Identification of myofibrillar substrates for μ-calpain

To identify myofibrillar substrates of μ-calpain under post-mortem conditions, a combination of SDS–PAGE, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used. Purified myofibrils were incubated with μ-calpain under post-mortem-simulated conditions for two or four days at 4 °C. The resulting protein changes were analyzed by SDS–PAGE and 2DE. The μ-calpain-mediated protein changes were identified by peptide-mass mapping using MALDI-TOF MS and revealed that desmin, actin, myosin heavy chain, myosin light chain I, troponin T, tropomyosin 1, tropomyosin 4, thioredoxin and CapZ are all degraded in vitro by μ-calpain. The findings that actin and myosin heavy chain are substrates of μ-calpain were rather surprising, as it has previously been reported that these proteins are resistant to μ-calpain degradation. However, both actin and myosin heavy chain are poor substrates compared with desmin.     

Proteolytic activity of Penicillium chrysogenum and Debaryomyces hansenii during controlled ripening of pork loins

The role of micro-organisms on the ripening process of dry-cured ham, particularly with respect to proteolysis, is not clear. This is partially due to the lack of an adequate system to study changes on a sterile control meat product for long ripening times. Using a meat system based on sterile pork loins ripened under aseptic conditions for 106 days, the contribution to the proteolysis of two micro-organisms isolated from dry-cured ham has been established. Changes were studied by SDS-PAGE of sarcoplasmic and myofibrillar proteins, capillary zone electrophoresis (CZE) of low ionic strength-soluble nitrogen compounds, and HPLC of free amino acids. Debaryomyces hansenii Dh345 did not show any significant proteolytic activity. However, Penicillium chrysogenum Pg222 showed high proteolytic activity on myofibrillar proteins resulting in an increase in soluble nitrogen compounds. For this, P. chrysogenum Pg222 should be considered to be used as starter culture in meat products made using long ripening times.     

Proteomic analysis of semimembranosus and biceps femoris muscles from Bayonne dry-cured ham

The aim of the study was to delineate and compare the proteomic maps of two muscles of dry-cured ham: the biceps femoris and the semimembranosus. For this purpose, we used two-dimensional electrophoresis on a subcellular muscle fraction: insoluble protein in low ionic strength buffer. After protein identification by MALDI-TOF mass spectrometry and bioinformatic analyses, we found differences in expression levels in the two muscles. Seventy-three proteins or fragments were differentially expressed: 43 were over-represented in semimembranosus and 30 in biceps femoris. Although the study was performed on the insoluble protein fraction in low strength ionic buffer, protein and fragment identifications by mass spectrometry showed that most of the proteins were involved in energy metabolism. The differences observed between the two muscles can be explained by the differences in salt and moisture content in the course of dry-cured ham processing.     

Proteomic profile of dry-cured ham relative to PRKAG3 or CAST genotype, level of salt and pastiness

Two-dimensional electrophoresis was used to compare dry-cured biceps femoris insoluble protein fraction according to genotype (PRKAG3Ile199Val and CASTLys249Arg/Ser638Arg) as well as salt and pastiness level. The PRKAG3 affected mainly muscle metabolic enzymes, indicating its possible influence on muscle metabolism with heterozygotes Ile/Val appearing different from both homozygous genotypes. The effect of CAST was smaller, affecting the quantity of one actin fragment. Dry-cured ham salt and pastiness level affected a wide variety of protein spots including metabolic enzymes, plasma proteins, chaperones and myofibrillar proteins, including protein fragments, indicating the connection with proteolysis. Pastiness was associated with salt content, reflected also by the fact that many spots were affected by both factors. Despite the absence of extreme pastiness (or low salt samples), some protein spots (actin, MHC fragment, desmin fragment) exhibited important differences in intensity according to pastiness (and salt level) suggesting they could be used as potential quality markers.     

SDS-PAGE of salted anchovies (Engraulis encrasicholus L) during the ripening process

 Changes in the muscle proteins during the ripening of salted anchovies have been examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis. The chemical changes occurring in the muscle proteins of anchovy during the ripening process were related to the degradation of the myofibrillar structure of the muscle. In spite of a considerable protein degradation the ripened salted anchovy maintained its structure and was easily cut into fillets. Hydrolysis of muscle proteins was significant during the first 6 weeks. Proteins of molecular weight higher than 35 kDa were more likely to be hydrolysed. Myosin heavy chains were the most sensitive myofibrillar protein. α-Actinin, actin and tropomyosin were more resistant to enzymatic degradation. Due to protein build-up of peptides resulting from the breakdown of the higher molecular proteins, it was difficult to measure changes in the myosin light chains and troponin sub-units. Received: 28 December 1999 / Revised version: 9 March 2000     

Postmortem meat quality and sex affect textural properties and protein breakdown of dry-cured ham

Texture measurements by instrumental texture profile analysis (TPA) and protein degradation analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were performed on 30 dry-cured hams resulting from four different post-mortem meat qualities categories (PSE, RSE, RFN and DFD). The main differences were observed in dry-cured hams from PSE and RFN meat qualities. Penetration force (80%), hardness, springiness, cohesiveness and chewiness, were significantly lower (P<0.05) in PSE than in RFN quality classes. The rate of the ripening process was affected as a higher proteolysis and absence of fragments at 150 and 85 KDa in PSE in relation to RFN classes, and with an intermediate proteolysis of RSE and DFD classes. The effect of sex was observed as a significant (P<0.05) low hardness in the hams obtained from female pigs. The duration of the ripening, for a better uniformity in dry-cured ham production, should be adapted to the initial pH and to drip loss parameters of the raw material.     

Proteolytic activity of lactic acid bacteria strains and fungal biota for potential use as starter cultures in dry-cured ham

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.     

不同地域典型干腌火腿肌原纤维蛋白的氧化特性及体外消化性对比

为比较几种典型干腌火腿的可消化吸收性,以巴马火腿、金华火腿、宣威火腿、帕尔玛火腿为原料,研究4 种干腌火腿的肌原纤维蛋白表面疏水性、巯基和二硫键含量、超微结构、蛋白降解程度、消化前后粒径及体外消化率。结果表明：金华火腿肌原纤维蛋白的表面疏水性显著高于巴马、宣威、帕尔玛火腿（P＜0.05）。微观结构分析显示金华火腿肌原纤维蛋白交联和聚集程度最低,肌原纤维排布更为伸展;帕尔玛火腿聚集程度最大,呈现明显聚集状。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳谱图显示金华火腿肌原纤维蛋白在体外消化过程中大幅降解,促进了肌原纤维蛋白粒径的降低。体外模拟消化结果表明,经胃蛋白酶、胰蛋白酶及α-凝乳蛋白酶作用后,金华火腿肌原纤维蛋白水解速率最高（P＜0.05）;帕尔玛火腿肌原纤维蛋白水解速率最低（P＜0.05）。这表明疏水基团的暴露和蛋白结构的伸展为消化酶提供了更多的识别位点,促进了金华火腿中肌原纤维蛋白的水解。综上,肌原纤维蛋白的氧化程度显著影响蛋白的体外消化率。     

Proteomic and peptidomic insights on myofibrillar protein hydrolysis in a sausage model during fermentation with autochthonous starter cultures

The hydrolysis of myofibrillar proteins during fermentation of sausage models by an autochthonous starter culture was investigated. In order to provide a whole map of the generated products, proteomic and peptidomic were used and complemented with the amino acid profile. Beaker sausages (BS) were used as models which were inoculated or not with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 as starter cultures. The hydrolysis of actin, myosin light chain 1/3 (MLC 1/3), myosin regulatory light chain-2 (MRLC-2) and myosin heavy chain (MHC) was evidenced by two-dimensional gel electrophoresis (2-DE). In addition, a total of 33 peptides arisen from troponin T, MRLC-2 and particularly from actin were identified by LC–MS/MS. These results showed that the starter culture significantly enhanced the proteolysis of the proteins named above, even when the endogenous enzymes induced a clear breakdown. L. curvatus CRL705 highly enriched both peptide pattern and amino acid concentrations. When the autochthonous starter culture was inoculated, although proteolysis was remarkably reinforced, a reduction in peptide and amino acid composition was observed. Regarding actin primary structure, three regions of this protein were highly susceptible to degradation by the starter culture. Additionally, the essential role of exopeptidases – from meat and bacteria – in diversity of actin peptides during fermentation was shown. This study improved the knowledge of the proteolysis of myofibrillar proteins and the involved enzymes, as well as, completed the previously reported degradation of sarcoplasmic proteins by the same autochthonous starter culture. The singular peptides and amino acids pattern generated might contribute to the uniqueness of produced fermented sausages while they may be used as quality markers.     

云南腾冲雪鸡肌肉蛋白质组学研究

腾冲雪鸡是云南省腾冲县的一个地方优良家鸡品种,具有羽毛洁白、肉骨乌黑、肉质好、抗病性强等优良特性.本研究以140日龄腾冲雪鸡为对象,采用双向电泳(DE)分析其腿肌与胸肌中蛋白表达差异,并以基质辅助激光解析电离-串联飞行时间质谱(MALDI-TOF/TOF MS)鉴定腿肌与胸肌中差异蛋白质点.结果表明,经双向电泳图谱分析所获得的肌肉组织蛋白点分布于pH4.0～7.0之间,蛋白点清晰,图谱分辨率较好,其中腿肌中检测到1421±34个蛋白点,胸肌中检测到1329±25个蛋白点,对比分析腿肌和胸肌组织的DE图谱,发现有110个点存在明显的表达差异,在腿肌中高表达的点有56个,在胸肌中高表达的点有54个;选取的蛋白质点经鉴定为核纤层蛋白A、磷酸葡萄糖变位酶1、β-烯醇酶、超氧化物歧化酶、肌酸激酶M型、乙二醛酶1、原肌原蛋白1,以及一些假设蛋白和非特征性蛋白.     

Identification of small troponin T peptides generated in dry-cured ham

Troponin T (TnT) is a myofibrillar protein present in striated muscle that develops important structural and regulatory functions. This protein is easily degraded during post-mortem ageing but details of the TnT hydrolysis occurring during much longer processes, e.g. the dry-cured processing, as well as the specific sequences of the generated fragments, still remain undetermined. In the present study, the isolation and identification of 27 peptides, generated from TnT during dry-cured processing, are reported for the first time. This study has been developed using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) and it evidences the extensive degradation of TnT occurring during the processing of dry-cured ham. Some of the cleavage sites, corresponding to these sequenced fragments, confirm the sites reported by other authors both in vitro and during post-mortem ageing of meat.     

两种电泳法分析丁香提取物对猪肌原纤维蛋白氧化位点的控制

研究猪肌原纤维蛋白（myofibrillar protein,MP）经胰蛋白酶水解后的肌球蛋白头部（S1）和尾部（Rod）受羟自由基的氧化程度以及丁香提取物对蛋白氧化位点的影响。选择未氧化的MP、氧化的MP及与丁香提取物结合后氧化的MP进行一维电泳、双向电泳实验和质谱鉴定。一维电泳结果表明,丁香提取物对S1部位的保护作用更加明显,Rod和S1分解生成的小片段主要是通过二硫键与肌动蛋白发生了交联。双向电泳和质谱鉴定结果证实了氧化后增加的蛋白,主要是肌球蛋白或肌动蛋白等MP中主要蛋白的亚基或碎片;丁香提取物的添加能够有效地控制这些碎片的产生,从分子水平上证实了丁香提取物抑制蛋白氧化的效果。肽段质谱鉴定结果显示,羟自由基诱发的MP氧化主要修饰的氨基酸部位为甲硫氨酸和半胱氨酸。本研究表明在肉制品加工与贮藏中添加丁香提取物可以改善肉制品的功能特性。     

Textural Degradation of Cooked Fish Meat Gel (Kamaboko) by the Addition of an Edible Mushroom, Judas' Ear (Auricularia auriculajudae (Fr.) Quel)

Commercial makers of kamaboko, a traditional Japanese preparation of gelled cooked fish meat, have observed that when slivers of the edible Judas' ear mushroom (Auricularia auricula-judue (Fr.) Quel) are incorporated into the gel without being cooked first, the gel tends to have an unsatisfactory texture. Experiments with casein as substrate have already shown that this mushroom has a proteinase. We showed that the proteinase acted on fish myofibrillar proteins, including myosin, actin, and tropomyosin. In kamaboko containing Judas' ear mushroom uncooked, fish proteins were hydrolyzed and gel strength decreased. The results showed that the decrease in gel strength was caused by the hydrolysis of fish proteins by the proteinase.     

生姜蛋白酶和猕猴桃蛋白酶对干腌羊火腿蛋白质降解的影响

为了促进干腌羊火腿肌肉蛋白质降解,加快其风干成熟进程,缩短加工周期,提高羊火腿的品质,在干腌羊火腿中添加生姜蛋白酶和猕猴桃蛋白酶。以带骨鲜羊后腿肉为试验材料,分别设计一个对照组和两个试验组;对照组不采取处理,而试验组分别添加0.05%的生姜蛋白酶和猕猴桃蛋白酶,并在相应条件下进行风干成熟,检测干腌羊火腿的总氮(Total nitrogen,TN)含量、非蛋白氮(Non-protein nitrogen,NPN)含量和蛋白质降解指数(Proteolysis index,PI)等蛋白质降解指标,并通过SDS-PAGE(聚丙烯酰氨凝胶电泳)分析了干腌羊火腿肌肉蛋白质的降解情况。结果表明,与对照组相比,猕猴桃蛋白酶处理组和生姜蛋白酶处理组的TN含量风干成熟后分别增加了1.2倍和1.3倍、NPN含量分别增加1.5倍和1.7倍、PI分别上升1.2倍和1.3倍;SDS-PAGE分析结果表明,生姜蛋白酶降解肌肉蛋白的效率较猕猴桃蛋白酶强。通过蛋白质将指数和SDS-PAGE电泳结果可知,生姜蛋白酶对肌肉蛋白的降解程度比猕猴桃蛋白酶大。     

THE STRUCTURE AND PROPERTIES OF MYOFIBRILLAR PROTEINS IN BEEF, POULTRY, AND FISH

The myofibrillar proteins myosin, actin, tropomyosin, and troponin are involved in the contraction of muscle. They are also an integral part of the unique, highly organized structure of muscle tissue. The chemistry and biochemistry of these proteins has been studied both in vivo and in vitro by scientists from many different fields. The same proteins are of great importance to food scientists because of their role in meat and meat products. This review summarizes some of the important physicochemical and biochemical properties of these proteins that may be important for food scientists.     

Proteolytic and lipolytic modifications during the manufacture of dry-cured lacón,a Spanish traditional meat product: Effect of some additives

The extractability of sarcoplasmic and myofibrillar proteins, the myofibrillar proteins and their degradation products, classical nitrogen fractions, free amino acids, acidity of the fat, and free fatty acids were determined throughout the manufacturing process of dry-cured lacón, a traditional dry-salted and ripened meat product made in the northwest of Spain from the foreleg of the pig, following a similar technological process to that of dry-cured ham. The effect of the use of additives (glucose, sodium nitrite, sodium nitrate, sodium ascorbate and sodium citrate) on the proteolytic and lipolytic changes was also studied.     

鸡胸肉肌原纤维蛋白的提取及凝胶特性的研究

研究从鸡胸肉中提取肌原纤维蛋白，探讨不同pH值对肌原纤维蛋白含量的影响。研究结果：在pH7．0时，蛋白含量最大为69．74％，通过SDS—PAGE凝胶电泳分析表明，其主要成分为肌球蛋白、肌动蛋白和肌动球蛋白及其他一些小分子肌原纤维蛋白碎片；在离子强度0．6、pH7．0时提取的肌原纤维蛋白所制备的凝胶的保水性、硬度、胶粘性最大。     

双向电泳分析宣威火腿蛋白质组方法的建立

以宣威火腿为对象,采用三氯乙酸-丙酮沉淀法提取宣威火腿肌肉蛋白,对蛋白定量后,进行一向等电聚焦电泳和二向聚丙烯酰胺凝胶电泳,电泳后的凝胶以考马斯亮蓝染色,ImageMaster 2D Platinum软件分析凝胶上的蛋白质点,切取1个蛋白质点,经胰蛋白酶消化后使用基质辅助激光解析电离-串联飞行时间质谱(MALDI-TOF/TOF MS)获取肽质量指纹图谱,并用Mascot软件分析,以建立宣威火腿的蛋白质组学研究方法。结果表明：经双向电泳图谱分析所获得的宣威火腿蛋白点分布于pH4.0～7.0之间,共发现1749个蛋白点,蛋白点清晰,图谱分辨率较好;切取的蛋白质点经鉴定为Gamma-2-肌动蛋白,Mascot检索分值为129分(阈值为67分)。可见,成功建立双向电泳分析宣威火腿蛋白质组的方法。     

云南盐津乌骨鸡与武定鸡肌肉蛋白质组学差异研究

选取云南两种具有代表性的地方优质鸡种盐津乌骨鸡和武定鸡为研究对象,采用双向电泳(2DE)分析其腿肌与胸肌中蛋白表达差异,并以基质辅助激光解析电离-串联飞行时间质谱(MALDI-TOF/TOF MS)鉴定腿肌与胸肌中差异蛋白质点。结果表明,经双向电泳图谱分析所获得的肌肉组织蛋白点分布于pH4.0~7.0之间,其中盐津乌骨鸡腿肌、胸肌中分别检测到(1421±34)、(1329±25)个蛋白点;武定鸡腿肌、胸肌中检测到(1585±19)、(1516±28)个蛋白质点。对比分析盐津乌骨鸡与武定鸡的DE图谱,发现有178个点存在明显的表达差异,在盐津乌骨鸡中高表达的点有72个,在武定鸡中高表达的点有106个。选取的蛋白质点经鉴定主要为核纤层蛋白A、磷酸葡萄糖变位酶1、β-烯醇酶、原肌球蛋白1、三磷酸腺苷合成酶、C-反应蛋白前体、B链α-晶状体蛋白、肌酸激酶M型、乙二醛酶1、磷酸甘油酸激酶 PGK1、慢骨肌肌钙蛋白T、蛋白酶体非ATP酶调节亚单位14、热休克蛋白β-1超氧化物歧化酶、以及一些假设蛋白和非特征性蛋白。     

Myofibril-Bound Serine Proteinase (MBP) and its Degradation of Myofibrillar Proteins

Proteolysis of a myofibril-bound serine proteinase (MBP) from carp Cyprinus carpio on myofibrillar proteins and their gel formation ability were investigated. MBP readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBP. The optimum degradation temperatures of MBP to myosin heavy chain in myofibril and kamaboko gel were 55°C and 60°C, respectively. The degradation effects of MBP on actin, α-actinin and tropomyosin were studied by the immunoblotting method. Because of its myofibril-bound and myofibrillar protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.