Myosin heavy chain degradation during post mortem storage of Atlantic cod (Gadus morhua L.)

Post mortem proteolytic degradation of fish fillets leads to textural changes like muscle softening and gaping. In this study proteolytic degradation of myosin heavy chain (MHC) was monitored during storage of muscle and of isolated myofibrils at different temperatures and pH-values by the use of MHC-specific antibodies. The ability of cathepsin D to associate to myofibrillar proteins was also studied. Muscle stored at 6 °C and isolated myofibrils stored at 0 °C, 6 °C and 20 °C were degraded at pH 6.3 or lower. Cathepsin D could be found associated with extensively washed myofibrils. Inhibition of cathepsin D during storage affected the observed MHC-degradation at pH 5.5, but not at pH 6.3. This indicates that cathepsin D to a less extend than formerly believed, is responsible post mortem degradation of MHC.     

Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine

This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency‐high power ultrasounds treatment or according to the linear function after multiple freezing‐thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology.     

Comparative action of cathepsins D, B, H, L and of a new lysosomal cysteine proteinase on rabbit myofibrils

Specific action of cathespins D, B, H, L, and of a new high Mr (molecular weight relative to hydrogen) cysteine proteinase, on rabbit muscle myofibrils was studied at pH 5·7 by following changes affecting their ATPase activities, their calcium sensitivity, their effect on the ultrastructure, as well as the electrophoretic pattern of the contractile proteins in the presence of SDS. With regard to the MgCa-enhanced ATPase activity, all these proteinases had a very similar effect. A decrease in this activity was thus noted concomitantly with a shift of the straight-line graph obtained when plotting the present acto-myosin ATPase activity versus KCl concentrations. Cathepsins D, B, L and the high Mr cysteine proteinase induced a decrease in both the calcium ATPase activity of myosin and the calcium sensitivity of myofibrils. On the contrary, the Mg-EGTA-dependent ATpase activity was increased. Except for cathepsin H, extensive hydrolysis of proteins occurred in myofibrils treated with each of the lysosomal proteinases tested. However, different specificities could be distinguished. On the one hand, cathepsins D and B affected mainly myofibrillar protein running above and below actin, respectively, on SDS-polyacrylamide gel electrophoresis; on the other hand, the high Mr cysteine proteinase exhibited broader specificity since most of the proteins were hydrolyzed irrespective of their Mr. Myofibrils incubated with cathepsins B and the high Mr cysteine proteinase showed ultrastructural modifications at the level of Z-line, M-bands and A-bands. Myofibrils treated with cathepsin D and cathepsin H appeared almost unaltered. On the basis of these characteristics, cathepsin H hardly affected myofibrils. These results provide evidence for the involvement of the lysosomal proteinases in the meat ageing process and are discussed in regard to the changes occurring at the myofibrillar level during conversion of muscle into meat.     

The effects of Ca ions, EGTA and storage time on myofibrillar protein degradation, levels of Ca(2+)-dependent proteases and cathepsins B, H, L and D of ostrich skeletal muscle

The effect of Ca ions and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) on myofibrillar protein degradation showed that when ostrich iliotibialis lateralis muscle was incubated with 10 mM EGTA at 2–4 °C for 24 hr, the activity of extracted cathepsin H was unchanged compared with a buffer-incubated sample. Ca⁺⁺ had no effect on extracted cathepsin H activity, while that of Ca²⁺-dependent protease (CDP) decreased significantly (p < 0.05). Ca²⁺-treatment enhanced post-mortem changes observed in myofibrillar protein patterns (production of fragments around 30 K) that were not observed in EGTA-incubated myofibrils. The effect of storage time on shear force, CDP activity, cathepsin B, D, H and L activities and the SDS-PAGE pattern of myofibrils showed a time-dependent reduction in CDP activity. Of the cathepsins studied only cathepsin H showed a reduction (40%) in activity. The most prominent component appearing on storage at 2–4 °C had a M_r of 27 K. The incubation of myofibrils with CDP mimicked the post-mortem changes. CDP may be responsible for some of the post-mortem changes observed, although shear force measurements suggest these changes do not lead to significant tenderisation.     

The function of endogenous cathepsin in quality deterioration of grass carp (Ctenopharyngodon idella) fillets stored in chilling conditions

Changes of textural properties and cathepsin activity of grass carp fillets during storage in superchilling (?1.5 ± 0.2 °C) and ice (0.2 ± 0.1 °C) was investigated, and the function of cathepsin in quality deterioration of grass carp fillets was discussed. Results showed that shear force of superchilled and ice‐stored fillets decreased by 50% and 55% after 6 days of storage, respectively. The cathepsin activies in different fractions changed significantly during storage at both conditions. Cathepsin B, B+L activities in sarcoplasma, myofibrillar and heavy mitochondrial fraction significantly increased during the early 3 days postmortem, accompanied by remarkable reduction of corresponding activity in lysosome. Cathepsin D activity in sarcoplasma and myofibrillar significantly increased during 6 days of storage with corresponding decrease in lysosome and heavy mitochondria. Correlation analysis showed that changes of shear force of grass carp fillets were significantly correlated with integration of cathepsin B, D and B+L throughout storage time (r² > 0.94). As derived from the first‐order exponential decay model, the enzyme efficiencies (k_a) of cathepsin B and B+L were more than twofold higher than those of cathepsin D, suggesting the major role of cathepsin B and B+L in textural deterioration of grass carp fillets in chilling storage.     

Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins

The modori phenomenon during surimi production is caused by endogenous proteinases, especially cathepsin L and myofibril-bound serine proteinase (MBSP). Cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) was purified to homogeneity by ammonium sulphate fractionation and a series of column chromatographies and revealed a single band with molecular mass of 30 kDa on SDS–PAGE. Peptide mass fingerprinting (PMF) obtained three fragments with 48 amino acid residues, which were highly identical to cathepsin L from other fish species. Its optimal pH and temperature were 5.5 and 55 °C, respectively. Meanwhile, MBSP was purified from the skeletal muscle of blue scad, and the roles of cathepsin L and MBSP in the degradation of myofibrillar proteins were compared. The results indicated that MBSP is more effective than cathepsin L in promoting the degradation of myofibrillar proteins, especially myosin heavy chain (MHC), suggesting that MBSP plays a more significant role.     

Lipid and Protein Changes Related to Quality Loss in Frozen Sardine (<Emphasis Type="Italic">Sardina pilchardus</Emphasis>) Previously Processed Under High-Pressure Conditions

This research focuses on biochemical changes related to quality loss in frozen (?18 °C for 9 months) sardine (Sardina pilchardus) previously subjected to high-pressure (HP) processing (125–200 MPa). The inhibition (p < 0.05) of lipid hydrolysis development (lower free fatty acid formation and lipase activity), observed in frozen sardine as a result of the previous HP treatment, increased with the pressure level applied. Several parameters including peroxide value, thiobarbituric acid index, fluorescent compounds, and polyenes showed that the applied HP conditions prior to sardine freezing did not increase lipid oxidation. Also, HP did not induce a substantial modification of acid phosphatase and cathepsins B and D activities, and the electrophoretic patterns of sarcoplasmic and myofibrillar protein fractions did not change. However, HP processing led to a decrease in myofibrillar protein content in frozen pressure-treated fish, an effect that was higher in 175- and 200-MPa treated samples. In conclusion, this research showed that pressure treatments in the 125–200-MPa range with holding time of 0 min cause only minor modifications in biochemical indicators of deterioration throughout the subsequent frozen storage of samples for up to 9 months. This study shows the need to optimize HP conditions, particularly in the case of applications combining HP treatments, frozen storage, and thawing to obtain products with high quality and commercial viability.     

Relationship between proteolytic changes and tenderness in prerigor lactic acid marinated beef

A meat tenderising procedure involving injection of a lactic acid solution into prerigor muscle was investigated using beef M pectoralis profundus. The distribution of lysosomal enzymes in subcellular fractions, densities of myofibrillar protein bands after SDS‐PAGE and shear force were measured in non‐injected, 0.5 M and 1.0 M lactic‐acid‐injected samples during a 21 days ageing period. The activities of cathepsin B + L and β‐glucuronidase in the soluble fraction increased with level of lactic acid and with time post‐mortem (P < 0.001). Lactic acid and storage decreased densities of SDS‐PAGE bands migrating at the position of myosin heavy chain (MHC) and α‐actinin and increased densities of a 150 kDa band (P < 0.01). SDS‐PAGE of isolated perimysium cleaved with CNBr showed proteolytic cleavage of collagen after prolonged storage. Lactic acid injection significantly reduced shear force (P < 0.001). The cathepsin B + L activity in the soluble fraction correlated to shear force (r = −0.8), the degradation of MHC and α‐actinin (r = −0.88 and −0.90) and the generation of the 150 kDa fragment (r = 0.90) but not to the generation of a 31 kDa fragment (r = 0.05). A major part of the tenderness improvement after lactic acid injection was complete at 24 h post‐mortem, and was therefore due to a rapid process, eg pH‐induced swelling of the muscle structure. The data on enzyme activities and protein degradation, however, suggested that the action of lysosomal cathepsins also contributed to textural changes. © 1999 Society of Chemical Industry     

基于组织蛋白酶催化的不冻液冻结中华管鞭虾中肌原纤维蛋白氧化分析

对比分析利用不冻液(-18℃和-30℃)冻结后冻藏与直接置于冰箱(-18℃和-30℃)冻结后冻藏的中华管鞭虾肌原纤维蛋白氧化指标变化以及组织蛋白酶B、H、L活性变化,通过肌原纤维微结构观察,分析酶活性对肌原纤维蛋白水解产生的作用,并探讨其与肌组织结构的相关性。结果表明：采用-18℃和-30℃不冻液冻结的中华管鞭虾的盐溶性蛋白含量(92.7 mg/g和97.8 mg/g)均明显高于-18℃和-30℃冰箱缓冻组(72.40 mg/g和77.90 mg/g);-30℃不冻液组的表面疏水性最低(400.6μg);两种冻结方式下-30℃组Ca²⁺-ATPase活性均高于-18℃组;总巯基含量变化趋势与Ca²⁺-ATPase活性变化趋势一致。冻藏前期(0～60 d),低温不冻液冻藏能较好地抑制中华管鞭虾组织蛋白酶活性,使其蛋白结构保持良好;冻藏后期(60～120 d),其对于酶活性的抑制作用减弱。综合以上结果,在一定冻藏期内,不冻液冻结比冰箱冻结能够更好减弱冻藏过程中中华管鞭虾的肌原纤维蛋白的氧化和组织蛋白酶活性,且不冻液的冻结温度越低,冻结速率越高,组...     

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.     

Oxidation in HiOx-packaged pork Longissimus muscle predisposes myofibrillar and sarcoplasmic proteins to N-nitrosamine formation in nitrite-curing solution

The effect of meat protein in situ oxidation on the formation of N-nitrosodiethylamine (NDEA) was investigated. Fresh minced pork was untreated (Con) or treated with 700 mg/kg α-tocopherol (Toc) or 300 mg/kg tea polyphenols (PPE), packaged in a HiOx atmosphere (78.8% O₂, 18.8% CO₂, 2.4% N₂), then stored at 2 ± 1 °C for up to 10 days. Crude myofibrillar (MP) or sarcoplasmic (SP) protein (20 mg/mL) extracted from stored meat was reacted with 43 μM sodium nitrite at 80 °C for 1 h. Lipid oxidation was totally inhibited in PPE pork but increased in Con and Toc samples after 10 days. There was significant protein oxidation (losses of sulfhydryls, formation of protein carbonyls) in both MP and SP in all samples during storage. However, the Con group suffered more extensive protein oxidation than Toc and PPE and produced more NDEA (P < 0.05), indicating that protein oxidation promoted nitrosation.     

乳清抗氧化肽对冷藏猪肉糜肌原纤维蛋白功能性及品质的影响

研究乳清抗氧化肽对冷藏猪肉糜肌原纤维蛋白功能性及品质的影响。实验分为6组,第1组为空白对照,第2组加入质量分数20%未水解乳清蛋白,第3~5组中分别加入质量分数10%、15%、20%的乳清抗氧化肽冻干粉,第6组加入质量分数0.02%的丁基羟基茴香醚(butyl hydroxyanisd,BHA)。在肉糜4℃冷藏0、1、3、5、7 d时分别测定浊度、Ca~(2+)-ATPase活力、乳化稳定性、挥发性盐基氮(totalvolatilebasicnitrogen,TVB-N)含量、蒸煮损失率、表面疏水性以及肌原纤维蛋白溶解性的变化。结果表明:添加质量分数15%的乳清抗氧化肽能有效抑制冷藏肉糜肌原纤维浊度的升高,抑制Ca~(2+)-ATPase活力的下降,减少TVB-N的产生,抑制肌原纤维蛋白乳化稳定性和溶解性的降低。添加质量分数20%的乳清抗氧化肽则在抑制生肉糜蒸煮损失和猪肉糜肌原纤维蛋白表面疏水性增加方面效果最佳,其作用与质量分数0.02%BHA相当。上述结果表明,乳清抗氧化肽具有改善冷藏猪肉糜肌原纤维蛋白功能性及品质的作用。     

The effect of active caspase-3 on degradation of chicken myofibrillar proteins and structure of myofibrils

The objective of this study was to investigate the potential contribution of caspase-3 to meat postmortem tenderisation by examining the role of caspase-3 in the degradation of myofibrillar proteins and disruption of myofibril structure in vitro. Myofibrillar protein prepared from chicken muscle was incubated with EDTA or EDTA plus caspase-3 at 25 °C for 16 h and used for detecting muscle protein degradation and ultrastructure of myofibril. Results revealed that caspase-3 reproduced the degradation patterns of titin, nebulin and α-actinin during postmortem storage of meat, but caused little proteolysis of desmin and no appearance of 28–30 kDa peptides. Meanwhile, caspase-3 also induced the weakening in the I band adjacent to Z-lines, which occurred during meat postmortem ageing. Therefore, caspase-3 could account only for a part of the myofibrillar protein degradation observed in naturally aged meat and is likely involved in postmortem tenderisation of meat together with other endogenous proteases.     

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.     

冷冻期间猪肌内脂肪氧化及肌纤维蛋白凝胶性能的差异分析

该试验以猪肉为研究对象,在-18℃冷冻一定周期(0、1、4、8、12周)后,对其肌内脂肪氧化程度和肌原纤维蛋白的凝胶性能(溶解度、凝胶保水性、凝胶白度及强度)进行测定.结果表明:随冷冻时间的延长(12周),猪肌内脂肪的TBARS值由0.07 mg/kg蛋白升至0.22 mg/kg蛋白;与新鲜原料猪肉相比,经12周冻藏后...     

Yield, composition and rheological characteristics of cheddar cheese made with high pressure processed milk

High Pressure (HP) treatment of milk prior to cheese-making was shown to increase the yield of cheese due to increased protein and moisture retention in cheese. Cheeses were made with raw milk or milk treated with high temperature short-time (HTST) pasteurization, and HP treatments at two levels (483 and 676 MPa) at 10 °C, 483 MPa HP at 30 °C, and 483 MPa HP at 40 °C. Cheese yield, total solids, protein, fat and salt contents were evaluated, and fat and protein recovery indices were calculated. Cheeses from HP treatments of 676 MPa at 10 °C and 483 MPa at 30 °C exhibited wet yields of 11.40% and 11.54%, respectively. Protein recovery was 79.9% for HP treatment of 676 MPa at 10 °C. The use of slightly higher pressurization temperatures increased moisture retention in cheese. Visco-elasticity of cheeses was determined by dynamic oscillatory testing and a creep-recovery test. Rheological parameters such as loss (G″) and storage (G′) moduli were dependent on oscillation frequency. At high (173 rad/s) and low (2.75 rad/s) angular frequencies, cheeses made from milk treated at 483 MPa at 10 °C behaved more solid-like than other treatments. Creep tests indicated that cheeses from milk treated with 483 MPa HP at 10 °C showed the smallest instantaneous compliance (J_o), confirming the more solid-like behavior of cheese from the 483 MPa at 10 °C treatment compared to the behavior of cheeses from other treatments. Cheeses made with pasteurized milk were more deformable, exhibited less solid-like behavior than cheeses made with HP treated milk, as shown by the J_o value. With more research into bacteriological implications, HP treatment of raw milk can augment Cheddar cheese yield with better curd formation properties.     

冻藏时间对猪肉中肌原纤维蛋白氧化程度的影响

对在-18℃下贮藏不同时间(0,1,4,8,12周)的原料猪肉进行肌原纤维蛋白提取,对其羰基、总巯基、自由氨基、表面疏水性和内源性色氨酸荧光进行测定。结果表明:随着贮藏时间的延长,羰基含量显著升高(P<0.05),总巯基、自由氨基、表面疏水性和内源性色氨酸荧光含量整体上呈下降趋势(P<0.05);贮藏时间与羰基、总巯基、自由氨基或表面疏水性之间具有显著相关性。因此,即便在冻藏条件下,肌原纤维蛋白也会出现一定程度的氧化,导致原料肉品质下降。     

Influence of meat exudates on the quality characteristics of fresh and freeze-thawed pork

The influence of the accumulated exudates released from pork loin of itself on the quality characteristics of fresh and freeze-thawed pork during cold storage was investigated. Pork loins were divided into four groups (fresh pork with exudates, fresh pork without exudates, freeze-thawed pork with exudates and freeze-thawed pork without exudates) and stored at 1.0 °C for 7 days. Exudate amount increased due to freeze-thawing and with storage, and most quality traits such as drip loss, cooking loss, tenderness, lightness, redness, and moisture content were affected by freeze-thawing (p < 0.05). Freeze–thaw increased drip loss but decreased moisture content, cooking loss, tenderness, lightness and redness of meat (p < 0.05). Microbial growth was solely affected by exudate removal and the removal of initial exudates decreased microbial growth (p < 0.05). Exudates were positively correlated with total protein content and total plate count but negatively correlated with pH and cooking loss. Therefore, removing meat exudates and avoiding freeze can slow down the quality deterioration of pork during cold storage.     

Changes on enzymatic activity and on sarcoplasmic and myofibrillar proteins of frozen-stored hake (Merluccius merluccius) pre-treated by high pressure

High-pressure (HP) pre-treatments were applied on European hake (Merluccius merluccius) followed by a frozen accelerated experiment (−10 °C). A central composite design ranging pressure levels (150–450 MPa) and frozen storage time (0–150 days) was used, being evaluated the enzymatic activities and muscle proteins. Acid phosphatase and calpain activities decreased after 150 days of frozen storage (58%/56% and 38%/56% for non-/HP-treated samples, respectively). Cathepsin B showed higher reductions (98%) for longer storage times. Furthermore, HP and frozen storage did not affect significantly cathepsin D activity, only slightly decreasing at 169 MPa. Furthermore, HP seemed to not affect myofibrillar proteins, while sarcoplasmic proteins were clearly affected by HP and frozen storage time, resulting in a reduction of about 53% or 23% for 431 or 450 MPa, respectively. Thus, HP could be used to lowering the deleterious effect of proteases on frozen European hake.     

不同类型电场辅助冰温贮藏对生鲜猪肉保水性的影响

为研究不同类型电场辅助冰温贮藏对生鲜猪肉保水性的影响,以猪背最长肌为研究对象,对比分析在交变电场辅助冰温、静电场辅助冰温及普通冰温（对照组）贮藏条件下生鲜猪肉水分含量、贮藏损失、蒸煮损失、横向弛豫时间T2、肌原纤维蛋白表面疏水性及结构的变化。结果表明：交变电场和静电场处理组生鲜猪肉水分含量和不易流动水弛豫峰面积比例P21显著高于对照组（P＜0.05）,贮藏损失、蒸煮损失、肌原纤维蛋白表面疏水性及自由水弛豫峰面积比例P22显著低于对照组（P＜0.05）,但交变电场处理组和静电场处理组各指标之间无显著差异。综上,输出电压为3 300～4 000 V所产生的交变电场和输出电压为3 800 V所产生的静电场辅助冰温贮藏均可延缓肌原纤维蛋白降解及不易流动水向自由水转化,从而提高生鲜猪肉的保水性,降低贮藏过程中的汁液损失。