Post-Mortem Changes in Subcellular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 1. Calcium Accumulation and Adenosine Triphosphatase Activities

SUMMARY– Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum and light sarcoplasmic reticulum fractions were isolated by differential centrifugation of homogenates from normal and pale, soft, exudative (PSE) porcine muscle at various times post-mortem. Calcium uptake was measured using a solution containing⁴⁵Ca⁺⁺. The oxalate-stimulated calcium accumulating ability of the subcellular fractions declined 5-10 fold between 0 and 24 hr post-mortem. The major portion of this decline occurred in the first hour after death in fractions from PSE muscle but was more gradual in the normal fractions. The ATPase activities of normal and PSE fractions obtained at death did not differ significantly. These activities increased with time post-mortem in most normal fractions but decreased in those from PSE muscle. The subcellular site of ATP hydrolysis post-mortem was discussed. The results obtained point to the potential importance of the relaxing, factor in muscle post-mortem.     

Post-Mortem Changes in Subcelllular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 2. Electron Microscopy.

SUMMARY— Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum, and light sarcoplasmic reticulum fractions were isolated from homogenates of normal and pale, soft, exudative (PSE) porcine muscle at 0 and 24 hr post-mortem and examined by electron microscopy. No differences were observed between normal and PSE myofibrils obtained at death. PSE myofibrils prepared at 24 hr post-mortem had more granular appearing filaments and wider Z lines than normal myofibrils at 24 hr. The PSE heavy sarcoplasmic reticulum fraction obtained at death had a higher proportion of granular material than the same fraction from normal muscle. Several structural differences between the other PSE and normal fractions were also observed, especially at 24 hr postmortem. This study indicated that the composition of the subcellular fractions changed with time post-mortem and that this change should be considered when analyzing biochemical data from these fractions. However, the differences observed could not explain the large changes in calcium accumulating ability that have been shown to occur post-mortem.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE. 9. Effect of Temperature and pH on Tropomyosin-Troponin and Purified α-Actinin from Rabbit Muscle

SUMMARY– A study was done on the effects of in vitro storage of purified α-actinin, troponin, tropomyosin, and the tropomyosin-troponin complex on the activity of these protein fractions in the ATPase and superprecipitation assays. Storage was done at various combinations of temperatures between 0 and 40°C and pH values between 5.7 and 7.0. Even after 40 hr of storage, activities of purified tropomyosin and the tropomyosin-troponin complex were not affected by any combination of temperature and pH included in this study, but activities of purified α-actinin and troponin were almost completely lost after 16 hr at 40°C and pH 5.7. Storage for 40 hr at low pH (5.7) and low temperatures (0°C) did not affect the activity of either α-actinin or troponin, but 40 hr of storage at high temperatures (40°C) and neutral pH caused some loss in activity for both these proteins. This loss of activity caused by 40°C, pH 7.0 storage was much more noticeable in the case of troponin than in the case of α-actinin. Storage periods of 40 hr or longer were required before any loss of α-actinin activity could be detected at pH 7.0 and 40°C. Since most meat animal carcasses are chilled soon after exsanguination and attain muscle temperatures of 25°C or lower before the pH falls below 6.2, it is probable that α-actinin and tropomyosin-troponin activity remain almost unchanged in meat handled through normal market channels. However, myofibrillar tissue in those porcine animals whose musculature undergoes a very rapid post-mortem decline in pH so that values of 5.7 or less are reached while muscle temperatures are still 37°C or higher may lose much of its α-actinin and tropomyosin-troponin activity during the first 24 hr post-mortem.     

SOME CHARACTERISTICS OF THE NAD(P)H-DEPENDENT LIPID PEROXIDATION SYSTEM IN THE MICROSOMAL FRACTION OF CHICKEN BREAST MUSCLE

Incubation of the microsomal fraction isolated from chicken breast muscle in the presence of NADPH, ADP and Fe⁺⁺⁺ ions was shown to result in lipid peroxidation. NADH was able to replace NADPH as the source of reducing equivalents but was less efficient. The pH optimum for malonaldehyde (MDA) production was found to be around pH 6. 7. Oxygen uptake by the system was shown to have a pH optimum of 5.5. Oxygen uptake was approximately linearly dependent on temperature but malonaldehyde production exhibited a sharp increase between 25°C and 37°C. Inhibitor studies and the virtual absence of cytochromes indicate that this system is not similar to that isolated from rat liver microsomes. The activity of the lipid peroxidation system was maintained after storage for 7 days at pH 7.25 and 4°C. At pH 5.6 a reduction in activity was noted after 7 days.     

The effect of post-mortem conditions on the extractability and adenosine triphosphatase activity of myofibrillar proteins of rabbit muscle

Summary. 1. Washed myofibrils from rabbit muscle have been heated at pH values between 4.8 and 5.6 and temperatures between 35°C and 42°C. It has been found that, under these conditions, myofibrils lose their Ca²⁺ activated adenosine triphosphatase, their Mg²⁺ activated adenosine triphosphatase and also become less extractable in M KCl–30 mM sodium glycerophosphate, pH 6.2.
2. The reactions follow first-order kinetics and the rates are dependent on pH and temperature. The first order rate constants, enthalpies and entropies for the three reactions are sufficiently near each other to suggest that all three reactions are occurring simultaneously.
3. When a muscle is allowed to go into rigor at 37°C the extractability in M KCl–30 mM sodium glycerophosphate is reduced after 4 hr at 37°C when the pH of the muscle has reached 5.55. At the same time the Ca²⁺ adenosine triphosphatase activity falls but the Mg²⁺ adenosine triphosphatase does not. The latter is reduced by prolonging the period at 37°C to 6 hr.
4. It is suggested that there is present in muscle, undergoing rigor at 37°C, myosin which does not bind to actin and is readily denatured. When bound to actin, myosin in the myofibril is more resistant and denatures only after long exposure to a temperature of 37°C.     

Inactivity of μ-Calpain Throughout Postmortem Aging of Meat

ABSTRACT: In order to determine the involvement of μ-calpain in the tenderization of meat during postmortem aging, μ-calpain was prepared from porcine skeletal muscle and its activity was measured under various conditions, using casein and myofibrils as the substrate. μ-Calpain was inactive at pH 5.89 and below 15 °C. Even when the reaction time was extended to 10 h, it was entirely inactive at pH 5.62 and 5 °C. In the early postmortem stage, when the pH value of muscle is above 6.0, μ-calpain was considered to be inactive due to the low concentration of sarcoplasmic calcium ions. μ-Calpain is, therefore, irrelevant to the tenderization of meat throughout postmortem aging.     

A "Cold Shortening" Effect in Avian Muscle

SUMMARY— Experiments were conducted to determine the effect of post-mortem temperatures between 0 and 20°C on the degree of shortening in isolated pectoralis major muscles of chickens and turkeys. A "cold shortening" effect in these muscles is described and compared to post-mortem pH, average sarcomere length of isolated myofibrils, and relative solubility of myofibrillar and sarcoplasmic proteins.
The degree of muscle shortening at each temperature after various periods post-mortem indicated that shortening was essentially complete after 3 hr in chickens and 5 hr in turkeys. Shortening in muscles stored at 0°C was significantly greater (P < .01) than in the 12–18°C temperature range. Shortening was greatest in muscles stored at 20°C. The degree of gross shortening observed was directly related to the average sarcomere length of isolated myofibrils. Post-mortem decline in pH was not significantly correlated (P > .05) with shortening. Extractability of myofibrillar and sarcoplasmic proteins after 5 hr at either 0 or 16°C was determined and found to be unrelated to the degree of post-mortem shortening.     

Monitoring Myosin Degradation During Conditioning in Chicken Meat Using an Immunological Method

ABSTRACT: Changes of chicken breast myosin during storage at 2°C and 37°C were monitored immunochemically. Anti-myosin subfragment-1 (S-1) monoclonal antibody, which recognized epitopes within the 27 kDa fragment of S-1, and the anti-myosin rod polyclonal antiserum, were prepared. Myosin degradation products were not detected in muscle extracts stored for 3 weeks at 2°C. In contrast, storage at 37°C brought about the degradation of myosin heavy chain to immunologically detectable small fragments. While, myosin rod produced during the conditioning period was not decomposed into any small filaments. Namely, storage of muscle at 37°C resulted in minor amounts of myosin heavy chain degradation, with initial conversion to rod and S-1 fragments, and subsequent breakdown occurred in the S-1 region only. Immunoblot assay also suggested that the pattern of changes in myosin heavy chain in muscle incubated at 37°C was similar to that produced by in vitro digestion with cathepsin D.     

DIGESTION OF MYOFIBRILLAR AND SARCOPLASMIC FRACTIONS FROM MENHADEN MUSCLE BY THE ACTION OF MENHADEN (Brevoortia spp.) AND WHITE CROAKER (Micropogonias furnieri) TRYPSINS

Trypsins from the pyloric caeca of menhaden (Brevoortia spp.) and croaker (Micropogonias furnieri) were used for hydrolysis of both myofibrillar and sarcoplasmic fractions from menhaden muscle. Digestion of muscle proteins was carried out at 37C for 20 min with the pH maintained at either 3, 4, 5, 6, 7, 8 or 9; or for 60 min at the pH's of 5, 7, 8 or 9 at 30C and 37C. The hydrolytic action was evaluated based on the concentration of peptides solubilized. Solubility after digestion of myofibrillar, sarcoplasmic fraction and hemoglobin was optimum at basic pH. The sarcoplasmic fraction was solubilized more than the myofibrillar fraction by both fish trypsins. Similar results were obtained with both crude pyloric extracts and purified trypsins from menhaden and white croaker. Thus, the preparation of the crude pyloric caeca may be preferentially used, because of its low cost as well as the simplicity of the preparative procedure .     

EFFECT OF A PORCINE PANCREATIC COLLAGENASE ON MUSCLE CONNECTIVE TISSUE

SUMMARY– Porcine and bovine pancreas as well as porcine spleen, liver and kidney were analyzed for possible collagenolytic activity. Both the porcine and bovine pancreas extracts possessed collagenolytic activity at pH 5.5 and 37°C. The ability of the pancreas extract to break down connective tissue was not due to tryptic or chymotryptic activity, but was due to a combination of collagenolytic and elastolytic activity. The collagenase was partially purified using Sephadex chromatography and was shown to degrade collagen into small soluble peptide fragments.     

EFFECT OF POSTMORTEM AGING ON CHICKEN BREAST MUSCLE SARCOPLASMIC RETICULUM

FSR (fragmented sarcoplasmic reticulum) isolated from chicken breast muscle (Pectoralis major) at 0 hr, 48 hr and 7 day postmortem was purified using linear density gradient centrifugation. The Ca⁺⁺accumulating ability of the FSR was found to increase with postmortem aging. No loss in ATPase activity was noted nor was any significant change observed in the SDS-gel electrophoresis patterns of the proteins with postmortem aging. The FSR from the aged muscle contained a higher proportion of phospholipids These studies indicate that the Ca⁺⁺ sequestering properties of sarcoplasmic reticulum from chicken breast muscle are not impaired during postmortem aging.     

THE EFFECTS OF DELAYED PICKING AND HIGH-TEMPERATURE CONDITIONING ON THE TEXTURE OF HOT- AND COLD-BONED BROILER BREAST MEAT

Four trials were conducted to evaluate the effects of delayed picking for 30 min versus normal picking, high temperature conditioning at 32°C versus low temperature conditioning at 0°C, and hot boning versus cold boning on broiler breast meat tenderness, pH, sarcomere length and R-values. None of the treatments affected cold-boned shear values. Within the hot-boned group, delayed picking significantly increased shear value (14.5 kg) compared with normally picked samples (12.5 kg). Conditioning at 32°C significantly lowered shear values (12.5 kg) compared with 0°C (14.6 kg). High-temperature conditioning lowered muscle pH immediately after evisceration, but delayed picking had no effect. Following 24 h aging, treatments had no effect on muscle pH or R-value. These results indicate that 32°C conditioning temperature and early picking significantly reduced meat toughness, but not to a commercially practical degree.     

Rigor State, Freeze Condition, pH, and Incubation Temperature and Their Influence on Color Development and Extract Release Volume in Ovine Muscle Homogenates

SUMMARY— The effect of pH and incubation temperature on the development of filtrate color, and a modified extract release volume (ERV) were studied in unfrozen and frozen, pre- and post-rigor ovine muscle homogenates.
Buffer-pH, incubation temperature and the interaction between these two factors had a highly significant effect (P < 0.01) on filtrate color and ERV. Rigor state and the interactions, rigor state × buffer-pH, freeze state × buffer-pH, and freeze state × incubation temperature had a highly significant effect (P<0.01) upon ERV after 24 hr incubation.
Pale colored filtrates developed in homogenates that were buffered at pH 5.2 and 5.6 and incubated at 20, 30, and 40°C. There was a significant correlation between ultimate pH and color scores for the filtrates obtained from homogenates incubated at 10, 20, 30, and 40°C but there was no relationship between the two variables when the homogenates were incubated at 0 and 5°C. The ERV decreased significantly (P < 0.01) with increasing incubation temperatures and pH of buffer. Pre-rigor homogenates at low pH levels released significantly smaller (P < 0.01) amounts of extract than did post-rigor tissue. Incubation temperature influenced the magnitude of the correlation between ultimate pH and ERV.     

EFFECT OF ULTIMATE pH UPON THE WATER-HOLDING CAPACITY AND TENDERNESS OF MUTTON

SUMMARY— Ultimate pH values in the musculature of sheep ranging from 5.6–7.0 have been obtained by using pre-slaughter injections of epinephrine. A high speed centrifugal method was used to measure the water-holding capacity of raw M. semitendinosus (ST), semimembranosus (SM) and biceps femoris (BF). Results showed a high correlation with ultimate pH. Cooking losses end the amounts of centrifugally expressed juice were determined for the SM and BF cooked for 1 hr at either 65°C or 90°C. Cooking losses at 65°C decreased linearly with increasing pH while the losses at 90°C showed little change up to o raw meat pH of ca. 5.9, then decreased linearly with increasing pH. The amount of juice centrifugally expressed from the cooked meat, which has a high positive correlations with organoleptic juiciness, increased linearly with pH. Tenderness of the cooked SM and BF muscles was measured using o Warner-Bratzler shearing device and an Instron Universal Testing Machine: both gave high objective-subjective correlations. Instron measurements have high negative linear correlations with ultimate pH for both the 65°C and 90°C cooked samples. Hardness of these muscles, cooked at 65°C or 90°C. decreased approximately three-fold as ultimate pH increased from 5.6–6.9. Results obtained using the Warner-Bratzler device showed linear regressions with a significant quadratic component for one muscle at both 65°C and 90°C.     

Alterations of Bovine Sarcoplasmic Proteins as Influenced by High Temperature Aging

SUMMARY— Effects of high temperature aging upon certain characteristics of bovine I. dorsi muscle were studied. Paired wholesale ribs of carcasses were obtained subsequent to slaughter. The left rib of each pair was held at 30°C for 24 hr, then stored at 3°C. Analogous right ribs were immediately stored at 3°C. A sampling schedule of 0, 1, 2, 3, 4, 7 and 10 days was followed.
There were minor variations in moisture, pH, tyrosine-tryptophan indices of non-protein nitrogenous compounds and expressible moisture ratios between treatments and with time. These differences were not statistically significant.
Up to three days storage, extractability of water soluble protein was greatest from muscles held at the elevated temperature. After the third day, however, extractability was greater for muscles held at 3°C.
Color differences between muscles treated via the two storage temperatures were marked. Absorbance ratios (422.280 mp) of extracts showed that muscles held at the high temperature had higher extractable levels of oxymyoglobin than ribs held at 3°C. This difference remained apparent throughout the aging period.
Results of DEAE-cellulose ion exchange chromatography of the sarcoplasmic proteins showed only minor variations in profiles between the two aging treatments. Alterations did appear with time. Profile alterations did not appear related to anticipated increases in tenderness.     

Fermentation of calcium-fortified soymilk with Lactobacillus: effects on calcium solubility, isoflavone conversion, and production of organic acids

ABSTRACT:  The objective of this study was to enhance calcium solubility and bioavailability from calcium-fortified soymilk by fermentation with 7 strains of Lactobacillus , namely, L. acidophilus ATCC 4962, ATCC33200, ATCC 4356 , ATCC 4461 , L. casei ASCC 290, L. plantarum ASCC 276, and L. fermentum VRI-003. The parameters that were used are viability, pH, calcium solubility, organic acid, and biologically active isoflavone aglycone content. Calcium-fortified soymilk made from soy protein isolate was inoculated with these probiotic strains, incubated for 24 h at 37 °C, then stored for 14 d at 4 °C. Soluble calcium was measured using atomic absorption spectrophotometry (AA). Organic acids and bioactive isoflavone aglycones, including diadzein, genistein, and glycetein, were measured using HPLC. Viability of the strains in the fermented calcium-fortified soymilk was > 8.5 log₁₀ CFU/g after 24 h fermentation and this was maintained for 14-d storage at 4 °C. After 24 h, there was a significant increase ( P < 0.05) in soluble calcium. L. acidophilus ATCC 4962 and L. casei ASCC 290 demonstrated the highest increase with 89.3% and 87.0% soluble calcium after 24 h, respectively. The increase in calcium solubility observed was related to lowered pH associated with production of lactic and acetic acids. Fermentation significantly increased ( P < 0.05) the level of conversion of isoflavones into biologically active aglycones, including diadzein, genistein, and glycetein. Our results show that fermenting calcium-fortified soymilk with the selected probiotics can potentially enhance the calcium bioavailability of calcium-fortified soymilk due to increased calcium solubility and bioactive isoflavone aglycone enrichment.     

The effect of temperature on the drip,denaturation and extracellular space of pork longissimus dorsi muscle

Pork longissimus dorsi muscles were cut across the muscle into slices as soon as possible after slaughter. The slices were held at temperatures ranging from 10 to 37°C during the first 24 h post-mortem. 1–2% drip was obtained from slices held at 10°C and increased to 10–14% at 37°C. As the amount of drip increased, the protein concentration of the drip fell from about 150 mg/ml to 100 mg/ml. Some of this decrease could be attributed to the denaturation of sarcoplasmic protein, which amounted to a maximum of 19% in the samples with the highest drip. It is also suggested that diffusion of fluid from the myofilaments or sarcoplasmic reticulum could dilute the sarcoplasm. Denaturation of the myofibrillar proteins, as measured by their ATPase activity, was also observed but this was measurable only at holding temperatures above 30°C. The extracellular space which was 2–5% of the muscle section when the holding temperature was 10°C and drip was low, increased to a maximum of 16–25% at 37°C when drip was high. It is suggested that this is a contributory factor to the increase in drip with increasing temperatures.     

Factors affecting solubilisation and oxidation of proteins during equine metmyoglobin-mediated lipid oxidation in extensively washed cod muscle

Lipid and protein changes in extensively washed cod muscle were studied at pH 5.6 and 2 °C for 47 h in the absence and presence of horse metmyoglobin (metMb). MetMb significantly increased lipid peroxides and TBARS formation. MetMb addition reduced solubility of proteins, such as myosin heavy chain and its fragments, and slowed down the enzymatic release of soluble proteins and protein fragments, e.g. fragments from collagen.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

Sarcoplasmic Reticulum Ca2+-ATPase and Cytochrome Oxidase as Indicators of Frozen Storage in Cod (Gadus morhua)

ABSTRACT: Activities of 2 membrane-bound enzymes, Ca²⁺-ATPase from the sarcoplasmic reticulum and cytochrome oxidase from the inner mitochondria membrane, were measured during frozen storage of cod. Enzyme activities were higher in cod muscle samples frozen at −30°C than at −20°C. Freezing-induced activation of both enzymes was observed and the activation was amplified by ice storage prior to freezing. Sensory evaluation conducted at 9 mo of frozen storage showed differences between the sensory properties of cod frozen immediately after catch and frozen after 3 d of storage on ice. These results indicated that the enzymes might be useful as indicators of quality changes by frozen storage.