treatments: (i) injected whole @ 15%w/w with salt-phosphate solution; (ii) injected and re-formed; (iii) injected with added flavouring and re-formed. The treated muscles were compared to whole uninjected controls. All injection treatments reduced shear force values of cooked samples and in most cases these reductions were reflected in sensory panel tenderness and chewiness ratings. For example, shear values for Supraspinatus were 83N/g in control samples and 50 in whole injected samples, while corresponding sensory panel tenderness ratings were 3.6 and 5.2. Enhanced samples did not differ from controls in sliceability or in colour and binding ratings, indicating that enhancement combined with re-forming can give an acceptable roast beef product. There were no differences in drip loss and very few differences in colour L*, a* and b* values for raw samples between any of the treatments. Addition of beef stock did not result in higher flavour ratings by sensory panels. Whole injected samples scored higher for flavour than both control (p<0.01) and injected+re-formed (p<0.05) samples.