Sarcoplasmic Protein Fractions Research Articles

High pressure induced changes on sarcoplasmic protein fraction and quality indicators

The combined effect of pressure and mild temperature treatments on bovine sarcoplasmic proteins and quality parameters was assessed. M. longissimus dorsi samples were pressurised in a range of 200-600 MPa and 10-30 degrees C. High Pressure Processing (HPP) induced a reduction of protein solubility (p<0.001) compared to non-treated controls (NT), more pronounced above 200 MPa. HPP at pressures higher than 200 MPa induced a strong modification (p<0.001) of meat colour and a reduction of water holding capacity (WHC). SDS-PAGE analysis demonstrated that HPP significantly modified the composition of the sarcoplasmic protein fraction. The pressurisation temperature mainly affected protein solubility and colour; a smaller effect was observed on protein profiles. Significant correlations (p<0.001) between sarcoplasmic protein solubility and both expressible moisture (r=-0.78) and colour parameters (r=-0.81 to -0.91) suggest that pressure induced denaturation of sarcoplasmic proteins could influence to some extent WHC and colour modifications of beef. Changes in protein band intensities were also significantly correlated with protein solubility, meat lightness and expressible moisture. These results describe the changes induced by HPP on sarcoplasmic proteins and confirm a relationship between modification of the sarcoplasmic protein fraction and alteration of meat quality characteristics.

Effects of washing methods on gel properties of chicken surimi prepared from spent hen breast muscle

In this study, 2 myofibrillar protein extraction methods were compared for the preparation of chicken surimi using spent hen breast muscle. One method involved 1 washing with 0.1, 0.5, or 1% sodium chloride solution followed by 2 washings with distilled water, with homogenization and centrifugation of the breast muscle after each washing (new method; NM). The other method used only distilled water for the extraction, and washing was repeated 3 times followed by homogenization and centrifugation of the breast muscle after each washing (conventional method; CM). The redness values of the batter as well as the cooked gel were significantly lower (P < or = 0.05) by NM as compared with CM. In addition, the SDS-PAGE of NM sarcoplasmic protein fractions showed phosphorylase bands with increased staining intensity as compared with CM, indicating that brightness was related to the sarcoplasmic protein fractions. Overall, the data implied that sodium chloride solution was more appropriate for the myofibrillar protein extraction of spent hen breast muscle than the commonly used distilled water method.

Effect of starter culture on proteolytic changes during processing of fermented beef sausages

Abstract Fermented beef sausages inoculated with four different starter cultures (Pediococcus acidilactici, Lactobacillus curvatus, Lactobacillus sake, or Streptomyces griseus) were evaluated for proteolysis during process stages (prefermentation, fermentation, drying and heating). Increases (p ⩽ 0.05) in the nonprotein nitrogen (NPN) fraction were found at sequential stages of processing, while starter cultures had no major effects on NPN content. Concentrations of most free amino acids increased (p ⩽ 0.05) during fermentation and drying, and culture effects were found for differences among concentrations of some individual free amino acids. From SDS–PAGE analysis of sarcoplasmic and myofibrillar protein fractions after fermentation and drying, myosin heavy and light chains, actin and troponin were degraded during processing. However, starter culture effects were absent from SDS–PAGE protein patterns.

A proteomic approach to study local chicken breeds characterization

Aim of this study is to apply a proteomic approach for characterization of local chicken breeds. The experiment involved a total of 29 males of Pépoi, Padovana, and Ermellinata local chicken breeds. Sarcoplasmic protein fractions of breast muscle were analysed by bidimensional electrophoresis. Image analysis followed by statistical analysis enabled to differentiate groups of individuals on the similarities of protein expression. Individuals were distinguished into clusters and groups, corresponding to the breed of origin. Results evidenced a possible utilisation of proteomic approach in the field of breed characterization studies as an alternative to genomic analyses performed using molecular markers, both for breed and product traceability purposes.

EFFECT OF HYDRODYNAMIC PRESSURE PROCESSING AND AGING ON SARCOPLASMIC PROTEINS OF BEEF STRIP LOINS*

ABSTRACT This study evaluated the effects of hydrodynamic pressure processing (HDP) and aging on the sarcoplasmic proteins of beef strip loins. Loins (n = 12) were halved at 48 h postmortem and assigned to HDP or control treatments. Following treatment, each half was divided into three portions for aging (0, 5 or 8 d). Samples were removed for Warner‐Bratzler shear force (WBSF) determination and sarcoplasmic protein isolation. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis demonstrated that aging significantly influenced the composition of the sarcoplasmic protein fraction and that HDP influenced protein bands corresponding to 143, 65, 44, 36 and 19 kDa. Changes in sarcoplasmic protein composition were significantly correlated to WBSF (r = −0.58 to 0.45). Sarcoplasmic protein solubility decreased with both HDP (P &lt; 0.05) and aging (P &lt; 0.0001). Changes in solubility were significantly correlated to SDS‐PAGE band intensities (r = −0.53 to 0.60). Data suggest that HDP and aging cause changes to sarcoplasmic proteins that may be indicators of proteolysis and tenderization.PRACTICAL APLICATIONSResults from this study demonstrate that postmortem aging and hydrodynamic pressure processing of beef influences the sarcoplasmic protein profile of the muscle. The relationships between these changes and Warner‐Bratzler shear force measurements suggest that sarcoplasmic protein changes may be potentially useful as tenderness indicators.

Characterisation of microbial deep spoilage in Iberian dry-cured ham

The purpose of this work was to investigate the micro-organisms involved in overlooked “bone taint” spoilage of dry-cured Iberian hams. The physico-chemical characteristics of spoiled hams with 12 and 24 months of ripening, showing initial signs of alteration, were analyzed and their correlations with microbial counts studied. The spoilage potential of different microbial groups was assessed by the relationship between the microbial counts and the proteolysis level of spoilage as observed in the degradation of myofribrillar and sarcoplasmic protein fractions and in the changes in free amino acids. Non-enteric gram-negative bacteria (NEGN) were the dominant microbial group, showing a positive correlation with the moisture of spoiled hams. The Catalase-positive cocci (GPCP) growth was favoured by high NaCl concentrations in the spoiled hams, whereas the counts of Enterobacteriaceae were negatively affected by high NaCl concentration. The highest proteolytic microorganisms were the Gram-negative microbial groups playing Enterobacteriaceae a major role in the undesirable changes of the texture properties of the spoiled hams. With respect to the sensorial analysis, a synergy between NEGN and GPCP was observed in most of the strongly spoiled samples.

Microbial Ecology of the Soppressata of Vallo di Diano, a Traditional Dry Fermented Sausage from Southern Italy, and In Vitro and In Situ Selection of Autochthonous Starter Cultures

The microbial ecology of "soppressata of Vallo di Diano," a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus, S. succinus, and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus, L. sakei, and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.

Gel Color and Texture of Surimi-like Pork from Muscles at Different Rigor States Post-mortem

Gels were made from surimi-like pork (SLP) made from muscles obtained at 1, 24 and 72 h post-mortem. The SLP from pre-rigor muscle had higher pH and moisture percentage compared to in- or post-rigor muscles. Also, SLP from pre-rigor muscle showed higher concentration of water-soluble protein that was washed out during the process. Gel from post-rigor muscle exhibited higher a* and b* value, and also resulted in higher Chroma and lower hue values. The dark color of gel from post-rigor muscle was related to higher concentration of sarcoplasmic protein in SLP and denser structure in the gel matrix. SDS-PAGE showed higher intensity of the phosphorylase in the sarcoplasmic protein fraction from pre-rigor muscle. Gel from post-rigor muscle showed higher hardness and sensory firmness, and the greater firmness was related to higher concentration of protein in SLP, and a compact network with smaller pockets in the gel matrix.

Changes in the muscle proteome after compensatory growth in pigs

Sixteen female pigs (Duroc x Landrace x Large White) were divided into 2 groups, which had either free access to the diet (control group) or were feed-restricted from d 28 to 80 and then had free access to the diet (compensatory growth group). The sensory analysis showed that the pigs exhibiting compensatory growth produced meat with increased tenderness compared with control pigs (P < 0.05). To gain further knowledge of the influence of compensatory growth on meat tenderness, the sarcoplasmic protein fraction of muscle tissue was studied at the time of slaughter and 48 h postmortem using proteome analysis. At slaughter, 7 different proteins were found to be affected by compensatory growth: HSC70, HSP27, enolase 3, glycerol-3-phosphate dehydrogenase, aldehyde dehydrogenase E2, aldehyde dehydrogenase E3, and biphosphoglycerate mutase. The HSC70 and HSP27 both belong to the heat shock family and are known to play a role during muscle development. Hence, they may be affected by compensatory growth and increased protein turnover. Forty-eight hours after slaughter, 8 different proteins were found to be affected by compensatory growth: myosin light chain (MLC) II, MLC III, sulfite oxidase, chloride intracellular channel 1, 14-3-3 protein gamma, elongin B, and phosphohistidine phosphatase 14. The changes observed on MLC II and MLC III could be a consequence of enzymatic cleavage in the neck region of the globular myosin head domain that causes the release of MLC II and MLC III from the actomyosin complex. It has previously been hypothesized that compensatory growth results in an increased postmortem proteolysis; thus it was presumed that the intensity of some protein fragments would be affected by compensatory growth. However, the peptides that were found to be affected at 48 h postmortem were all full-length proteins. The 14-3-3 protein gamma has been proposed to play a role in the contraction of muscle during rigor and may thereby have an effect on meat tenderness. This study reveals some very interesting changes in the muscle proteome affected by compensatory growth, which may be useful in understanding the relationship among compensatory growth, protein turnover, and meat tenderness.

Myosin, sarcoplasmic and mitochondrial protein synthesis rates in rats adapted to a high protein diet

High protein diets have been shown to produce either no effect or a decrease in mixed muscle protein synthesis rates in rats and humans. However, no data regarding their effect on specific muscle protein fractions is available, despite their possible different metabolic regulation. In this context, we aimed at determining the fractional synthesis rates (FSR) of myosin, sarcoplasmic and mitochondrial protein fractions in gastrocnemius muscle of growing rats adapted to a high protein diet (HP, 49% of energy, n=16) for 14d or a normal protein diet (NP, 13% of energy, n=16). After the 14-d diets, rats were killed in the fasted or the fed state after a flooding dose of 13C-valine. NP and HP rats exhibited the same body weight, gastrocnemius mass, hydration and protein content. FSR and absolute synthesis rates of mixed muscle protein were neither significantly influenced by diet (NP or HP) nor by nutritional state (fasted or fed). There was also no effect of diet or nutritional state on the FSR of myosin fraction, sarcoplasmic or mitochondrial protein, which all ranged from 6 to 9%/d. Unlike splanchnic protein, which synthesis rates are greatly sensitive to an increased chronic protein intake, muscle protein fractions are not affected by dietary changes. This finding is in agreement with the lack of lean tissue anabolism usually accompanying high protein diets and suggest a very efficient system redistributing or oxidizing excess amino acids.

Variations in metabolite contents and protein denaturation of the longissimus dorsi muscle in various porcine quality classifications and metabolic rates

The purpose of the present study was to investigate the variations in metabolite contents and muscle protein denaturation in various porcine quality classifications and metabolic rates. A total of 226 crossbred pigs were evaluated. Samples were classified based on muscle pH 45 min and R-value into fast or normal glycolysing group. Drip loss and lightness ( L*) were used to assign samples as PSE, RSE, or RFN pork. Normal-glycolysing PSE pork contained an exceptional amount of initial glycogen and the high level of lactate at 24 h postmortem (PM). The initial levels of metabolites related closely with muscle pH, R-value and protein solubility at 45 min PM. The fast glycolysing group exhibited severe protein denaturation during the early postmortem period, and among them, fast-glycolysing PSE pork exhibited most pronounced protein denaturation. Via examination of each sarcoplasmic protein fraction, it was found that the bands for fructose-6-phosphate kinase, creatine kinase, triosephosphate isomerase, and myokinase were generally more evident as fainter bands in the fast-glycolysing pigs.

The impact of feeding β2‐adrenergic agonists on rainbow trout muscle β2‐adrenoceptors and protein synthesis

The β2‐adrenergic agonists (β2‐AAs) clenbuterol (CLEN) and ractopamine (RACT) were fed to rainbow trout Oncorhynchus mykiss for 30–37 days and red and white muscle β2‐adrenoceptor (β2‐AR) binding characteristics and mRNA expression were assessed in parallel with fractional protein synthetic rates. Feeding CLEN or RACT had no significant effect on any body or physiological variables measured. There were no significant differences in the number of binding sites (Bmax) while a significant increase in the Kd was observed for the β‐ARs of red and white muscle membranes isolated from β2‐AA‐fed rainbow trout. No change in β2‐AR mRNA levels was observed with β2‐AA feeding, implying that these β2‐AAs do not act at the transcriptional level at least for the β2‐AR. The β2‐AA treatments, however, did significantly increase red and white muscle fractional protein synthesis rates in whole protein, myofibrillar protein and sarcoplasmic soluble protein fractions. Although not conclusive, this study supports a direct link between rainbow trout muscle β2‐ARs and muscle protein synthesis.

Study of the proteolysis and cathepsin D activity of commercial dry-cured Iberian and Serrano hams

Physicochemical characteristics, proteolysis, and cathepsin D activity of the Semimembranosus and Biceps femoris muscles from commercial Iberian and Serrano hams were evaluated. Total nitrogen, sodium chloride, fat, and amino acid nitrogen contents were the only parameters of physicochemical composition and nitrogen fractions that enabled Iberian ham to be differentiated from Serrano ham in the two muscles considered. A wide variety of polypeptides in sarcoplasmic and myofibrillar protein fractions were found in the Semimembranousus and Biceps femoris muscles from Serrano and Iberian commercial samples. The sarcoplasmic proteins present in both muscles from both ham varieties had molecular weights of 80, 50, 45 (creatine kinase), 30 (phosphoglycerate mutase), 18, 16 (myoglobin), and 9 kDa. The common myofibrillar proteins in both muscles from both ham varieties had molecular weights of 102 (a-actinin), 90, 80, 60, 50, 35 (Tropomyosin), 30, 20 (C-troponin), 15 (Myosin light chain III), and 11 kDa. The protein profile showed that, in general, the myofibrillar protein fraction underwent more intense proteolysis than the sarcoplasmic protein fraction, giving rise to a larger number of low-molecular-weight peptides. Residual cathepsin D activity in the samples was very low, with no differences between either the muscle tissues or the ham varieties, indicating that the hams are relatively stable, as regards to proteolysis, once they have reached the marketplace.

Contribution of starter cultures to the proteolytic process of a fermented non-dried whole muscle ham product

Porcine longissimus dorsi muscles were cured by brine injection. Curing brine containing 15% (w/v) NaCl, 1.33% (w/v) glucose, 750 ppm sodium nitrite, and appropriate levels of either Lactobacillus sakei LAD, L. sakei LAD plus Kocuria varians FT4 (formally Micrococcus varians), L. sakei LAD plus papain and GDL (glucono-delta-lactone) plus K. varians FT4, was injected to the muscle at a pumping rate 15% w/v. The effect of these treatments on the proteolysis in the ham system was compared to a control ham, produced without starter culture and containing GDL acidulant to control pH and antibiotics to reduce the contribution of background microflora. Hydrolysis of sarcoplasmic and myofibrillar protein fractions was evaluated by SDS-PAGE and reverse phase-HPLC. Hams with different treatments were also investigated for differences in amino acid profile, protein and non-protein nitrogen level, colour, pH, water activity and moisture and microbiological evolution. There was no significant difference in the gross compositional analysis of any of the treatments compared to the control. There was no significant difference ( p>0.05) in the protein content, non-protein nitrogen level, SDS-PAGE and free amino acid analysis between the control ham and ham inoculated with proteolytic starter culture. However, it was observed that hams containing starter cultures exhibited decreases in certain peptide fractions and corresponding increases in some free amino acids compared to the uninoculated control. It can be concluded that, while the principle mechanisms resulting in the proteolysis of this non-dried ham product involve the activity of endogeneous cathepsins, the addition of proteolytic starter cultures influence the amino acid profile thereby potentially enhancing the sensorial attributes of the ham.

Fractionation and Properties of Sarcoplasmic Proteins from Oil Sardine (Sardinella longiceps): Influence on the Thermal Gelation Behavior of Washed Meat

ABSTRACT: The properties of total sarcoplasmic proteins from oil sardine (Sardinella longiceps)have been assessed. Ammonium sulfate fractionation of total sarcoplasmic proteins yielded 5 different fractions. The UV‐ab‐sorption spectra of all 5 sarcoplasmic proteins fraction showed a peak in the wavelength range of 275 to 280 nm indicating aromatic amino acid residues in its composition. The SDS‐PAGE of different fractions showed components in the molecular weight range of 97 to 29 kD. The thermal stability in the temperature range of 40° to 80 °C of different fractions were varied in nature. The flow profile of different fractions indicated pseudo‐plastic behavior and minimum structural impairment for fraction V at 30 and 40 °C, respectively. The gelation profile of washed sardine meat in combination with different sarcoplasmic proteins fraction revealed fraction V could enhance the storage modulus values during heating regime.

Effect of temperature and glassy states on the molecular mobility of solutes in frozen tuna muscle as studied by electron spin resonance spectroscopy with spin probe detection.

The mobility of solutes in frozen food systems (tuna muscle, sarcoplasmic protein fraction of tuna muscle, and carbohydrate-water) has been studied using the temperature dependence of the shape of electron spin resonance (ESR) spectra of the spin probe 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL). The spin probe was incorporated into the tuna meat from an aqueous solution of TEMPOL or by contact with a layer of TEMPOL crystals. The melting/freezing of freeze-concentrated solutes in frozen tuna meat was observed to take place over a range of temperatures from -25 to -10 degrees C. Lower temperatures gave ESR powder spectra due to the decreased mobility of the spin probe, and the temperature dependence of the mobility of the spin probe did not show abrupt changes at the glass transition temperatures of the systems. The mobility of nonglass forming solutes is concluded to be decoupled from the glass forming components. Similar behavior was also observed for TEMPOL in frozen, aqueous carbohydrate systems. The temperature dependence of the mobility of TEMPOL in the frozen systems was analyzed using the Arrhenius equation, and the logarithm of the Arrhenius preexponential factor tau(a) was found to be linearly correlated with the activation energy for all of the tuna and carbohydrate samples, indicating a common molecular mechanism for the observed mobility of TEMPOL in all of the systems. The linear correlation also suggests that the observed mobility of TEMPOL in the frozen aqueous systems is dominated by enthalpy-entropy compensation effects, where the mobility of TEMPOL is thermodynamically strongly coupled to the closest surrounding molecules.

Changes in composition and functional properties of proteins and their contributions to Nham characteristics

Changes in composition and functional properties of proteins during fermentation of Nham, a Thai-fermented sausage, were studied. An alkaline-soluble fraction constituted a major protein component of Nham. The amount of each protein fraction in Nham varied, depending on the fermentation time. As fermentation proceeded, the progressive decrease in sarcoplasmic and myofibrillar protein fractions was accompanied by an increase in the alkaline-soluble fraction and non-protein constituents ( P<0.05). Slow pH lowering to pH 4.6 during fermentation as a result of bacterial growth and accumulation of lactic acid affected the molecular conformation of the muscle proteins and resulted in changes in protein functional properties. The acid produced resulted in changes in solubility, water-binding capacity, textural properties, and color characteristics. Proteolysis of Nham proteins occurred during fermentation, resulting in increases in TCA-soluble peptides and free α-amino acids, which may contribute to the taste and aroma of Nham.

The question of high- or low-temperature glass transition in frozen fish. Construction of the supplemented state diagram for tuna muscle by differential scanning calorimetry.

The thermal behavior of fresh tuna muscle, rehydrated freeze-dried tuna muscle, and tuna sarcoplasmic protein fraction was studied by three types of differential scanning calorimetry (DSC): conventional DSC, alternating DSC, and sensitive micro-DSC. The relationship between glass transition temperature, T(g), and water content was established. Only a low-temperature glass transition was detected for fresh tuna and freeze-dried tuna rehydrated to high water contents, whereas for sarcoplasmic protein fraction both a low-temperature and an apparent high-temperature glass transition were detected for samples of high water content. Construction of the supplemented state diagrams for whole tuna muscle and for tuna sarcoplasmic protein fraction confirmed the low-temperature transition to be glass transition of the maximally freeze-dehydrated phase. The apparent upper transition of sarcoplasmic protein fraction was shown not to be a glass transition but rather to originate from the onset of melting of ice, and the temperature of this event should be denoted T(m)'. The glass transition temperature and the concentration of the maximally freeze dehydrated tuna muscle are -74 degrees C and 79% (w/w), respectively.

Acceleration of curing period of pastrami manufactured from buffalo meat: Chemical and microbiological properties.

Usually the curing period of meat used for pastrami manufacture is three weeks at room temperature. In the present work a trial aimed to accelerate the curing period of pastrami (dry cured meat) prepared from buffalo lean round muscles by heat treatment (approximately 71 degrees C--internal; for partial drying) was carried out to investigate the probable changes during processing and in quality parameters of the end product. Results showed that pH value and the residual NaNO2 content decreased, while lactic acid and conversion in meat pigment percentages were increased. The determined data indicated remarkable decrease of moisture content during aging and curing periods, while the NaCl content in the investigated product was noticeably increased. During pastrami processing soluble proteins were decreased; the sarcoplasmic protein fraction denatured more rapidly than myofibrillar protein. On the other hand, the non-protein nitrogen and the insoluble protein nitrogen were increased gradually during ageing and after the heat treatment step. Slight variations in their values were observed during ripening of pastrami (complete curing and drying in air at room temperature up to 6 days). Heat treatment caused apparent decrease in Aerobic Plate Count (APC). Salmonella and Coliform bacteria were not detected through meat curing and in the end product. The suggested heat-treated pastrami was of good quality and nearly similar to the traditional sample.

Specific enzyme detection following isoelectric focusing as a complimentary tool for the differentiation of related Gadoid fish species

The presence of specific enzymes in the sarcoplasmic protein fraction of eight gadoid fish species has been investigated following isoelectric focusing (IEF) in the 3.5–9.5 pH range. Lactate dehydrogenase (LDH) and glycerol-3-phosphate-dehydrogenase (G-3-PD) allowed the differentiation of most of the gadoids tested, even in the case of closely related species belonging to the genus Merluccius. Investigation of arginine kinase and creatine kinase afforded non-specific enzyme patterns in the species tested. Adenylate kinase and malate dehydrogenase did not give specific or reproducible enzyme patterns. Analysis of LDH and G-3-PD activities in commercial IEF gels was shown to be a reliable and reproducible technique for distinguishing closely related Merluccius spp. and other gadoid fish species.