Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle

The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat.     

Effects of camptothecin, etoposide and Ca2+ on caspase-3 activity and myofibrillar disruption of chicken during postmortem ageing

Recently, a novel consideration has focused on the potential relationship of apoptosis and the protease caspases and the underlying mechanism for meat postmortem tenderization. In this study, apoptosis inducers, camptothecin and etoposide as well as Ca(2+) were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during 7 days of ageing. All three treatments resulted in significantly higher caspase-3 activities during storage (p<0.05), with the natural substrates, whereas Western blotting analysis of the α-spectrin cleavage product, 120 kDa peptide (SBDP 120), showed that Ca(2+) was more effective than either camptothecin or etopside, and all were most active up to day 3 (p<0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product, especially during the first 3 days (p<0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected, even at day 7. The findings revealed that the three apoptosis inducers could increase myofibrillar dissociation and proteolysis during the first 3 days of chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat.     

The effect of myofibrillar/sarcoplasmic protein ratios on the properties of round scad muscle protein based film

The effect of the ratios of myofibrillar protein (MP) to sarcoplasmic protein (SP) from round scad (Decapterus maruadsi) muscle on the properties of the resulting films was investigated. Tensile strength (TS) of films decreased with increasing SP content (p < 0.05). Films prepared from MP/SP ratio of 10:0 (w/w) exhibited the highest TS (p < 0.05). Elongation at break (EAB) of films prepared with SP content greater than 30% had the decreased EAB (p < 0.05). Water vapor permeability (WVP) of films increased when SP content increased up to 20% and decreased with increasing SP content up to 30% (p < 0.05). Solubility of films decreased but protein solubility increased with increasing SP contents (p < 0.05). The a*-value and ΔE* of film increased with increasing SP content. Films with all MP/SP ratios exhibited the negligible transmission to the light in UV range. Therefore, it is suggested that the type and ratio of proteins in fish muscle, both SP and MP, influenced the properties of film from round scad muscle. Results suggested that the removal of sarcoplasmic protein from fish muscle by thorough washing was an effective means to improve the mechanical properties as well as color of the fish muscle protein-based film.     

低温贮藏对鸡胸肉肌原纤维蛋白结构及热诱导凝胶性能的影响

目的 研究4℃(冷藏)与-18℃(冷冻)贮藏条件对鸡胸肉肌原纤维蛋白(myofibrillar protein, MP)结构及热诱导凝胶性能的影响。方法 通过测定总巯基、钙离子ATP酶(calcium-ATPase, Ca2+-ATPase)活性、表面疏水性、凝胶质构、保水性等指标衡量MP结构及凝胶特性的变化,并从分子间作用力及蛋白质构象角度阐述贮藏条件对MP热诱导凝胶机制的影响。结果 在4℃与和-18℃贮藏环境下,随着贮藏时间的延长,MP总巯基含量减少,表面疏水性上升, Ca2+-ATPase酶活性降低。其次, MP热诱导凝胶中离子键及氢键作用力也随贮藏时间的延长而呈下降趋势,而疏水作用力呈上升趋势;凝胶中的α-螺旋和β-折叠含量呈下降趋势,而β-转角与无规则卷曲含量呈上升趋势。结论 低温贮藏期间MP结构发生劣变,蛋白凝胶结构由有序转向无序结构,导致凝胶质构、保水性下降;相较4℃贮藏,-18℃能够更好地长时间维持MP结构和凝胶特性。     

Changes in phosphorylation of myofibrillar proteins during postmortem development of porcine muscle

A gel-based phosphoproteomic study was performed to investigate the postmortem (PM) changes in protein phosphorylation of the myofibrillar proteins in three groups of pigs with different pH decline rates, from PM 1 to 24 h. The global phosphorylation level in the group with a fast pH decline rate was higher than that in the slow and intermediate groups at early PM time, but became the lowest at 24 h. The protein phosphorylation level of seven individual protein bands was only significantly (p < 0.05) affected by PM time, and two protein bands were subjected to a synergy effect between PM time and pH decline rate. A total of 35 non-redundant highly abundant proteins were identified from 19 protein bands; most of the identified proteins were sarcomeric function-related proteins. Myosin-binding protein C, troponin T, tropomyosin and myosin regulatory light chain 2 were identified in the highly phosphorylated protein bands with the highest scores. The results indicate that the phosphorylation pattern of myofibrillar proteins in PM muscle is mainly changed with PM time, but only to a minor extent influenced by the rate of pH decline, suggesting that the phosphorylation of myofibrillar proteins may be related to the meat rigor mortis and quality development.     

超声技术对肌原纤维蛋白结构的影响及其在肌肉食品中的应用现状

近年来,随着人们对食品加工技术的不断研究,超声技术作为一种新型食品物理加工技术,在肉制品加工应用中取得了显著的进展。本文综述了超声技术对肌肉组织和蛋白结构等微观品质的调控作用及其对肉制品质构、色泽、风味、保水性等宏观品质的影响,详细阐述了超声技术在不同肉制品加工工艺场景下超声技术的应用进展,包括原料肉的解冻、嫩化、凝胶化和乳化特性的提升以及腌制剂的渗透、腥臭味的掩埋等方面。此外,本文进一步探讨了超声技术与变压滚揉、腌制剂、微波、浸渍冷冻等其他技术的联合应用在改善肉制品加工品质方面的潜在作用,为超声技术在肌肉食品加工领域的深层次应用提供了重要的理论参考。     

The effects of transglutaminase on the functional properties of the myofibrillar protein concentrate obtained from beef heart

The aim of the present study was to evaluate the effect of bacterial transglutaminase on the functional properties of the myofibrillar protein concentrate from beef heart. The degrees of hydration and aggregation and emulsifying properties were studied. The degree of polymerization of the myofibrillar proteins depended on the enzyme concentration and setting time; the best results in terms of functional properties were obtained with 0.3 g transglutaminase/100 g protein with 60 min setting at 35 °C. This investigation confirms that transglutaminase may be used for the production of myofibrillar protein aggregates with enhanced functional properties.     

Microbial transglutaminase-induced structural and rheological changes of cationic and anionic myofibrillar proteins

This study investigated the effects of microbial transglutaminase (TG) on structural changes in porcine myofibrillar protein (MP) under varying pH (2.0–6.0) and two ionic strength conditions (0.1 M versus 0.6 M NaCl). Lowering the pH below the isoelectric point (pI) of myosin induced protein unfolding as revealed by surface hydrophobicity and differential scanning calorimetry. Although the MP solubility at the low ionic strength (0.1 M NaCl) was maximal at pH 3.0, both SDS-PAGE profiles and dynamic rheology indicated TG could not cross-link MP under this condition. Based on the carboxyl group content, the TG-catalyzed deamidation was dominant at a pH lower than the pI of myosin (pH 5.0) while cross-linking occurred at higher pH. Moreover, deamidation had no effect on rheological properties of MP. The results indicate that the TG reaction was governed by the pH of substrate protein, and the reaction intensity was related to the solubility of protein.     

Effect of lactic or acetic acid on degradation of myofibrillar proteins in post‐mortem goose (Anser anser) breast muscle

The effects of lactic acid (LA) and acetic acid (AA) on changes in myofibrillar proteins of post‐mortem goose breast muscle marinated for 24 h at 5 °C were studied. Purified myofibrils were prepared from 0.1 M LA or AA samples and controls (non‐marinated samples) after 0, 1, 3, 7 or 14 days of storage at 5 °C. The changes in myofibrillar proteins of goose muscle were examined by SDS‐PAGE. Goose breast muscle marinated in LA and AA exhibited degradation of myosin heavy chains. The appearance of ∼95 and ∼27 kDa components and the disappearance of titin and nebulin were also more rapid than for control muscle. These results suggest that acid marination enhanced the post‐mortem proteolysis of goose breast muscle. © 2000 Society of Chemical Industry     

Surface hydrophobicity and functional properties of myofibrillar proteins of mantle from frozen-stored squid (Illex argentinus) caught either jigging machine or trawling

The surface hydrophobicity and functional properties of actomyosin from the mantle of frozen squid caught either by jigging machines (AME1) or by trawl (AME2) were investigated. Two components of 155 and 55 kDa were present in the gels at zero time of storage. Degradation of the myosin heavy chain and increase in the 155 kDa component occur earlier in AME2. Irrespective of the catch method used, no significant (P>0.05) changes in protein solubility were observed. The reduced viscosity of both AME1 and AME2 decreased up to months 3 and 5 of frozen storage, respectively. At the beginning of storage, the superficial hydrophobicity of AME2 was 30% higher than that of AME1. So 1-anilino-8-naphthalene sulfonic acid of AME2 significantly increased during 3-5 months of storage period and that of AME1 at the end of storage. The emulsion activity index (EAI) of AME2 significantly (P<0.05) increased during the first month and decreased after 3 months of storage. EAI of AME1 decreased at month 3 and remained unchanged thereafter. Emulsion stability (ES) of AME2 showed a behavior that was similar to its IAE and that of AME1 remained unchanged.     

The effect of acidification on myofibrillar proteins

Isolated myofibrillar proteins of mutton, beef and chicken were treated with an acidulent to give various pH values and stored at 5°C for 20 h before analysing the proteins using sodium dodecylsulphate polyacrylamide gel electrophoresis. It was found that protein degradation occurred below pH 4·5, with a decrease in band intensity of all major myofibrillar proteins, particularly myosin heavy chain, and the appearance of new bands at approximately 140 and 70 kd. The degradation had an optimum around pH 3·0 and was inhibited by a high temperature pre-treatment or the presence of the endopeptidase inhibitors pepstatin A and leupeptin. Results are discussed in terms of the action of acid proteinase enzymes.     

Analysis of fish and squid myofibrillar proteins by capillary sodium dodecyl sulfate gel electrophoresis: actin and myosin quantification

 A capillary electrophoretic method for the separation and quantification of fish and squid myofibrillar proteins was developed. The method uses sodium dodecyl sulfate and β-mercaptoethanol for solubilization and analysis of myofibrillar protein subunits. The separation of the different polypeptides is achieved by the sieving effect of the gel inside the capillary. A calibration curve for myosin heavy chain and actin UV absorbance quantification of these proteins was developed. Received: 2 November 1999 / Revised version: 16 February 2000     

Changes in myofibrillar proteins during processing of pastirma (Turkish dry meat product) produced with commercial starter cultures

The effects of different commercial starter cultures (Staphylococcus carnosus, S. carnosus + Lactobacillus pentosus and Staphylococcus xylosus + Lactobacillus sakei), on myofibrillar proteins were investigated using differential scanning calorimetry (DSC) during the processing of pastirma. The stage of pastirma production significantly decreased the thermal stabilities of myosin and actin. Actin was less affected than myosin. The myofibrillar fraction of pastirma was hardly denaturated by S. carnosus, but more pronounced denaturation was obtained with S. carnosus + L. pentosus.     

Changes in the ultrastructure and texture of prawn muscle (Macrobrachuim rosenbergii) during cold storage

Changes in the ultrastructure and proteolytic activity of prawn muscle were determined during storage at 5 °C, in order to better understand changes in physical and sensory texture measurements. Progressive deterioration of myofibril structure was observed during refrigeration of prawn for 14 d. The loss of density and order in Z-line alignment was first detected after 3 d of storage. Progressive disruption of Z-line, I-bands and M-lines was observed after 4-6 d of storage. Muscle degradation included pronounced disruption of the mitochondria as revealed by swollen cristae, loss of cristae material, and membrane breakage. Along with ultrastructural changes, decreased shear force values and mean textures scores were measured. An initial shear force value of 18.21 N/g decreased to 14.50, 12.46, and 10.79 N/g on days 3, 6, and 14, respectively. Mean texture scores indicated that prawn muscle maintained firm texture during 0-3 d of storage, and became soft during 4-6 d of storage. After 6 d of storage, the prawn texture was very soft. Increased deterioration of the muscle ultrastructure coincided with the increase of proteolytic microorganisms and salt soluble muscle proteins found by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is concluded that the weakening of the ultrastructure is related to proteolytic activity and results in a more soft texture in prawns.     

Proteolytic pattern of myofibrillar protein and meat tenderness as affected by breed and aging time

The effects of breed and aging time (1, 7, 14, 21 days) were evaluated on meat tenderness and on proteolysis in 24 young bulls from Romagnola × Podolian crossbreed, Podolian and Friesian breed. Shear force decreased with aging in all breeds and showed the highest values at 1 and 7 days in Podolian meat. Myofibrillar fragmentation index significantly increased in Podolian meat throughout aging whereas in Friesian and in Crossbreed meat it increased only in the first week. Proteolysis was investigated by SDS-PAGE and 2-dimensional electrophoresis showing a different quantity and expression profile of myofibrillar proteins among breeds. In all breeds a decrease of troponin-T and an increase of troponin-T derived polypeptides during aging were observed. The highest decrease of troponin-T together with the presence of fragments of MHC in Podolian meat during aging was an outcome of a more extensive proteolysis in this breed. Data suggest that tenderness and proteolytic changes during aging are related to animal's breed.     

Extreme pH treatments enhance the structure-reinforcement role of soy protein isolate and its emulsions in pork myofibrillar protein gels in the presence of microbial transglutaminase

Alkali (pH₁₂) and acid (pH_1.5) pH-treated soy protein isolates (SPI) were incorporated (0.25–0.75% protein) into sols of myofibrillar protein (MP, 3%, in 0.6 M NaCl at pH 6.25) with or without 0.1% microbial transglutaminase (TG) to investigate the potential as meat processing ingredients. Static and dynamic rheological measurements showed significant enhancements of MP gelling ability by the inclusion of pH_1.5-treated as well as preheated SPI (90 °C, 3 min). A 7-h incubation with TG accentuated the gel-strengthening effect by these SPI samples. The B subunit in 11S of SPI was the main component manifesting structure reinforcement in the mixed gels. The MP gelling properties were also greatly improved (P < 0.05) by the addition of 10% canola oil emulsions stabilized by pH-treated SPI. The principal force in the MP gels incorporated with pH-treated SPI was hydrophobic patches; in the presence of TG, cross-linking of previously dissociated A and B subunits of 11S was also intimately involved.     

Effect of microwave heating time on the gel properties of chicken myofibrillar proteins and their formation mechanism

The present work was conducted to illustrate the mechanism of gel formation of myofibrillar proteins (MPs) under different microwave heating times. The results showed that the denaturation enthalpy (ΔH) of the MPs significantly decreased when the heating time increased from 3 to 9 s and then completely disappeared as the heating time progressed, indicating that the MPs gradually denatured and subsequently aggregated with increasing heating time, which was further verified by the changes in the secondary structure, electrophoretic bands, and gel properties (e.g., water holding capacity and textural profiles) of the MPs. Microstructural images indicated that the MP gel formed under 12 s had the most compact network, indicating that extended microwave heating time could induce quality deterioration of MP gels. Moreover, the hydrophobic forces, electrostatic forces, and disulphide bonds of the MPs gradually intensified with increasing microwave heating time, suggesting that both non-covalent and covalent bonds could promote molecular denaturation and subsequent aggregation of MPs. In addition, correlation analysis revealed that the changes in the molecular conformation of MPs induced by different microwave heating times could effectively regulate the formation of MP gels and their related properties.     

冷冻-解冻肌原纤维蛋白结构评价技术研究进展

肌肉类食品在加工和运输中常常经历冷冻-解冻的过程,严重破坏肌原纤维蛋白的结构与功能特性,进而降低肌肉食品的食用价值。因此,评价肌原纤维蛋白冷冻-解冻过程中结构特性的变化对于监测冻融肌肉食品的加工食用价值具有重要意义。本文针对现代检测技术在评价冻融肌原纤维蛋白结构特性方面的研究进展和应用情况进行了系统总结和分析,主要介绍了光谱学技术、显微镜技术、核磁共振技术、蛋白组学和分子动力学模拟等技术的研究应用现状。此外,比较了上述不同检测技术的优缺点,并对现代检测技术在评价肌原纤维蛋白品质特性方面的未来发展方向进行了展望,未来需进一步优化相关检测技术,使其操作更简单、更易实施,以期在冷冻肉品生产和加工工业化中推广应用并提供技术支持,旨在为提高冷冻肌肉食品品质提供科学参考。