The effect of temperature on the activity of μ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.     

Effect of stress-induced high post-mortem pH on protease activity and tenderness of beef

Forty-four Swiss Brown young bulls were stressed by regrouping unfamiliar animals before slaughter. M. longissimus thoracis (6-9th ribs) of carcasses were analysed for post-mortem pH, protease activities (m- and α-calpain, calpastatin and cathepsin B + L), Warner-Bratzler shear force and sensory tenderness and juiciness. Muscles were classified into three groups, according to ultimate pH values: > 6.3, 6.3-5.8 and < 5.8. The most significant difference related to high pH was a higher activity of m-calpain at 7th day post mortem. It was also found that meat showing the highest pH was significantly more tender and juicy. Sensory tenderness was highly correlated with activity of m-calpain at 7th day post mortem (r = 0.776) and with ultimate pH (r = 0.708). It is concluded that high ultimate pH induced by stress significantly increases m-calpain activity, and this results in a greatly enhanced tenderisation of beef meat.     

牛肉成熟过程中氧化对钙激活酶活性及降解的影响

为了探究牛肉宰后成熟过程中氧化对钙激活酶活性及降解的影响,在牛屠宰后立即取其背最长肌,注射不同浓度氧化剂（0 mmol/L H2O2＋5 mmol/L NaCl ;50 mmol/L H2O2＋5 mmol/L NaCl;200 mmol/L H2O2＋5 mmol/L NaCl;500 mmol/L H2O2＋5 mmol/L NaCl）。真空包装后于4 ℃成熟。分别在0、1、3、7、14 d时取样,通过活性电泳和蛋白免疫印迹进行分析钙激活酶的活性和降解情况。结果表明：在牛肉的宰后成熟过程中,随着氧化程度的提高,μ-钙激活酶活性逐渐减弱,降解程度逐渐增强,而m-钙激活酶活性几乎未发生变化。因此,氧化促进了μ-钙激活酶的降解并抑制了其活性。     

Activities of calpastatin, μ-calpain and m-calpain are stable during frozen storage of meat

The stability of μ-calpain, m-calpain and calpastatin activity during frozen storage of pork was studied in two experiments. In experiment 1, pork longissimus muscle was stored at either -20 or -80°C, and the samples were assayed at 2-3 weeks interval for calpain activity and calpastatin activity using a m-calpain stock solution stored at 4°C. No effects on calpain activity at either temperature were observed for up to 123 days of storage. Calpastatin activity was stable the first few weeks of storage, where after it decreased up to 143 days of storage independently of meat storage temperature. At day 143, calpastatin activity was also assayed using a newly purified stock solution of m-calpain giving a calpastatin activity equal to the activity measured day 0 using the original m-calpain stock solution. The m-calpain stock solution was unstable during storage at 4°C and the activity decreased in a linear manner and was highly related to the observed decrease in calpastatin activity during storage. In experiment 2, meat was stored as in experiment 1 and was assayed at 2-3week intervals for calpastatin activity using a m-calpain stock solution stored at either 4 or -80°C. As in experiment 1, the measured activity of calpastatin decreased during storage using m-calpain stock solution stored at 4°C and this decrease was highly correlated to the decrease in the activity of the m-calpain stock solution. The activity of the m-calpain stock solution stored at -80°C was constant during storage period of 153 days and likewise was the calpastatin activity measured using this stock solution. The relation between measured calpastatin activity and storage time of m-calpain stock solution was tested by adding, to a calpastatin assay, up to 10μL of a partly inactivated m-calpain solution. A negative relationship was observed between added inactivated m-calpain and measured calpastatin activity which suggests that the inactive m-calpain molecules mask some of the binding sites on calpastatin and thereby prevent some of the active m-calpain molecules from binding to calpastatin. This would underestimate the measured calpastatin activity. In conclusion, the calpains as well as calpastatin are stable during frozen storage of meat, and the observed decreased in calpastatin activity is due to instability of the m-calpain stock solution used in the calpastatin assay.     

Post-mortem kinetics of meat tenderness and the components of the calpain system in bull skeletal muscle

Eight strip loins (M. longissimus dorsi) from pasture fed Friesian bulls were aged at 15 °C for a range of times from 1 to 120 h. pH declined from 6.29 (SE 0.119) one hour post slaughter to an ultimate pH of 5.48 (SE 0.013). The activities of the components of the calpain system (μ-calpain, m-calpain and calpastatin) were determined after separation on a DEAE-sephacel column. There was a dramatic decline in μ-calpain activity post slaughter with a complete disappearance within 48 h. The rates of decline in m-calpain and calpastatin activity were slower with 30% and 50% remaining 120 h post slaughter, respectively. The rapid decline in μ-calpain activity relative to the calpastatin activity is likely to reduce the degree of tenderisation and ultimate tenderness of the meat.     

Proteolysis and tenderisation in reindeer (Rangifer tarandus tarandus L.) bull longissimus thoracis muscle of varying ultimate pH

The effect of dietary α-tocopheryl acetate supplementation on the uptake of α-tocopherol in ewe plasma, lamb plasma, milk, organs and muscles was investigated. The oxidative stability and colour in fresh M. longissimus dorsi and frozen M. longissimus dorsi, M. psoas major and M. gluteus medius were also investigated. Ewes (n = 12) were selected and scanned to assess pregnancy. They were divided into two groups (n = 6). The control group was fed a diet containing 20 mg α-tocopheryl acetate/kg feed/day and the supplemented group fed a diet containing 1000 mg α-tocopheryl acetate/kg feed/day, for 9 weeks ante-parturition and 3 weeks post-parturition. The lambs were weaned at 3 weeks and fed supplemented or basal feed for 10 weeks before slaughter. Plasma α-tocopherol increased significantly (p < 0.01) in ewes in the 9 weeks ante-parturition, and lamb plasma taken just before slaughter was significantly (p < 0.01) higher for the supplemented group than the basal group, following 13 weeks of supplementation. Milk α-tocopherol levels were significantly (p < 0.01) higher from ewes fed the supplemented diet at parturition and for the three weeks of supplementation post-parturition (p < 0.05). Supplementation increased the α-tocopherol levels in all tissues sampled. The α-tocopherol concentrations in M. longissimus dorsi and M. psoas major were also determined after frozen storage at -20 °C for 34 weeks. Frozen storage resulted in a significant (p < 0.01) reduction in mean α-tocopherol levels for M. longissimus dorsi but not M. psoas major. Dietary supplementation with α-tocopheryl acetate significantly (p < 0.05) increased the oxidative stability of lamb muscle. Surface colour (Hunter L, a, b) was found to be negatively correlated with metmyoglobin content. Supplementation reduced surface discolouration in refrigerated display under fluorescent light over a 6-7 day storage period. The effect was more pronounced in frozen displayed muscles than in freshly displayed samples.     

Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either μ-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin molecule were identified by N-terminal sequencing of the different proteolytic fragments. Desmin incubated with either m-calpain or μ-calpain was primarily cleaved in the head and tail region leaving the rod domain relatively intact even after prolonged incubation. Incubation with cathepsin B produces a sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved in degradation of desmin early post-mortem, targeting the non-helical region of the desmin molecule and resulting in depolymerisation and initial disorganisation of the intermediate filament structures of the muscle cell.     

Determinants of tenderisation in beef Longissimus dorsi and Triceps brachii muscles

Tenderisation of bovine Mm. longissimus dorsi and triceps brachii and factors impacting tenderisation were studied. Mm. longissimus dorsi and triceps brachii of 12 Friesian-Holstein cows (age 3-11 years; 212-349 kg carcass weight) were sampled at various times post mortem (p.m.) for determination of pH, temperature, fibre type and morphology, connective tissue distribution, SDS-PAGE of myofibrillar proteins, Warner-Bratzler shear force, sarcomere length and osmolality. The stretched position of the M. triceps brachii (sarcomere length 2.35 ± 0.24 μm) resulted in a relatively low shear force at 1 day p.m. (6.2 ± 0.9 kg/cm(2)) with further storage having little additional effect. M. longissimus dorsi entered rigor in a more contracted state (sarcomere length 1.65 ± 0.11 μm), resulting in a relatively high shear force at 1 day p.m. (10.3 ± 2.3). Stepwise linear regression was used to calculate the best 1- to 3-variable equations for shear force of M. longissimus dorsi at 1, 7 and 14 days p.m. and the decrease in shear force between 7 and 14 days p.m. Shear force at 1 day p.m. appeared to be determined mainly by the speed of pH- and temperature-decline. Proteolysis of myofibrillar proteins and animal age appeared to be the main determinants for shear force at 1 and 14 days p.m. The average surface area of type I fibres could explain part of the variation in the decrease in shear force between 1 and 14 days p.m.     

Measurement of autolysis in beef muscle homogenates

Homogenates of beef longissimus dorsi muscle have been used to study the effect of pH, temperature and Ca(2+) ions on post-mortem autolysis. Measurements of myofibrillar proteins were made after SDS-polyacrylamide gel electrophoresis and it was found that the intensity of the troponin T band could be used as an indicator of autolysis. At pH values below 6·0, the addition of EDTA increased the rate of loss of troponin T: above pH 6·0, the rate of loss of troponin T was accelerated by Ca(2+) ions. It was concluded that there were at least two proteolytic enzyme systems involved-cathepsin B at low pH and a calcium-activated factor at high pH.     

Dehydrogenase Enzymes Associated to Glycolysis in Beef Carcasses Stored at 0 °C

After the death of an animal, cell metabolism is controlled locally. The post-mortem oxygen depletion increases the glycolytic activity and lactate production. However, many mechanisms of post-mortem metabolic regulation have not been fully investigated in beef carcasses. In this work, we studied the post-mortem glycolytic behavior (including lactate dehydrogenase) and three dehydrogenase associated to glycolysis (glycerophosphate dehydrogenase, glucose 6-phosphate dehydrogenase, and glycerol dehydrogenase) by using cytochemistry techniques in three fast-twitch muscles (M. longissimus dorsi, M. semimembranosus, and M. cutaneus trunci) of carcasses stored at 0 °C. Our results indicate that glycolysis depends on the type of muscle. The post-mortem glycolytic flux and lactate dehydrogenase activity of M. cutaneus trunci was the lowest of the three muscles studied. Of the other dehydrogenases analyzed, only glycerophosphate and glycerol dehydrogenase showed clear cytochemical reaction. Glucose 6-phosphate dehydrogenase was not used by muscles very much. The glycerophosphate dehydrogenase was the strongest enzymatic activity correlated to the post-mortem glycolytic flux. In addition, a relationship between glycerophosphate dehydrogenase and glycerol dehydrogenase was detected by using a multiple regression model. This phenomenon was studied by using bioinformatics tools, suggesting that glycerophosphate dehydrogenase could oxidize the glycerol in bovine fast-twitch muscles.     

Influence of early pH decline on calpain activity in porcine muscle

This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation. From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3 h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline. The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24 h. Calpain activity and autolysis from 1 to 72 h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation.     

注射水和氯化钙溶液对宰后猪肉肉色及其稳定性的影响

目的：探讨注射水和氯化钙溶液对宰后冷藏期间猪背最长肌肉色及其稳定性的影响。方法：猪背最长肌于宰后1.5h注射肉质量分数5%的水和200mmol/L氯化钙溶液,分别测定其冷藏期间肉色a*值、总色素含量、高铁肌红蛋白(MetMb)相对百分含量、MetMb还原酶活性等指标。结果：注射氯化钙溶液能降低肉中a*值、总色素含量、MetMb还原酶活性和乳酸脱氢酶-B(LDH-B)活性,增加MetMb相对百分含量和丙二醛(MDA)含量。注水能降低肉中总色素含量和MetMb还原酶活性,增加MetMb相对百分含量。结论：注射水和氯化钙溶液均能降低宰后冷藏期间猪背最长肌中总色素含量,增加MetMb相对百分含量,从而加快猪背最长肌的褪色,不利于其冷藏期间新鲜肉色的维持。     

猪背最长肌蛋白质在冷冻贮藏过程中的变化

为研究冻藏时间对猪背最长肌蛋白质特性的影响,本文主要采用凝胶电泳、差示量热扫描技术分析了猪背最长肌在-18 ℃冻藏0 d和1、3、6、9、12、15月后,其蛋白质的疏水性、巯基含量、降解情况、热稳定性的变化。结果表明:随冻藏时间的延长,蛋白质溶解度显著降低(p<0.05),肌原纤维蛋白(MPI)的表面疏水性和巯基含量升高,发生降解的MPI主要集中在67 kDa和20~29 kDa;肌球蛋白和肌浆蛋白的变性温度和焓值呈现逐渐降低的趋势,表明这两类蛋白的热稳定性降低。在冻藏的前3个月内,猪背最长肌的各项指标维持在较好的水平,从第6个月开始,所有指标的变化幅值增加,变化速率加快。研究发现,冻藏6个月以后,猪背最长肌蛋白质品质开始加速下降。     

The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging

The objective was to study the potential role of calpain 3 in postmortem myofibril breakdown and meat tenderization. We determined the temporal changes in calpain 3 protein in the ovine m. longissimus thoracis et lumborum (LTL, n=4) during post-mortem storage. Concurrently, we also determined the kinetics of tenderization level, changes in MFI, degradation of nebulin and desmin, and autolysis of calpain 1. The autolysis of calpains 1 and 3 were strongly correlated with the kinetics of tenderization and changes in MFI. The best correlation was between the appearance of the autolyzed calpains 1 and 3 and nebulin degradation. Taken together, the results indicated that calpains 1 and/ or 3 might be playing a key role in post-mortem tenderization of LTL via the proteolysis of specific muscle structural proteins such as nebulin. This is the first report that relates calpain 3 to myofibrillar protein degradation in post-mortem skeletal muscle.     

不同排酸方式对巴美肉羊肉品质的影响

随机选择相同饲养条件下的巴美肉羊,以非吊挂排酸和吊挂排酸两种方式进行对比实验,分别测定其不同部位0.5～48h pH值的变化规律和24～72h剪切力的变化规律。结果显示：非吊挂排酸羊股二头肌、背最长肌、臂三头肌均在宰后6h或8h的pH值显著小于吊挂排酸羊（P〈0.05）。非吊挂排酸羊在不同时间pH值没出现显著下降,吊挂排酸羊在不同时间pH值易出现显著下降。通过对巴美肉羊宰后24～72h时间内指定部位肌肉剪切力变化规律得出吊挂排酸羊3个指定部位剪切力总体要比非吊挂排酸羊剪切力低。在吊挂排酸方式下背最长肌剪切力最易出现显著变化。     

冷冻贮藏过程中氧化诱导牦牛肉肌原纤维蛋白结构的变化

探讨在普通包装和真空包装冻藏条件下的牦牛背最长肌和股二头肌的肌原纤维蛋白氧化变化。结果表明：羰基含量60 d时显著增加（P＜0.05）,除股二头肌普通包装组在90 d保藏时的羰基含量显著升高外,其他处理组都呈下降趋势（P＞0.05）;总巯基含量在冻藏60 d内总体上升（P＜0.05）,60 d之后呈下降趋势;采用真空包装的两个处理组的表面疏水性在冷冻贮藏90 d以后都比普通包装的低;背最长肌的Ca2＋-ATP酶活性比股二头肌的低,而K-ATP酶活性则无显著差异。背最长肌肌原纤维蛋白溶解性在整个实验周期中有显著变化（P＜0.05）,股二头肌肌原纤维蛋白溶解性的变化不显著（P＞0.05）。此外,冷冻贮藏期间的蛋白质条带发生了变化,肌球蛋白重链和肌动蛋白随着冻藏时间的延长都发生了不同程度的降解。该结果说明随着冷冻贮藏时间的延长,肌原纤维蛋白发生了氧化。且随时间延长,蛋白氧化越严重,表面疏水性和溶解性越低,总巯基含量、K-ATP酶活性和Ca-ATP酶活性含量越高。     

紫外照射和温度波动对冷鲜肉肉色稳定性的影响

以不同部位冷鲜猪肉(背最长肌、后腿肉、腰大肌)为对象,研究肉样在2种贮藏条件(实际销售贮藏条件:4℃-13 h-紫外照射,0℃-11 h-避光;实验室设置条件:4℃-24 h-紫外照射)下色泽、肌红蛋白含量、高铁肌红蛋白还原酶活等的变化,旨在为提高冷鲜肉在销售贮藏期间的肉色稳定性提供依据。结果表明:实验室条件下,3个部位(背最长肌、后腿肉、腰大肌)冷鲜猪肉的色泽感官评定、a*值、氧合肌红蛋白含量和高铁肌红蛋白还原酶活性等均显著优于实际销售条件(P<0.05),实验室条件下更利于不同部位冷鲜猪肉的贮存。另外,在实验室条件下,3个部位中猪背最长肌肉色最稳定,其次是后腿肉,腰大肌肉色稳定性最差。     

The effect of altered growth rates on the calpain proteolytic system and meat tenderness in cattle

The present study was conducted to determine the effects of growth pattern on the calpain system and meat tenderization. Twenty-four Friesian calves were randomly allocated to three treatment groups: FAST (fast growth rate), SLOW (severely restricted growth rate) and ALTER (restricted growth for 30 days followed by fast growth rate). Four animals from each group were slaughtered on day 32 or 45 after altering the growth rates. Samples of M. longissimus dorsi were rapidly frozen at slaughter for protein analysis by Western blotting. Restricted growth reduced the immunoreactivity of a calpastatin band (135 kDa) measured at 24 h postmortem. Immunoreactivity associated with the large subunit of μ- or m-calpain appeared to be unaffected by growth patterns. Shear force measurements taken after 14 days of conditioning were positively related to 135 kDa calpastatin at 24 h postmortem. In this study there was no clear relationship between shear force and growth pattern.     

Electrical stimulation of red deer (Cervus elaphus) carcasses - effects on rate of pH-decline, meat tenderness, colour stability and water-holding capacity

A total of 14 female red deer were included in a study on the effects of low voltage carcass electrical stimulation on meat tenderness, colour stability and water-holding capacity. Carcasses were randomly allocated to either electrical stimulation treatment (ES; 90-95 V unipolar pulses, 7.5 ms duration, 15 Hz for a duration of 55 s) or no electrical stimulation (non-ES) (n=7 in each group). Temperature and pH decline was recorded in M.m. triceps brachii, longissimus dorsi et lumborum (at the last rib; LD) and biceps femoris, at intervals from 0.5 to 20 h post-mortem. At 24 h post-mortem, LD from the left side were excised, vacuum packaged and refrigerated at -1.5°C. Glycogen concentrations, measured at 30 min post-mortem, and ultimate pH did not differ between groups. Compared to controls, ES increased the rate of muscle pH decline and produced lower shear forces at 1 day, 1 week and 3 weeks post-mortem, but these differences disappeared by 6 and 12 weeks post-mortem. Sarcomere lengths at 24 h post-mortem were unchanged by ES. After 1 week of refrigerated storage, ES significantly reduced display life (hours of Minolta a* value ? 12), but this difference disappeared at 3, 6 and 12 weeks of ageing. ES did not affect drip at any ageing time point. The present results demonstrate that the benefits of ES on tenderness are not permanent, and the procedure is not necessary for a long-term, chilled product. This study showed no detrimental effects of using electrical stimulation on meat colour stability or drip loss.     

Factors Affecting Collagen Solubility in Bovine Muscles

SUMMARY— Solubility of intramuscular collagen was studied as affected by chronological maturity in 15 bovine longissimus dorsi and 15 semimembranosus muscles and as affected by post-mortem contraction state in the semitendinosus of 7 animals. Collagen solubility decreased significantly with each advancing maturity group in both longissimus dorsi and semimembranosus muscles. Collagen solubility was also higher (P < 0.05) in the longissimus dorsi than in the semimembranosus, except in the E maturity group. It was also related to panel tenderness in both muscles (r = 0.77 and 0.81 (P < 0.01) for longissimus dorsi and semimembranosus muscles, respectively. However, within-maturity group correlations of solubility of collagen and tenderness were low and nonsignificent.
Collagen content did not differ significantly in longissimus dorsi muscles of animals of A, B, and E maturity groups; however, the semimembranosus had more collagen (P < 0.05) in E than in A and B maturity groups. Collagen content was not related (P > 0.05) to panel tenderness in either muscle (r =−0.42 and −0.48 for longissimus dorsi and semimembranosus, respectively). Neither collagen solubility nor collagen content was significantly affected by post-mortem contraction state. Furthermore, collagen solubility did not increase significantly with post-mortem aging up to ten days.