Potential of electrospray mass spectrometry for meat pigment identification

The potential of electrospray mass spectrometry (ESMS) to identify haem pigments from different species has been investigated. Purified haemoglobin and myoglobin from various sources (pig, beef, sheep and horse) were analysed by ESMS. The spectra showed ions corresponding to the molecular weights of the globin portions of the haemoproteins. When boiled and then analysed by ESMS, the globin chains remained intact but, on autoclaving for 1 h at 121°C, partial hydrolysis was observed, although the fragments could still be used to identify the origin of the haemoglobin. ESMS is a rapid, sensitive technique and may have potential as an analytical method for meat speciation.     

ELECTROSPRAY MASS SPECTROMETRY ANALYSIS OF MYOGLOBIN IN MINCED MEAT PRODUCTS

The heat stability of myoglobin in burgers was studied by Electrospray Mass Spectrometry (ESMS). Burgers containing mixtures of meat and other adjuncts were cooked and myoglobin was extracted with a urea solution. The extracts were analyzed by ESMS and the globin chain of myoglobin was the major peak as myoglobin dissociates to heme and globin during ionization. As time of cooking increased, the globin peak became broader with a tail at high mass indicating the presence of other unidentified components. Extracts from burgers made with one meat species showed one globin peak with a molecular weight characteristic of the species. However, when mixtures of beef-lamb and beef-pork were analyzed only beef-myoglobin globin was detected and no trace of globins from the other species could be detected. The presence of curing salt and glucose had an adverse effect, such that reproducible analysis of globins was not possible.     

Proteolytic degradation of connectin, a high molecular weight myofibrillar protein, during heating of meat

When homogenised muscle (pH 5·5) was heated at 55°C, connectin was extensively degraded, whereas actin and myosin heavy chains were apparently unaffected. It was concluded that carboxyl proteases (e.g. cathepsin D) were largely responsible, because the breakdown of connectin was inhibited by the addition of pepstatin. When whole muscle samples were heated at 50-70°C, degradation of connectin was inversely related to the ultimate pH of the source muscle, again indicating the role of carboxyl proteases. The greater activity of carboxyl proteases in tissues from older animals was apparently responsible for the more extensive degradation of connectin in muscle from older sheep. Because connectin is extensively degraded in cooked meat, it is unlikely to contribute to meat toughness.     

Evaluation of a commercial lateral flow feed test for rapid detection of beef and sheep content in raw and cooked meats

Meat species adulteration is a common problem in the retail market. This study investigated the feasibility of a commercial lateral flow immunoassay designed to detect ruminant muscle tissue in feedstuffs, such as "meat-and-bone meal" (MBM) for detection of beef and/or sheep flesh in meat mixtures, and developed a simple method for meat sample extraction. Laboratory adulterated samples including raw, cooked (100°C, 30min), and sterilized (121°C, 15min) beef-in-chicken, beef-in-turkey, and lamb-in-pork at 0 to 1.00% (w/w) adulteration levels were extracted by different solvents (tap water, NaCl, and PB-NaCl with and without EDTA; and a kit-provided "Extraction Solvent") using three mixing methods. The test rapidly (20min) detected 0.50% (w/w) bovine or ovine meat; Extraction Solvent was the most efficient extractant tested; EDTA coupled with heating (100°C, 10min) improved the assay sensitivity; and all the mixing methods achieved the same results. This immunoassay can be conveniently applied to detect low levels of beef/sheep meat in a wide range of meat products.     

Use of haemoglobin in foods-A review

Blood from domestic meat animals contains biologically valuable protein equivalent to 6-7% of the lean meat content in the carcass. The major part of the protein is haemoglobin in the erythrocytes which even in minute concentrations imparts a dark brownish colour to foods. Large scale use of haemoglobin in foods consequently requires that the protein is made colourless unless attempts are made to hide the colour by technological manipulation in processing. The haem pigment can be removed from the globin by extraction with acidified organic solvents, usually acetone, or absorption on certain agents like carboxymethylcellulose. Alternatively, the globin may be digested by proteolytic enzymes and the liberated amino acids separated from the haem by ultrafiltration or centrifugation. Another approach is oxidative destruction of the haem after inactivation of the catalase activity of the erythrocytes. Hydrogen peroxide is preferred as oxidative agent but care must be taken to limit its impairment of protein quality. Because of its high lysine content globin should be suitable for fortification of cereals. Together with the blood plasma globin it may be used to replace meat in cured meat products. Use of blood proteins in foods may, however, sometimes be opposed for psychological reasons.     

The effect of preheated tendon as a lean meat replacement on the properties of fine emulsion sausages

Tendon from beef hind leg muscles was used to replace some of the lean in a conventional emulsion formulation. The tendon was homogenized and either used raw or preheated for 2·5 h at a range of temperatures (50, 60, 70, 80°C) before use. Texture analysis and sensory evaluation were performed on cylinders of cooked sausage. Texture analysis was carried out on formulations which had 20% of meat protein replaced by 20% tendons which were raw or had been preheated to 50, 60, 70, or 80°C. Fracturability decreased by about 40% with raw tendon, but was restored to within 20% of the no-replacement control if the tendon had been preheated. Hardness was approximately doubled by replacement with raw tendon or tendon heated at 50°C. At temperatures higher than that, hardness returned to approximately no-replacement levels. For sensory evaluation (0-25% replacement; preheating at 70°C), sausages were assessed by a 12-member panel for texture, flavour and overall acceptability. All attributes decreased with increasing collagen content, the decrease being less marked with preheated tendon. Thus more connective tissue could be added for the same panel score if the tissue was preheated. Comparison of the texture profile and the panel scores for texture at the same lean replacement level suggested that reduced fracturability was the texture parameter that panellists objected to when heated tendon replaced some of the lean. Other researchers have shown that connective tissue preheated to 100°C before addition in emulsion sausages results in improved yields and better sensory attributes, but the present results show that temperatures as low as 60°C can be effective for beef tendon.     

Inactivation of Nisin by Glutathione in Fresh Meat

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to determine the fate of nisin in meat products. Nisin in 0.02N HCl was added to fresh and cooked meat and meat juice and samples were stored at 4 °C overnight. Residual nisin solutions and meat juice and meat extract supernatants were analyzed for antimicrobial activity and for nisin. Nisin was recovered from cooked meat extract and cooked meat juice; however, only nisin bound to a food component was detected in fresh meat extract. Mass spectra for raw meat and juice showed a signal 307 Da greater than the mass of nisin. Results indicated that nisin was likely inactivated in raw meat by an enzymatic reaction with glutathione.     

Use of species-specific antisera to adrenal heat-stable antigens for the identification of raw and cooked meats by agar gel diffusion and counter immunoelectrophoretic techniques

Rabbit antisera to adrenal heat-stable and ethanol-precipitable antigens of buffalo, cattle, sheep, goat and pig were used to develop an agar gel precipitation test and a counter immunoelectrophoretic method for the identification of homologous species in raw, partially heated and boiled meat extracts. Immunoabsorption was necessary to make the primary antisera species specific. The specific antisera can be recommended for identification of the species of origin of meats and their mixtures (5-10% adulteration) in raw, partially heated and cooked states even in the case of closely related species, viz cattle and buffalo, sheep and goat.     

Catalysts of lipid oxidation in meat products

In emulsions consisting of refined lard, egg white and corn starch haemoglobin, initially a mixture of the oxy and met forms, at levels similar to the haemoprotein contents found in fresh meat, was a far more powerful catalyst of lipid oxidation, as measured by TBA number, than inorganic iron compounds at levels appropriate to those found in meat. This was true over the pH range 4·5 to 7·5. When added and evenly distributed to exhaustively washed muscle fibres (WF), at levels appropriate to those found in meat, haemoglobin was again a powerful catalyst, but all forms of inorganic iron appeared to have little prooxidant activity. The rate of oxidation of the lipid was very dependent on the haemoprotein concentration, being maximal in the range 10(-4) to 10(-5) M. This equates to an approximate unsaturated lipid to haem molar ratio of several hundred to one, similar to the values reported for model linoleate haem systems. In heated systems the haemoprotein again appeared to be a more effective catalyst than inorganic iron at levels appropriate to those found in meat. It is concluded that the conflicting results as to the roles of haem pigments and inorganic iron in lipid oxidation found in the literature are due, at least in part, to the difficulty of evenly dispersing the catalysts in the washed fibres, especially if heat or freezing leads to subsequent phase separation, and that H(2)O(2), formed by autoxidation of the oxypigments, may be necessary for ferric haem pigments to be effective catalysts.     

Effect of water-soluble haem and non-haem iron complexes on lipid oxidation of heated muscle systems

The distribution of soluble iron between three main components—ferritin, haemoglobin plus myoglobin and a low molecular weight fraction—and the pro-oxidant activities of each fraction in heated water-washed muscle systems from beef, pork and mackerel was determined. Over 75% of soluble iron was associated with the haem fraction. Even so, in all meat systems, the low molecular weight fraction was the major catalyst for lipid oxidation. Ferritin contributed significantly to lipid oxidation in all systems. The addition of nitrite significantly inhibited oxidation in all systems.     

The effect of conditioning on the strength of perimysial connective tissue dissected from cooked meat

Tensile tests were carried out on ribbons of perimysial connective tissue dissected from slices of bovine semitendinosus muscles that had been conditioned or not conditioned and then cooked to a range of temperatures. A consistent reduction in the strength of the perimysia was seen in the conditioned samples, both in the raw meat and meat cooked to 50°C. At higher cooking temperatures (60-80°C), no effect of conditioning was seen. The content of collagen or total protein of mechanically extracted perimysia and the collagen content of the test pieces from conditioned and unconditioned muscles was not significantly different. It was concluded that conditioning decreases the breaking strength of the perimysial connective tissue in raw meat or in meat which is subsequently cooked to 50°C, but not in meat cooked to the temperatures normally employed by consumers. The tenderization observed in conditioned meat cooked to 60°C and above is, therefore, due to the weakening of muscle fibres within the fibre bundles.     

Catalysis of lipid oxidation in muscle model systems by haem and inorganic iron

The catalytic effect of haem proteins (haemoglobin and myoglobin) and inorganic iron (FeSO(4)) on lipid oxidation in a muscle model system was investigated. Haem proteins had a greater prooxidant effect than inorganic iron in raw and heated pork muscle residue when these prooxidants were present at levels approaching those in red meats. The rate of lipid oxidation catalysed by each prooxidant increased as the iron concentration increased over a range of 1-17 μg/g muscle residue. The relative prooxidant effects of haem protein and inorganic iron were not affected by the mode of addition of the prooxidants to the muscle residue (addition in a concentrated form or dispersed in water), or by the method of cooking (fast versus slow cooking).     

Effect of salt on oxidative changes in pre- and post-rigor ground beef

Pre- and post-rigor beef was ground and salt was added to give 0·0, 0·5, 2·0 and 4·0% NaCl (w/w). Samples were removed after 0, 24, 48, 72 and 96 h at 4°C and analyzed for pH, TBA numbers and percentages of reduced myoglobin (Mb), metmyoglobin (MMb) and oxymyoglobin (MbO(2)). After holding for 96 h the samples were cooked in a boiling water bath to an internal temperature of 80°C and held at 4°C for 48 h before TBA analysis. Pre-rigor grinding and salting reduced the post-mortem pH decline and the extent of meat discoloration as shown by the differences in the amount of MMb. The extent of lipid oxidation as measured by TBA numbers was not significantly different for the pre- and post-rigor ground salted meat samples, although salt accelerated oxidation during storage. Results demonstrated that pre-rigor grinding and salting of beef produces a more stable bright red color, which appears to be associated with a lower percentage of MMb and a higher ultimate pH in the pre-rigor salted meat.     

Effect of post-mortem temperature on beef tenderness

Beef adductor muscles were incubated for 4 h post mortem at 10°C and for 4 h and 6 h post mortem at 30°C, 37°C and 42°C. Half of the muscles were cooked just after incubation and the other half was first stored for two days at 4°C and then cooked. Meat kept for 4 h or 6 h at 42°C and for 6 h at 37°C and cooked at once had a significantly (p<0·05) lower shear force than meat kept for 4 h at 37°C, 4 h at 30°C, 6 h at 30°C or 4 h at 10°C. The respective significant differences were also found when the meat was cooked two days after incubation. Organoleptic evaluation showed that meat incubated for 6 h at 37°C or for 4 h at 42°C was not significantly more tender than other samples. However, meat kept for 6 h at 42°C was more tender (p<0·5) than the other samples. After two days of storage, meat incubated for 6 h at 37°C and for 6 h at 42° was more tender (p<0·05) than meat kept for 6 h at 30°C. It was concluded that high temperature conditioning at 37°C or higher for 6 h (4 h at 42°C) just after slaughter makes meat more tender than conventional cooling systems.     

A quick and simple method for the identification of meat species and meat products by PCR assay

The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-specific DNA fragments could be identified by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the six meats. The products showed species-specific DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identification is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were amplified from cooked meat heated at 100 or 120°C for 30 min, but horse DNA fragments could not be detected from the 120°C sample. Detection limits of the DNA samples were 0.25 ng for all meats.     

Myoglobin Analysis for Determination of Beef, Pork, Horse, Sheep, and Kangaroo Meat in Blended Cooked Products

This method allows the concurrent detection of several types of animal muscle meat in comminuted, cooked meat products. Meat proteins were dissolved in a solvent containing 8M urea and dithioerythritol and separated by electrofocusing on an ultra-thin polyacrylamide gel, containing urea. The proteins were then transferred to nitrocellulose by electroblotting and the blot was incubated with anti-human myoglobin serum, a linking antibody, peroxidase-antiperoxidase (PAP) immune complex, and enzyme-substrate. Detection of less than 10% pork, horse and sheep meat was possible in a beef-based meat product which had been heated to an internal temperature of 120°C for 5 min. The method is not suitable for detection of chicken and turkey meats.     

The antioxidant activities of nitrite and nitrosylmyoglobin in cooked meats

Low concentrations of nitrite (20 mg/kg) caused significant (p < 0·001) inhibition of lipid oxidation, measured by the TBA test, in a cooked muscle system and 50 mg/kg nitrite resulted in a highly significant (p < 0·001) reduction in TBA values. Similar antioxidant effects of nitrite were observed in heated water-extracted pork muscle systems catalysed by 5 mg/g metmyoglobin (Mb) or 5 mg/kg Fe(2+), Cu(2+) or Co(2+). The cured meat pigment, nitrosylmyoglobin per se exhibited significant (p < 0·05) antioxidant effects in pork muscle systems catalysed by Mb or metal ions. Progressive depletion of nitrite occurred during refrigerated storage of heated and unheated nitrite-treated pork muscle, muscle aqueous extract and in systems containing Mb, Cu(2+) ot Co(2+). Nitrite depletion occurred much more rapidly in Fe(2+)-containing systems and nitrite concentration had decreased to 5% of the original concentration immediately after heating. In addition, nitrite caused a significant (p < 0·05) reduction in the concentration of non-haem iron in heated aqueous-extracts of beef muscle, whereas, in nitrite-free extracts, a highly significant (p < 0·001) increase in the concentration of non-haem iron, probably due to heat denaturation of the haem structure with release of iron, was observed. Based on the results of this study, three co-operative mechanisms for the antioxidative activities in meat are proposed: (a) by the formation of MbNO which has antioxidant properties per se, (b) on heating, MbNO forms a stable complex, nitrosylhaemochrome, which blocks the catalytic activity of haem iron and also prevents release of haem iron as non-haem iron, which is a highly effective catalyst and (c) nitrite appears to 'chelate' non-haem iron-and possibly copper and cobalt-forming a stable complex, thus inhibiting catalytic activity.     

Cholesterol oxidation in frozen dark chicken meat: influence of dietary fat source, and α-tocopherol and ascorbic acid supplementation

A factorial design assessed the effect of dietary fat source (beef tallow, fresh and oxidized sunflower oils, and linseed oil), and α-tocopheryl acetate (α-TA) and ascorbic acid (AA) supplementation (225 and 110 mg/kg feed, respectively) on the cholesterol oxidation product (COP) content and 2-thiobarbituric acid (TBA) values in raw and cooked dark chicken meat vacuum packaged and stored at -20°C for 7 months. COP determination showed good linearity, recovery and precision. Dietary α-TA was highly effective in protecting raw or cooked meat from cholesterol and fatty acid oxidation, regardless of its degree of unsaturation. In contrast, AA supplementation was ineffective and even promoted oxidation in raw meat from broilers fed unsaturated fat diets that had not been supplemented with α-TA. Oxidation values (raw or cooked meat) from α-TA or α-TA+AA supplemented diets were not statistically different (P>0.05). TBA and COP values were significantly correlated in raw samples (r=0.6466, P=0.0001).     

Effect of dietary vitamin e on the oxidative stability of raw and cooked rabbit meat

The effect of dietary α-tocopheryl acetate supplementation (200mg/kg diet) on plasma and muscle levels of α-tocopherol and the oxidative stability of raw and cooked rabbit meat was determined. Two groups of 20 male hybrid rabbits were fed the experimental diets from 35 to 80 days of age. Feed intake, live weight, feed efficiency and qualitative traits of the carcass and meat were recorded. The α-tocopherol levels in plasma and muscle were significantly higher (p≤0·01) in the supplemented group, which also showed an increase in oxidative stability in both raw and cooked meat. The higher α-tocopherol level improved the physical traits of the meat, significantly reducing shear value and increasing water-holding capacity; n-3 fatty acids in raw and cooked meat increased (p≤0·05) and the thrombogenic index decreased (p≤0·05). Dietary vitamin E did not influence weight gain, feed intake and dressing yield.     

Effects of carbon dioxide on yield, texture and microstructure of cooked ground beef

The objective of the study was to find the effects of CO(2) gas on cooking loss, instrumental hardness and microstructural changes of ground beef heated to 70-83 °C. In two experiments, ground beef was stored for 4 days in 60% CO(2)/39.6% N(2)/0.4% CO and vacuum (1), or in 100% CO(2), 50% CO(2)/50% N(2), 20% CO(2)/80% N(2), 100% N(2) and vacuum (2). In an additional experiment, slices of beef semimembransosus muscles were stored for 10 days in 100% CO(2), 100% N(2) and vacuum. Cooking loss of ground beef patties was higher of all CO(2) treatments than non-CO(2) treatments (p<0.05). Storage of raw ground beef in CO(2) caused a concentration dependent decrease in raw meat pH of up to 0.12 units in 100% CO(2). In the beef slices, small CO(2) related fissures and pores were formed in the cooked meat. These changes in pH and microstructure probably contributed to the elevated cooking loss. The hardness of cooked ground beef was not affected by CO(2) exposure (p>0.05). Because CO(2) in concentrations of 20-100% is commonly used in industrial packaging processes for retail meat and meat trimmings, a reduction in cooking yield of 1-3% may have sensory and economic implications.