Eileen O'Neill

Characterization of proteolysis during the ripening of semi-dry fermented sausages

The respective contribution of indigenous enzymes and enzymes from starter bacteria to proteolysis in fermented sausages were determined by comparing the proteolytic changes occurring in sausages resulting from the presence of a proteolytic strain of Staphylococcus carnosus, i.e. S. carnosus MC 1 to the proteolytic changes occurring in control sausages containing glucono-δ-lactone (GDL) and an antibiotic mixture. Proteolysis was quantified by assaying for non-protein nitrogen (NPN) and free amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (RP-HPLC) were used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins as ripening progressed. The concentration of NPN and free amino acids increased in both sausages initially, but subsequently decreased towards the end of ripening in sausages inoculated with the starter culture. SDS-PAGE showed a similar pattern of proteolysis of sarcoplasmic proteins in both sausages, while of the two sausage types; the S. carnosus MC 1 inoculated sausages exhibited the most intense degradation of myofibrillar proteins, especially myosin and actin. RP-HPLC profiles of 2% trichloroacetic acid (TCA)-soluble peptides for the two sausage types were similar, with the production of numerous hydrophilic peptides. N-Terminal amino acid sequence analysis and sequence homology with proteins of known primary structure showed that six of the TCA-soluble peptides were released from the sarcoplasmic (myoglobin and creatine kinase) and myofibrillar (troponin-I, troponin-T and myosin light chain-2) proteins. In addition, the initial degradation of sarcoplasmic proteins was due to the activity of indigenous proteinases, while both indigenous and bacterial enzymes contributed to the initial degradation of myofibrillar proteins. Furthermore, indigenous enzymes were responsible for the release of TCA-soluble peptides, which, were further hydrolysed by bacterial enzymes.

Assessment of the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of hydrolysates of bovine brisket sarcoplasmic proteins produced by papain and characterisation of associated bioactive peptidic fractions.

The main objective was to investigate the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of sarcoplasmic proteins isolated from the brisket muscle (Pectoralis profundus) of 3 (Bos taurus) cattle and hydrolysed with papain for 24 h at 37°C. Sarcoplasmic protein hydrolysates were ultra-filtered using molecular weight cut off (MWCO) membranes and 10-kDa and 3-kDa filtrates were obtained. The total sarcoplasmic protein extracts and the 3-kDa filtrates were tested for angiotensin I-converting enzyme inhibitory (ACE-I) activities. The total hydrolysates, 10-kDa and 3-kDa filtrates were also tested for their associated antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe(2+) metal chelating ability assay. The peptidic content of the total hydrolysates, the 10-kDa and the 3-kDa filtrates were analysed using an ORBITRAP mass spectrometer, and mass spectral data obtained were analysed using TurboSEQUEST. Eleven peptides were characterised from the total hydrolysates, fifteen from the 10-kDa filtrate fractions, whilst nine peptides were characterised from the 3-kDa filtrate fractions. Similarities between the amino acid sequences of the peptides identified in this study and previously identified antioxidant and ACE-I inhibitory peptides detailed in the BIOPEP database were outlined.

Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Rabbit skeletal muscle troponin-T (200 μg ml(-1)) was incubated with μ-calpain (2 μl ml(-1)) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr(45)-Ala(46), Leu(69)-Met(70), Glu(220)-Lys(221) and Asn(231)-Val(232). The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met(151)-Gly(152), Asn(188)-Ile(189), Lys(223)-Arg(224), Arg(233)-Ala(234) and Ala(240)-Lys(241). In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.

Heat-induced gelation of actomyosin.

Rheological properties of actomyosin gels were markedly affected by protein concentration, pH and heating temperature. Gel strength increased with increasing protein concentration (30-60 mg ml(-1)) and heating temperature (55-75°C), but decreased with increasing pH (5·5-9·0). Low heating temperatures (50-55°C) favoured the formation of more cohesive actomyosin gels than the higher heating temperatures (60-75°C). Gels formed at low pH (5·5 and 6·0) were less cohesive than those formed at high pH (7·5-9·0). Addition of ATP and pyrophosphate (10 mm) prior to heating decreased gel strength and cohesiveness, whereas EDTA (1-5 mM) reduced gel strength but did not affect gel cohesiveness.

Molecular forces involved in the formation and stabilization of heat-induced actomyosin gels

Increasing concentrations of KCl, KF and NaCl from 0·2 to 1·0 m increased the compressive strength and cohesiveness of heat-induced actomyosin gels (0·6 m KCl pH 6·0). On the other hand, KBr, KSCN, KI, NH(4)Cl, MgCl(2) and CaCl(2) decreased gel strength and cohesiveness. Gel compressive strength increased with increasing urea concentration (0·4-2·4 m). Addition of the sulphydryl blocking agent N-ethylmaleimide and the reducing agents dithiothreitol and cysteine decreased the compressive strength and cohesiveness of actomyosin gels. Overall, the results indicate that hydrophobic interactions, disulphide and hydrogen bonding contribute to the formation and stabilization of actomyosin gels.

Harnessing the Potential of Blood Proteins as Functional Ingredients: A Review of the State of the Art in Blood Processing

Blood is generated in very large volumes as a by-product in slaughterhouses all around the world. On the one hand, blood generation presents a serious environmental issue because of its high pollutant capacity; however, on the other hand, blood has the potential to be collected and processed to generate high-added-value food ingredients based on its exceptional nutritive value and its excellent functional properties. In this paper, we review the current state of the art for blood processing, from collection to final recovery of protein isolates, the functional properties of blood, impact of processing on functional properties, and potential applications as food ingredients. Furthermore, future challenges are outlined for this underutilized and abundant product from the meat industry.

Alternative uses for co-products: Harnessing the potential of valuable compounds from meat processing chains

Opportunities for exploiting the inherent value of protein-rich meat processing co-products, in the context of increased global demand for protein and for sustainable processing systems, are discussed. While direct consumption maybe the most profitable route for some, this approach is influenced greatly by local and cultural traditions. A more profitable and sustainable approach may be found in recognizing this readily available and under-utilised resource can provide high value components, such as proteins, with targeted high value functionality of relevance to a variety of sectors. Applications in food & beverages, petfood biomedical and nutrition arenas are discussed. Utilization of the raw material in its entirety is a necessary underlying principle in this approach to help maintain minimum waste generation. Understanding consumer attitudes to these products, in particular when used in food or beverage systems, is critical in optimizing commercialization strategies.

The surface-active properties of muscle proteins

Abstract The surface-active properties of myosin, actomyosin, F- and G-actin at the air-solvent interface, at initial bulk phase concentrations in the range of 10 − 4 % to 10 − 2 % w/v, were determined by the drop volume method. Overall myosin was the most effective surface tension depressor, followed by actomyosin, F-actin and G-actin. The surface pressures attained after 40 min at an initial bulk phase concentration of 10 − 2 % w/v were 21·02, 19·27, 17·25 and 14·91 mNm − 1 , respectively. Furthermore, myosin effected the most rapid change in surface pressure during the initial 5-min period.

Optimization of Protein Recovery from Bovine Lung by pH Shift Process Using Response Surface Methodology

BACKGROUND Response surface methodology (RSM) was used in a sequential manner to optimize solubilization and precipitation conditions in the recovery of protein from bovine lung using pH shift. RESULTS Separate D-optimal designs were employed for protein solubilization and precipitation. Independent variables investigated for protein solubilization were time (10-120 min), temperature (4-20 °C), pH (8.0-11.0) and solvent/sample ratio (2.5-10). Variables for protein precipitation were time (0-60 min) and pH (4.25-6.00). Soluble protein yields ranged from 323 to 649 g kg-1 and the quadratic model for protein solubilization revealed a coefficient of determination R2 of 0.9958. Optimal conditions for maximum protein solubility were extraction time 140 min, temperature 19 °C, pH 10.8 and solvent/sample ratio 13.02. Protein precipitation yields varied from 407 to 667 g kg-1 , giving a coefficient of determination R2 of 0.9335. Optimal conditions for maximum protein precipitation were pH 5.03 and 60 min. Based on the RSM model, solubilization conditions were manipulated to maximize protein solubilization under reduced water and alkaline usage. These conditions were also validated. CONCLUSION Models for solubilization and precipitation using bovine and porcine lung were validated; predicted and actual yields were in good agreement, showing cross-species applicability of the results.

The effects of pH and heating on the surface activity of muscle proteins

Abstract The effects of pH and thermal denaturation on the surface active properties of muscle proteins at the air-solvent interface were determined by the drop volume method. The surface pressures attained after 40 min, π 40 , by myosin and actomyosin solutions heated at 45°C for 30 min were similar to those attained by the unheated protein solutions. However, the π 40 values were increased when the proteins were heated at 60°C prior to determining surface activity. The rate of surface tension decay was much faster following heating of myosin at 45°C compared with the unheated sample. Myosin heated at 60°C showed an initial induction period, which was followed by a very rapid rate of surface tension decay. The rate of surface tension decay for actomyosin heated at 45°C was similar to that of the unheated protein solution: however, heating at 60°C increased the rate compared to that of the control. The surface pressures attained after 40 min ( π 40 ) by myosin and Factin solutions were not significantly affected by the pH of the solutions. However, π 40 values for both G-actin and actomyosin were greater at pH 11·0 than at pH 5·6 or 7·0. For all the proteins studied, pH greatly affected the rates of surface tension decay, the rates being slowest at pH 5·6 and fastest at 11·0.