Chemical oxidation decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins

The objective of this study was to investigate the effect of chemical oxidation on proteolysis susceptibility of myofibrillar proteins. Myofibrils were prepared from pig M. longissimus dorsi and oxidised by a hydroxyl radical generating system. Protein oxidation level was measured by the carbonyl content, free thiol group content and bityrosine formation. Oxidised or non-oxidised myofibrillar proteins were exposed to papain and proteolysis was estimated by fluorescence using fluorescamine. Oxidation of myofibrillar proteins was dependent upon the oxidising agent concentration. Disulfide bridge and bityrosine formation indicated that oxidation by OH° can induce protein polymerization. Electrophoretic study showed that myosin was the protein most sensitive to oxidation. Results showed a direct and quantitative relationship between protein damages by hydroxyl radical and decreased proteolytic susceptibility. Electrophoretic observations suggest that polymerization and aggregation may explain in part decreased susceptibility of myofibrillar proteins to proteolysis.     

Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size

When skim milk at pH 6.55 was heated (75 to 100 degrees C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30-35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of beta-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6.5 to pH 6.7.     

Functional and biological activities of casein glycomacropeptide as influenced by lipophilization with medium and long chain fatty acid

This study determined the functional and different biological activities of casein glycomacropeptide (GMP) after conjugation with fatty acids. Medium (i.e. caproic, lauric and myristic acid) and long (i.e palmitic and stearic acid) fatty acids were conjugated to GMP at the available amino group. Functionalities of lipophilized GMP conjugates included foaming and emulsifying activities, and biological activities for bacterial growth inhibition, cell damage and anti-invasion. Greater lipophilization of GMP was achieved with medium chain fatty acids (p < 0.05), which resulted in reduced GMP solubility regardless of fatty acid conjugate. Foaming activity of GMP was lost after lipophilization, but emulsification activity of GMP was enhanced (p < 0.05). A parallel increase in growth inhibition of Salmonella spp. coupled with anti-invasion of Salmonella enteritidis (Inv A) into mammalian cells was induced by lipophilization of GMP with long chain fatty acid. Our results show that GMP modified by lipophilization with specific fatty acids provides improved functionality as a surfactant with enhanced antibacterial activity towards gram negative bacteria.     

Partial characterization of protease from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein

Proteases are one of the most important groups of industrial enzymes, with considerable application in the food industry. The aim of this work was to study a novel protease produced by the thermophilic fungus, Thermoascus aurantiacus, through solid-state fermentation (SSF). The enzyme acted optimally at pH 5.5 and 60 °C; it was stable up to 60 °C for 1 h and in the pH range 3.0–9.5. To elucidate the enzyme’s proteolytic activity, its hydrolytic profile on bovine casein, an important protein in the food industry, was studied by enzymatic hydrolysis on skim milk, analyzed by gel electrophoresis (UREA-PAGE), which clearly showed that the protease does not have the same specificity as bovine chymosin.     

Raman spectroscopic study of heat-induced gelation of pork myofibrillar proteins and its relationship with textural characteristic

Structural changes, textural properties and their relationships in pork myofibrillar proteins (PMP) were studied by Raman spectroscopy, texture profile analysis (TPA) and principal component analysis (PCA). Raman spectroscopy analysis revealed the occurrence of secondary structural changes in myofibrillar proteins. Modifications in the amide I (1600-1700 cm(-1)) and amide III (1200-1300 cm(-1)) regions indicated a significant (p<0.05) decrease in α-helix content, accompanied by a significant (p<0.05) increase in β-sheets, β-turns and random coil content. Texture property changes were also determined by TPA. All these features contributed to the formation of strong, irreversible heat-induced gels. The application of a dimensionality reducing technique such as PCA proved to be useful to determine the most influential properties of heat-induced gel. Significant (p<0.05) correlations were found between these structural changes and the textural characteristic (hardness) in the pork myofibrillar proteins system by PCA.     

Interaction of fish myoglobin and myofibrillar proteins

ABSTRACT:  Interactions between fish myoglobin (Mb) and myofibrillar proteins were investigated in a Mb-natural actomyosin (NAM) model at 4 °C. Increases in metmyoglobin (MetMb) formation and the relative content of bound Mb were observed, as were decreases in whiteness and Ca²⁺-ATPase activity ( P < 0.05). During the first 6 h of incubation, Mb bound preferably to myosin at domains other than the head portion, as evidenced by measurable ATPase activity. The potential binding of Mb to myosin heads occurred after 24-h incubation as evidenced by the marked decrease in Ca²⁺-ATPase activity of the NAM–Mb mixture when compared to that of NAM alone ( P < 0.05). The interaction between fish Mb and myofibrillar proteins was more pronounced with increased storage time; formation of high-molecular-weight aggregates (> 206 kDa) also increased with time. Electrophoretic study revealed that disulfide bonds were not involved in Mb–NAM interactions.     

Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels

The structure of muscle injected with specific cysteine protease inhibitors was examined to determine whether inhibitors cause denaturation and the degradation post-mortem of myofibrillar proteins was followed using SDS electrophoresis. Given the central role of calcium in theories of tenderisation the level of free calcium was measured during the early post-mortem period. The protease enzyme inhibitor E-64 was injected into the m. longissimus et thoracis lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). Muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). Muscle samples were selected from eight portions of the LTL (1-day post-mortem, from six different carcasses) for examination by transmission electron microscopy. Matching light images of myofibrils were obtained after determination of myofibrillar fragmentation. Free calcium concentration was determined for all samples (n=191) using an ion selective electrode excluding those 'at death'. Light images of myofibrils from treated samples showed normal striations and no evidence of denaturation or aggregation compared to control samples. This also applied to the samples processed for examination by electron microscopy. Appearance of the 30-kDa subunit increased with time (P<0.001) post-mortem. The interaction between ageing and stimulation had an effect (P<0.001) on the amount of a protein designated M1. The amount of M1 measured pre-rigor was greater for stimulated muscle, but the rate of decline was also greater through to day 2 post-mortem. Proteolysis was very rapid in the first 24 h post-mortem in ovine muscle. Ageing had an effect (P<0.001) on the free calcium concentration, which increased as muscle aged. As a covariate pH also had an effect (P< 0.05). Based on a non-linear model when the concentration of free calcium reached a plateau (～110 μM) the predicted pH was 5.5 (ultimate). From the qualitative observation of images and the levels of free calcium in injected muscle there is no support for the view that the inhibitors bind to sarcomere proteins, occupying sites to which calcium might bind. The levels of free calcium do not provide support for the view that m-calpain has a role in post-mortem tenderisation, but do suggest along with results of protein degradation that activation of μ-calpain is likely to occur before the pH drops to 6.2-6.1.     

Evaluating the profile of myofibrillar proteins and its relationship with tenderness among five styles of dry-cured hams

In order to investigate the relationship between profile of myofibrillar proteins and tenderness among 2 kinds of Chinese hams (Jinhua and Xuanwei) and 3 kinds of European hams (Iberian, Serrano and Parma), shear force, myofibril fragmentation index (MFI), SDS-PAGE, carbonyls content and Raman spectroscopy were investigated. The shear force and salt content of Chinese hams were significantly higher than that of European hams, while moisture content was lower than that of European hams (p < 0.05). MFI values and SDS-PAGE profile revealed that the degradation of myofibrillar proteins in Chinese hams was lower than in European hams. In addition, Chinese hams showed significantly higher carbonyls content and β-sheet content compared with European hams, indicated that proteins aggregation intensively inhibited the degradation of myofibrillar proteins in Chinese hams. These results indicated that the higher shear force in Chinese style hams could be attributed to the lower moisture content and limited proteolysis.     

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.     

Ultrasound-assisted generation of ACE-inhibitory peptides from casein hydrolyzed with nanoencapsulated protease

BACKGROUND: Bioactive peptides generated from milk proteins are eminent ingredients for functional foods and nutraceuticals. Amongst several approaches to release these peptides, hydrolysis of milk proteins with proteolytic enzymes is a promising choice. It is, however, required to inactivate the enzyme after a predetermined time, which leads to impurity of the final product. Immobilization of enzyme molecules can overcome this problem as it simplifies enzyme separation from the reaction mixture. A fungal protease from Aspergillus oryzea was encapsulated within nanoparticles yielded via silicification of polyamidoamine dendrimer template generation 0. It was used to hydrolyze the dominant milk protein (casein) in the absence or presence of sonication. The production of angiotensin converting enzyme (ACE)‐inhibitory peptides was monitored during hydrolysis. RESULTS: Sonication did not affect maximum ACE‐inhibitory activity but shortened the process sixfold. Ultrafiltration permeate of the centrifugal supernatant of casein solution hydrolyzed during sonication inhibited ACE activity as efficiently as the supernatant obtained from it. CONCLUSION: The protease from Aspergillus oryzea encapsulated within nanospheres is suitable for generation of ACE‐inhibitory peptides from casein. The nanoncapsulation procedure is simple, rapid and efficient. This may enable the industrial production of functional products from milk. Copyright © 2011 Society of Chemical Industry     

Tenderization and fragmentation of myofibrillar proteins in bovine longissimus dorsi muscle using proteolytic extract from Sarcodon aspratus

The objective of this study was to examine the Sarcodon aspratus extract including protease how to affect tenderness of the bovine longissimus dorsi muscle. In addition, we investigated myofibrillar protein fragmentation, particularly in myosin, and its influence on meat tenderness. Beef loin chunks were marinated with 0.5 g/100 g, 1 g/100 g, and 2 g/100 g powdered S. aspratus extract, 0.2 g/100 mL papain, and distilled water (control), respectively. Although tenderness of meat is increased by adding S. aspratus extract, differences in meat quality traits, such as muscle pH and meat color, were small and not considered to have practical importance between the control and enzyme-treated samples. Furthermore, the S. aspratus extract influenced the myofibril fragmentation index (MFI) as well as protein solubility. The changes in MFI and protein solubility were due to the myofibrillar protein degradation. Through Western blotting, we found that the S. aspratus extract, as well as papain, caused fragmentation of the myosin heavy chain, but the mushroom extract induced more fragmentations of myofibrillar proteins, and caused more tender meat.     

NaCl对添加丝氨酸蛋白酶的肌原纤维蛋白凝胶特性的影响

以养殖大黄鱼作为研究对象,探究NaCl对添加了丝氨酸蛋白酶的肌原纤维蛋白凝胶特性的影响.首先对养殖大黄鱼肉经过提取分离纯化得到的丝氨酸蛋白酶进行研究,探讨其最适温度、最适pH以及NaCl对丝氨酸蛋白酶活性的影响.并进一步研究NaCl对含有丝氨酸蛋白酶的肌原纤维蛋白凝胶的质构特性、持水性、白度、拉曼光谱、荧光分析、微观结...     

Extraction and partial characterization of proteolytic activities from the cell surface of Lactobacillus helveticus Zuc2

Proteolytic activities were extracted from a dairy Lactobacillus helveticus strain and partially characterized. A first cell envelope proteinase (CEP) was extracted using a high ionic strength buffer, both in the presence and in the absence of Ca²⁺. Moreover, cell treatment by 5 M LiCl allowed for the selective removal of the S-layer protein and CEP, suggesting an enzyme ionic linkage to the cell envelope similar to that observed for the Slayer structure. The enzyme specificity against α_s1-CN (f1-23) showed unusual activity on the Lys₃-His₄ bond compared with other proteinases of the same species. A second proteinase appeared to be linked to the cell membrane because it was extractable only after membrane disgregation by detergents. Its specificity against CN fractions and α_s1-CN (f1-23) was different from that of the first CEP; moreover, the measured activity was lower than that of CEP.     

Functional properties and biological activities of perilla seed meal protein hydrolysates obtained by using different proteolytic enzymes

Food Science and Biotechnology - In this study, we aimed to determine the potential functional properties and biological activities of the hydrolysates of perilla seed meal (PSM), which is a...     

Resistance of purified seed storage proteins from sesame (Sesamum indicum L.) to proteolytic digestive enzymes

Sesame has been increasingly associated with food allergy. The main seed storage proteins of sesame (the 2S albumin and the 7S and 11S globulins) were purified and subjected to proteolysis with pepsin, trypsin and chymotrypsin. The degree of proteolysis obtained was monitored by SDS–PAGE, followed by densitometry of the main bands. The 2S albumin was found to be stable to proteolysis, being extremely resistant to pepsin, and relatively resistant to trypsin and chymotrypsin. The 7S and 11S proteins were relatively labile to pepsin. Acidic polypeptides from the 11S protein were more susceptible to proteolysis than the basic polypeptides. Both 7S and 11S proteins generated what appeared to be stable polypeptides after proteolysis with trypsin and chymotrypsin. The results are discussed in relation to similar studies on related seed storage proteins, available structural information, and the potential allergenicity of the sesame proteins.     

Gelation of mixed myofibrillar/wheat gluten proteins treated with microbial transglutaminase

Gluten (Glu) and an acid-extracted protein fraction (AF) from wheat flour were mixed (1:1 ratio) with myofibrillar protein (MP) and treated with microbial transglutaminase (MTGase) to observe the effect on heat-induced gelation. Dynamic rheological properties and thermal denaturation patterns of treated samples were measured, respectively, with an oscillatory rheometer and a differential scanning calorimeter. The storage modulus (G′) of control MP sample (no MTGase), with a value of 533 Pa at end of heating (77 °C), was not affected (P > 0.05) by Glu nor by AF. However, mixed protein samples after the MTGase treatment produced higher gel elasticity values that differed (P < 0.05) between samples (1355, 1700 and 1875 Pa at 77 °C for MP, MP/AF, and MP/Glu, respectively). The MP sample underwent three endothermic transitions (peaking at 61.5, 68.0, and 78.5 °C) during thermal scan. The treatment with MTGase and/or addition of Glu or AF tended to lower the temperature for the first transition but raised the temperature for the third transition, suggesting possible interactions of the muscle with non-muscle proteins.     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages.Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.     

Improving gelling properties of myofibrillar proteins incorporating with cellulose micro/nanofibres

The effects of cellulose microfibres (CMFs, Average size: 100 ± 5 μm) and cellulose nanofibres (CNFs, Average size: 60 ± 3 nm) on the properties of myofibrillar protein (MP) gels from duck breast meat were studied. The results demonstrated that CMFs and CNFs were mostly connected to MP by non-covalent bonds, the diffusion and cross-linking of MP molecules was promoted, and a denser and more complete gel network was formed. With the increases of CMFs and CNFs concentration (0–10%), the hardness was increased by 13.15% and 19.78% for CMFs10% and CNFs10% gels, respectively, and the elasticity was increased by 40% and 80%, respectively. At the same concentration (0–10%), the increase in gel hardness, viscoelasticity and immobilised water content was greater in the CNFs-MP group than in the CMFs-MP group. The CNFs-MP group had a tighter gel network, and CNFs had a better potential to improve the gelation performance of MP.     

Effects of pork collagen on thermal and viscoelastic properties of purified porcine myofibrillar protein gels

The objectives of this study were to examine the thermal, water-binding and viscoelastic properties of mixed protein systems containing purified myofibrils from porcine semimembranosus (MP) and pork collagen (PC) during gelation and subsequent cooling. MP:PC mixtures (100:0, 90:10, 80:20, 70:30, 60:40, 50:50) normalized to 4% protein were evaluated. No significant differences in thermal characteristics of these mixtures could be detected using differential scanning calorimetry. A primary peak was observed near 66?°C. Using small-strain oscillatory testing, the rheological properties during gelling and cooling were quantified. Storage modulus (G') increased upon heating for all treatments, but the rate of gel firming and the G' value at 85?°C were significantly lower (P<0.05) as PC was added to the mixed protein system. Upon cooling, gels revealed a significantly lower (P<0.05) rate of gel firming and significantly lower (P<0.05) G' value at 5?°C in samples with 20% inclusion of PC and higher. Addition of PC yielded a significant linear (R(2)=0.65; P<0.01) increase in the water-holding capacity (WHC) of the gels, indicating that the matrix formed in MP:PC gels had a greater ability to entrap water than that of the control MP gels. The inclusion of 10% PC resulted in gels with significantly higher (P<0.05) WHC and similar firmness when compared with gels comprised of MP as the only protein source.