Effects of muscle pH on pressure-induced changes in the sarcoplasmic reticulum

The effect of muscle pH on high pressure treatment of rabbit longissimus dorsi muscles has been studied by the use of pre-slaughter injections of epinephrine in order to vary the glycogen content and thus the extent of post-slaughter glycolysis in the muscles. Following pressure treatment the pH of the muscles varied between 5·9 and 7·1 while the control muscles varied between 6·6 and 7·1.

The effects of the high pressure treatment on the ATPase activities, protein yield and protein composition of the sarcoplasmic reticulum (SR) were measured. The basal ATPase activity was not affected over this pH range while the extra ATPase activity, protein yield and protein composition of the SR were affected. The effects increased with decreasing pH.     

Effect of high pressure on the regulation of phosphorylase activity in pre-rigor rabbit muscle

The effects of high pressure (150 MPa) on the regulation of phosphorylase activity in pre-rigor rabbit muscles have been studied at 35° and 0°C. At 35°C muscle contracts, phosphorylase is activated and the muscle pH falls to 5·8 in 2 min. Coinciding with these changes, phosphorylase phosphatase activity falls rapidly, while phosphorylase kinase, although active for longer, loses its activity as the pH falls. Both of these enzymes are completely inactivated after 5 min under pressure, while phosphorylase still retains 80% of its activity under these conditions. The effects of pressure on the activities of these enzymes in white and red muscles of rabbits were compared, with a greater effect being observed in white muscles. At 0°C, muscles subjected to high pressure did not contract, but at this temperature the three enzyme activities (phosphorylase, phosphorylase kinase and phosphorylase phosphatase) were all lost at a greater rate than at 35°C, although the pH of the muscle did not fall below 6·5. The effects of high pressure treatment on isolated phosphorylase a and b and phosphorylase kinase were also studied at both 0° and 35°C and the results obtained closely paralleled those observed in whole muscle.     

Effect of pressure treatment on the sarcoplasmic reticulum of red and white muscles

The effects of high pressure (150 MPa) on the sarcoplasmic reticulum of red and white muscles have been studied. When whole muscle is pressurised either pre-rigor or post-rigor the major effect on the SR is the loss of extra ATPase activity and the loss of several proteins including the 100,000 dalton ATPase and calsequestrin. When isolated SR is pressurised the extra ATPase activity is lost but there is no protein degradation. Measurements of muscle pH and the influence of pH on pressurisation effects indicate active proteolysis in white muscle when it is pressurised. In all these studies the basal ATPase activity was relatively unaffected. The effect of pressure treatment on the yield of SR protein varied with different muscles, being greatest in the muscles which had the highest concentration of extra ATPase and calcium uptake activities. These muscles also reached the lowest pH during pressurisation, thus favouring proteolysis.     

High voltage electrical stimulation enhances muscle tenderness, increases aging response, and improves muscle color from cabrito carcasses

Our study examined high and low voltage electrical stimulation and postmortem storage as strategies to improve tenderness and lean color in cabrito carcasses. Boer cross (n=60) kids were assigned to either high (550 V), low (20 V), or no electrical stimulation treatments. No differences in muscle temperature were observed between treatments at any time measured. Muscle pH declined fastest in high voltage treated carcasses. High voltage electrical stimulation slightly increased (P<0.05) b^* and a^* in the M. gluteus medius and tended to increase L^* and b^* (P=0.06 and 0.11, respectively) values in the M. longissimus thoracis. Electrical stimulation had no effect on myofibril fragmentation at 1-, 3-, or 14-d postmortem or sarcomere length. High voltage electrical stimulation increased (P<0.05) tenderness at 1- and 3-d postmortem, but not at 14-d postmortem. Aging for 3 d did not affect tenderness regardless of stimulation treatment, but aging time for 14 d improved (P<0.05) tenderness. These data indicate that high voltage electrical stimulation and 14 d aging were effective in improving the tenderness of meat from cabrito carcasses.     

Electrical stimulation of rats: Part 2-The effect of electrical parameters on muscle tension and post-Mortem glycolysis

Anaesthetized rats were used as a model to determine the effect of changes in electrical stimulation parameters on the pH fall of the M. longissimus dorsi and M. triceps surae and tension development by the M. triceps surae. Stimulation 5 min before decapitation resulted in a reduced post-stimulation fall in pH. Tension development resulting from stimulation of the sciatic nerve decreased rapidly 5 min after decapitation and ceased 30 min after decapitation, whereas a current pathway through the muscle was still effective. Post-stimulation pH fall was less in the leg not in the stimulation pathway, indicating a lack of crossover effect from one leg to the other; but the difference diminished as stimulation voltage increased. For direct stimulation, 20 V produced a maximal response in the stimulated leg; at least twice that voltage was required for a maximal pH fall in the leg not in the stimulation pathway. The total resistance of the rat with a needle electrode at the nape of the neck and crocodile clips attached to the unskinned legs was 3400 ω, whereas with skinned hindlegs the resistance was 860ω. With this high skin resistance at least 40 V was needed for effective stimulation whereas, for low voltage stimulation of much larger animals, only twice this voltage is used. High contact resistance and unknown current density factors make simple correlations of animal size and voltage effects impossible. The optimal stimulation frequency for rats was 20–30 pulses a second. If the cranial electrode was positive, about 15% more tension was developed than when the cranial electrode was negative. With alternating-polarity pulses, there was a high-low tension response, which was more pronounced at low voltages but evened out as the muscles became exhausted. Alternating and cranial-positive pulses produced similar pH falls but cranial-negative pulses were inferior.     

Studies in electrical stimulation: Post-mortem decline in nervous response in lambs

A study has been made of the effects of electrical stimulation, either indirectly through the nerves or directly through the muscles, on glycolysis in lamb hind leg muscles. Responses were affected by delay before application and the stimulating voltage. At 5 min post mortem the same fall in pH during stimulation (ΔpH) and subsequent increased rate of pH fall (dp H/dt) occurred in muscles stimulated both directly and indirectly with 12 V. With 200V, ΔpH was 50% greater in muscles stimulated directly and 30% less if stimulated indirectly. At 30 min post mortem there was very little effect of stimulation at either voltage on glycolysis when muscles were indirectly stimulated, indicating that the nerves had lost their capacity to trigger muscles to contract. In contrast, if the legs were stimulated directly, maximum ΔpH and dpH/dt values were achieved with 200 V pulses. The significance of these findings to the application of electrical stimulation to commercial meat processing is discussed.     

Stunning and shackling influences on quality of porcine Longissimus dorsi and Semimembranosus muscles

Seventy-one barrows of three porcine genotypes (nn, Nn and NN genotypes with respect to halothane) were electrically stunned on the right side and randomly assigned to one of three treatments during the bleeding process (prone, PR; shackled left, SL; or shackled right, SR) to investigate the influence of both shackling and stunning on ultimate meat quality. PR carcasses had less side-to-side variation in 40 min pH than either SL or SR carcasses. Shackling contributed to a decreased 40 min pH in the semimembranosus (SM) and posterior longissimus dorsi (PL). The effects of stunning appeared to be additive when combined with shackling, tending to lower pH in the mid-longissimus dorsi (ML; P <- 0·10). The ‘free’ side of the shackled carcass also had significantly lower 40 min pH in the ML, PL and SM locations when compared to PR sides. Genotype had no influence on shackling since there was no treatment by genotype interactions. Despite significant effects on post-mortem metabolism (indicated by the differences in 40 min pH) shackling alone had little influence on overall ultimate meat quality of the carcass. Depending on the sampling location, soluble protein and L^* and b^* values varied among treatments. Shear force and a^* values were similar among treatments, but PR and SL carcasses had significantly lower expressible juice and drip loss compared to SR carcasses. These results suggest that, although shackling alone has little effect on meat quality, in combination with electrical stunning, shackling can lead to a decrease in meat quality.     

Effect of electrical stimulation on post mortem biochemical characteristics and quality of Longissimus dorsi thoracis muscle from buffalo (Bubalus bubalis)

High voltage electrical stimulation (700 V, 1400 V peak, pulses 1 s on/1 s off, 60 Hz, 2 A) on buffalo carcasses resulted in a significantly more rapid pH fall in Longissimus dorsi thoracis muscle when compared to non stimulated controls (p < 0.01), during the first 24 h after slaughter. The IMP/ATP ratio on the same period showed a much more rapid increase for the stimulated muscles (1.07 and 1.16 at times 1 and 2.5 h post mortem vs control values of 0.77 and 0.83, respectively). Sensory and instrumental evaluation of texture of meat cooled by two distinct processes showed that tenderness at 24 h post mortem was higher in the stimulated muscles compared to non-stimulated controls, irrespective of the cooling process adopted. High voltage stimulation significantly decreases cohesiveness, increases myofibril fragmentation; and SDS polyacrylamide gel electrophoresis of the myofibrillar proteins showed a weakening of Troponin T band during 6 days of ageing in non-stimulated control muscles, whereas electrical stimulation accelerated the process of ageing over 3 days. This is the first report on acceleration of conditioning in buffalo muscle and the conditions described here have a high potential for application in meat industry for buffalo slaughter.     

Activities of metabolic and contractile enzymes in 18 bovine muscles

Contractile and metabolic characteristics of 18 bovine muscles were studied using 10 animals of various ages (2-10 years), sexes (females, males and castrated males) and types (dairy, meat or crossbred). Contractile type was assessed by measuring myofibrillar Ca-Mg-activated ATPase activity. Metabolic type was appraised by determining phosphorylase, glycogen synthetase, hexokinase, haem iron as well as glycolytic and mitochondrial oxidative activities. 'Glycolytic potential' and buffer capacity (as change in lactate concentration as a function of change in pH) were measured. The results showed that myofibrillar ATPase activity was positively related to glycolytic activities and negatively related to oxidative activities. Ultimate pH was negatively correlated with myofibrillar ATPase activity and had little if any relationship to the glycolytic potential. Metabolic and contractile activities were essentially unaffected by sex, age or breed type.     

Characterization of Micrococcaceae isolated from Iberian dry-cured sausages

The populations of Micrococcaceae in different types of Iberian dry-cured sausages from central-west Spain were characterized and their technological and antimicrobial properties determined in order to evaluate their suitability as starter cultures in dry-cured sausage manufacture. Of a total of four hundred strains isolated from two manufacturers, one hundred and sixty-six were selected to evaluate nitrate reductase, proteolytic, lipolytic, and antimicrobial activities, and growth at different values of pH and water activity (a_w). Most of the strains were identified as Staphylococcus except for eight isolates assigned to Kocuria spp. The species most often isolated was Staphylococccus xylosus. Others were, in descending order of abundance, S. aureus, S. lugdunensis, S. saprophyticus, S. sciuri, S. chromogenes, and S. capitis. The distributions of the minority Staphylococcus species were different for the two manufacturers. All the investigated strains were able to grow at pH and a_w greater than 5.0 and 0.85, respectively, the values usually found in Iberian dry-cured sausages. Five S. xylosus strains showed antimicrobial activity against some indicator strains which were investigated. Seven strains with the best properties were pre-selected and tested for their lipolytic and proteolytic activities against pork fat and myofibrillar and sarcoplasmic pork proteins, respectively, and for their low biogenic amines production. Most of the strains showed proteolytic and lipolytic activities, but none produced histamine, tyramine, phenylethylamine, or spermine. Three strains, identified as Staphylococcus xylosus, possess useful properties which make them candidates for testing as starter cultures in pilot processing of Iberian sausages.     

Instrumental evaluation of pH effects on ability of pork chops to bloom

Color plays an important role in consumer purchase decisions of pork. For this reason, the objective of this study was to determine the usefulness of instrumental measures of bloom and bloom as a function of pH. Color was measured with two instruments on pork chops of varied ultimate pH (5.1–6.1) in a vacuum package and again after blooming. During bloom, Hunter a* increased while Minolta a values changed little. Bloom had no effect on either measure of lightness (L* or L). With bloom, Hunter Hue Angle decreased indicating an increase in red color, while Minolta Hue Angle measurement increased with bloom, implying a decrease in true red color. Although lightness was unaffected by bloom, pH had a significant effect on Hunter L* and Minolta L values. Increasing pH resulted in an increase of Hunter a*, but not Minolta a, in both unbloomed and bloomed chops. Hunter Hue Angle was consistently higher than Minolta Hue Angle regardless of bloom status or pH.     

Low voltage electrical stimulation of lamb carcasses: effects on meat quality

The effects of an early post mortem low voltage electrical stimulation (28 V, 60 Hz) on biochemical changes and on final tenderness in muscles Longissimus thoracis et lumborum and Semimembranosus from lamb carcasses were studied. It was shown that electrical stimulation accelerated the glycolytic process resulting in a significant fall in pH during the first 6 h post mortem in both muscles examined and in a significant reduction in adenosine triphosphate (ATP) content in muscle Longissimus thoracis et lumborum. The effect of electrical stimulation on tenderness was recorded by measuring shear force values 2 and 7 days post mortem. Tenderness was significantly improved by electrical stimulation for the Longissimus thoracis et lumborum both at 2 and at 7 days post mortem, while for Semimembranosus electrical stimulation significantly increased tenderness just at 7 days post mortem.     

Effects of electrical stimulation on post-mortem changes in the activities of two Ca dependent neutral proteinases and their inhibitor in beef muscle

Post-mortem changes in the activities of two calcium-dependent neutral proteinases and their inhibitor were studied in electrically stimulated and control beef Longissimus dorsi muscles.During meat conditioning in control muscles the activity of the high-calcium-requiring proteinase (mmCa ANP) was not affected while a decrease in the activities of the low-calcium-requiring proteinase (μmCa ANP) was observed.After electrical stimulation, the mmCa ANP activity was only slightly decreased, but the μmCa ANP and the inhibitor activities were drastically affected. An 80% decrease in μmCa ANP activity was observed 4 h post electrical stimulation. The post-mortem changes seem to be closely related to the post-mortem pH fall.If the Ca-requiring neutral proteinases are involved in the meat ageing mechanism, it eems that the μmCa ANP is more likely to cause most of the post-mortem changes in the myofibrillar proteins, taking into account the low level of intracellular calcium. But as its activity is drastically reduced during the rigor mortis process, most of the effect of μmCa ANP could occur at the very beginning of the post-mortem changes.     

Pathways of high and low voltage electrical stimulation in sheep carcasses

Using the neuromuscular blocking agent, curare, the involvement of the nervous system in the transmission of high (850 V peak) and low voltage (45 V peak) electrical stimulation in sheep carcasses was studied. Post-mortem metabolism in the Longissimus dorsi, Semitendinosus and Semimembranosus muscles was followed by measuring changes in pH, adenosine triphosphate, creatine phosphate, inorganic phosphate and hexose monophosphate. Stimulation was considered to be effective if it accelerated glycolysis. Low voltage stimulation of glycolysis was completely inhibited by curare, indicating that a functional nervous system is required for effective stimulation by low voltages. In contrast, high voltage stimulation of glycolysis was not affected by curare, indicating that high voltages exert their effect by depolarising the cell membrane directly.     

Low voltage electrical stimulation and post-mortem energy metabolism in beef

In the present study, the influence of low voltage electrical stimulation on glycolysis, concentrations of creatine phosphate and adenine nucleotides, as well as the relationship between metabolic activity and tenderness, was examined in bovine M. longissimus dorsi. During stimulation, about 50% of the creatine phosphate was consumed and the immediate pH drop was, on average, 0·21 units. The increased energy consumption resulted in a decrease in the halftime of ATP from 10 h in non-stimulated muscles to 5 h in electrically stimulated muscles. The total high energy phosphate turnover was found to be 2400 μmol/h/g during stimulation and 18 μmol/h/g immediately following stimulation compared with 9 μmol/h/g for the non-stimulated counterpart. No obvious abnormalities were found in the course of changes during energy metabolism following stimulation, besides the increased metabolic rate. Significant relationships were found between the degradation of several of the energy metabolites during stimulation and improvements in tenderness. It is concluded that, within the variation found in this study, the better the effect of electrical stimulation in increasing the metabolic rate, the better the resulting meat tenderness. Results also showed that the higher the shear force in the non-stimulated part, the better was the effect of electrical stimulation in improving the meat tenderness.     

Influence of myosin heavy chain isoform expression and postmortem metabolism on the ATPase activity of muscle fibers

The objective of this study was to determine the effects of postmortem muscle pH and temperature declines on the actomyosin ATPase activity of muscle fibers expressing different MyHC isoforms. Using a quantitative histochemical procedure to determine ATPase activity, the maximum actomyosin ATPase activity was determined on individual fibers classified by MyHC expression. Samples were collected from the red (RST) and white (WST) semitendinosus muscles at 3 min and 24 h postmortem from electrically stimulated (ES) and control (NS) pork carcasses. In samples taken at 3 min postmortem, type I fibers had the lowest ATPase activity staining and type 2X and 2B had the highest activity staining, with type 2A fibers intermediate. Postmortem time and carcass treatment did not influence the ATPase activity staining of type I muscle fibers. ATPase activity staining of 2A fibers was lower (p<0.001) in 24 h samples than in 3 min samples from ES carcasses. In 3 min and NS-24 h samples, RST type 2A fibers had lower (p<0.05) activities than type 2A fibers from the WST. In type 2X fibers, ATPase activity staining decreased (p<0.01) from 3 min to 24 h postmortem in ES carcasses. This decrease was more severe in WST 2X fibers compared to RST 2X fibers. ATPase activity staining in type 2B fibers did not decrease from 3 min to 24 h postmortem in NS carcasses. In ES carcasses, activity staining of 2B fibers decreased (p<0.0001) with time postmortem. The results of the experiment indicate that fibers expressing fast MyHC isoforms have a higher ATPase activity early postmortem than slow muscle fibers but are more prone to inactivation by a rapid pH decline.     

Meat color stability affected by barley variety fed in finishing diet to beef steers

Angus crossbred steers were assigned randomly to one of four finishing diets based on corn, Chinook, Logan, or H3 barley. Steers were harvested and after a 72-h chill, carcass quality and yield grade data were collected. Beef ribs were removed from 72 carcasses for further analysis. Ribs were aged in vacuum bag at 2 °C for 14 days. After aging three adjacent steaks (3.18 cm) were removed to determine color stability, tenderness, proximate analysis and pH. Diets fed to steers had no effect on quality and yield grade or tenderness of beef steaks. Steaks from steers fed Logan barley variety were significantly less red at 10 days of storage (Hunter a* = 24.06) than steaks from steers fed the other barley varieties (Chinook a* = 26.4; H3 a* = 28.05) or corn (a* = 26.14). Identification of barley varieties that affect color stability could result in designing diets specifically for improved color and increase the use of barley as a finishing grain.     

PROPERTIES OF COMMERCIAL MICROBIAL PROTEINASE PREPARATIONS

Twenty three commercial microbial proteinase preparations derived from various Bacillus or Aspergillus spp. or from Rhizomucor niveus were assessed for proteolytic activity on azocasein at pH 5.5 or 7.0, or specificity on sodium caseinate at pH 5.5 and semi-quantitatively assessed for esterase, lipase, trypsin, chymotrypsin, general aminopeptidase, phosphatase and glycosidase activities using the API-ZYM system. Selected preparations were further assayed for peptidase, esterase and lipase activities at pH 7.0. The proteolytic activity of the Bacillus preparations was greater at pH 7.0, while that of the Aspergillus and Rhizomucor preparations was greater at pH 5.5. All the Bacillus preparations contained one of two main proteolytic activities, thought to be either bacillolysin or subtilisin. Most of the Aspergillus preparations contained the same proteinase, thought to be aspergillopepsin I, but two preparations appeared to contain a different unidentified proteinase. The proteolytic specificity of the Rhizomucor preparation was different from the Bacillus or Aspergillus preparations; thought to be rhizopuspepsin. According to the results of the API-ZYM system, all preparations contained enzyme activities in addition to their main proteolytic activity, with the Aspergillus and Rhizomucor preparations containing the highest levels and widest range of activities. Generally preparations derived from Aspergillus contained the highest level of general, proline and endopeptidase activities, with the Bacillus preparations conspicuous by the absence of general and proline-specific peptidase activities, while the Rhizomucor niveus preparation contained little or no general or endopeptidase activity. Esterase activity was found in all of the preparations evaluated, with only two Aspergillus preparations containing lipase activity.     

Electrical stimulation of post-mortem glycolysis in the Semitendinosus muscle of sheep

Changes in pH and phosphorylase a content in control and electrically stimulated Semitendinosus muscles of sheep were studied. The results provided evidence of acceleration of glycolysis during slaughter and sampling. In muscle electrically stimulated soon after slaughter there was a transient increase in phosphorylase a during stimulation. The magnitude of this increase and of the fall in pH during stimulation both depended on the pH of the muscle at the time of stimulation and reduced to zero when this pH was about 6·3. There was little or no phosphorylase a in samples of muscle taken about 35 min post mortem or, at any time, in muscle electrically stimulated about 35 min post mortem. Possible reasons for the transient nature of the increase in phosphorylase a content during stimulation are discussed.     

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing

The aim of this study was to investigate the effect of water activity (a_w) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various a_ws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 μg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of β-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P < 0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a ~ 7.5-log₁₀ reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P < 0.05) when the peptone water concentration was decreased below 60% (a_w ~ 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r² = 0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low a_w results in protein stabilization, which prevents protein denaturation and cell death during HPP.