Studies of Desmin and α-Actinin Degradation in Bovine Semitendinosus Muscle

The degradation of desmin and α-actinin was studied in post-mortem bovine semitendinosus muscle. Using a desmin-specific monoclonal antibody, SDS-PAGE, and immunoblotting we show that desmin is easily degraded at 4°C during the aging process. Within 96 hr, fragments of degraded desmin are detected with our antibody probe. Further storage at 4°C results in an increase of proteolytic fragments and concomitant loss of intact desmin. By 3 wk post-mortem, little un-degraded desmin remains in the muscle. In contrast, α-actinin was degraded slowly at 4°C. Proteolytic fragments of α-actinin were not detectable with anti-α-actinin polyclonal antisera until the second week of incubation at 4°C. However, the degradation of α-actinin was accelerated when meat was incubated at 25°C or at 37°C.     

PREPARATION AND CHARACTERIZATION OF α-AMYLASE IMMOBILIZED ON COLLAGEN MEMBRANES

Crystalline pancreatic α-amylase was codispersed with hide collagen at pH 4.0 and tanned to form a membrane which degraded starch. The optimum pH for the codispersed membrane preparation was at pH 7.0 in contrast to the soluble enzyme which was as active at pH 8.0 as at pH 7.0. The immobilized enzyme responded maximally to 0.22M chloride whereas 0.02M chloride gave optimum rates for the soluble enzyme. The immobilized enzyme resisted thermal inactivation better than the soluble α-amylase. Raising the temperatures from 30 ° to 50 °C produced a 500% increase in rate for the bound enzyme. It was also demonstrated that membranes retained greater activity when stored in starch solution than in water. The effect of glutaraldehyde concentration on membrane activity was also studied.     

A "Cold Shortening" Effect in Avian Muscle

SUMMARY— Experiments were conducted to determine the effect of post-mortem temperatures between 0 and 20°C on the degree of shortening in isolated pectoralis major muscles of chickens and turkeys. A "cold shortening" effect in these muscles is described and compared to post-mortem pH, average sarcomere length of isolated myofibrils, and relative solubility of myofibrillar and sarcoplasmic proteins.
The degree of muscle shortening at each temperature after various periods post-mortem indicated that shortening was essentially complete after 3 hr in chickens and 5 hr in turkeys. Shortening in muscles stored at 0°C was significantly greater (P < .01) than in the 12–18°C temperature range. Shortening was greatest in muscles stored at 20°C. The degree of gross shortening observed was directly related to the average sarcomere length of isolated myofibrils. Post-mortem decline in pH was not significantly correlated (P > .05) with shortening. Extractability of myofibrillar and sarcoplasmic proteins after 5 hr at either 0 or 16°C was determined and found to be unrelated to the degree of post-mortem shortening.     

Molecular Properties of Post-Mortem Muscle. 3. Electron Microscopy of Myofibrils

SUMMARY— A study was made of the fine structure of myofibril suspensions prepared from seven heifers immediately after death and after various times post-mortem. Studies on myofibrils sampled immediately after death showed that sucrose isolation gave the best structural preservation as indicated by maintenance of Z-line structure. Although the appearance of resting muscle was maintained in both sucrose and KCI preparations, several myofibrils from the KCI-treated preparations showed stretched sarcomeres. Glycerol-treated myofibrils usually had shorter sarcomere lengths than myofibrils prepared with the other two solvents. Although fibrillar preservation seemed adequate when glycerol was used, Z-line structure was seldom well-preserved with glycerol.
Myofibrils from muscle sampled 24 hr post-mortem at 2°C were supercontracted with thick filaments pushed against or through the Z-line, and no trace of l-bands remained. Myofibrils from muscle sampled 24 hr post-mortem at 16°C were contracted, but to a much lesser extent than 2°C-24 hr myofibrils. Storage at 2°C for 312 hr after death resulted in myofibrils that were contracted and that were structurally in a much poorer state of preservation than their 16°C counterparts. The 16°C-312 hr myofibrils were slightly contracted as indicated by the absence of H-zones and the presence of prominent, although narrowed, I-bands. All observations showed that shortening accompanying rigor mortis caused changes in banding patterns similar, and probably identical, to those predicted by Huxley's sliding filament model for contracting muscle.     

Molecular and physical characteristics of squid (Todarodes pacificus) skin collagens and biological properties of their enzymatic hydrolysates

ABSTRACT:  The physicochemical properties of squid skin collagens and biological activity of their enzymatic hydrolysates were determined to produce more value-added materials. The amino acid compositions of the inner and outer squid skin collagens were similar, but distinct from that of bovine tendon collagen in respect to the higher levels of aspartic acid, arginine, threonine, and serine, and of the lower levels of alanine, proline, and hydroxyproline. SDS-PAGE patterns suggested that squid skin collagen consisted of at least 2 different polypeptides (α1 and α2 chains) and their cross-linked chains. The molecular weights of α1 and α2 chains of bovine tendon collagens were higher than those of the corresponding α1 and α2 chains of squid skin collagens. The denaturation temperatures of inner and outer skin collagens were 27.1 and 27.3 °C, respectively, which were about 9 °C lower than that of bovine tendon collagen. Water holding capacities of inner and outer squid skin collagens were relatively similar, but were significantly greater than that of bovine tendon collagen. The maximum hydrolysis of squid skin collagens was obtained as follows: for outer skin collagen, enzyme concentration, 3.5%; hydrolysis time, 83 min; pH 7.0; hydrolysis temperature, 60 °C, whereas for inner skin collagen, enzyme concentration, 3.2%; hydrolysis time, 83 min; pH 7.0; hydrolysis temperature, 60 °C. The enzymatic hydrolysates of outer and inner skin collagens were separated by Sephacryl S-100 column, resulting in the production of 3 fractions (F1, F2, and F3). F3 fraction exhibited higher antioxidant, tyrosinase inhibitory, and antielastase activities than the other fractions.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

Molecular Properties of Post-Mortem Muscle.

SUMMARY— Post-mortem changes in nucleoside triphosphatase activity of bovine myosin B have been studied by using several different modifiers with either 5 mM ATP or 5 mM ITP as substrate at ionic strengths (r/2) of 0.09, 0.19, or 0.52. Enzymic activity was determined by measuring the release of inorganic phosphate. There was very little difference in enzymic activity between myosin B isolated from prerigor, rigor (24 hr post-mortem) or post-rigor (312 hr post-mortem) muscle stored at either 2° or 16°C except that the specific activity of myosin B prepared from muscle stored for 12–24, hr post-mortem was higher than activity of myosin B prepared immediately after death. This increase cannot be explained in terms of rigor shortening, but suggests that a change in myosin conformation or in the nature of the actin-myosin interaction occurs in post-mortem muscle. If an actin-myosin interaction occurs during rigor mortis and if this association remains unchanged during extraction of myosin B, then the very low Mg⁺⁺-modified myosin B enzymic activities obtained at Γ/2 = 0.19 and 0.52 indicate that this interaction is not irreversible. Extraction in the absence of ATP produced a myosin B whose ATPase activity was markedly inhibited by trace amounts of Mg⁺⁺. This may be due to the absence of a-actinin in these myosin B preparations. No consistent differences in activation energies were found either at Γ/2 = 0.19 or 0.52 among the NTPase reactions of myosin B samples prepared from muscle after various times of post-mortem storage.     

EFFECT OF TEMPERATURE DURING POST-MORTEM GLYCOLYSIS AND DEPHOSPHORYLATION OF HIGH ENERGY PHOSPHATES ON POULTRY MEAT TENDERNESS

SUMMARY– Tests made on pectoralis major muscles having post-slaughter pH values ranging between 6.1–7.0, indicated that holding poultry meat at 30 and 37°C during the onset of rigor mortis caused toughness. This toughening effect of high temperature appeared to occur when the pH level of the meat dropped from a value of about 6.3 to its ultimate low value and the adenosine triphosphate content dropped below 40% of its initial concentration. Holding temperatures at 10, 15 and 25°C during the onset of rigor mortis, or cooling to 15°C before the pH value dropped to about 6.3 produced more tender meat. After completion of post-mortem glycolysis and dephosphorylation of high energy phosphates, high temperature had no deleterious effect on tenderness. These results indicate that dephosphorylation of adenosine triphosphate at high temperature affects the mode or extent of stiffening of the muscular tissue and prevents tenderization.     

The Effect of pH-Temperature Treatments on the Calcium Accumulating Ability of Purified Sarcoplasmic Reticulum

SUMMARY: Pig sarcoplasmic reticulum fragments obtained from the longissimus dorsi muscle at 0- and 24-hours post-mortem were purified by salt extraction and density gradient centrifugation. The calcium uptake activity of 0-hour purified preparations was more than 20-fold higher than that from 24-hr old muscles, but there was no significant difference between fractions for calcium activated ATPase activities. When observed electron microscopically after negative staining, the ultrastructures of the 0. and 24-hour membrane fragments were found to be essentially identical. Incubation of isolated sacroplasmic reticulum fragments at pH 7.2 and 37°C or pH 5.6 and 0°C caused negligible inhibitoin of their calcium accumulating ability. However, treatment at pH 5.6 and 37°C for 1 hr almost completely abolished the sarcoplasmic reticulum calcium uptake. Thus it appears that low muscle pH and high temperature may be responsible for the inactivation of the calcium accumulating ability of the sarcoplasmic reticulum that occurs in situ.     

Molecular Properties of Post-Mortem Muscle. 6. Effect of Temperature on Protein Solubility of Rabbit and Bovine Muscle

SUMMARY– Studies were conducted to investigate the effect of temperature on the actin-myosin interaction of rabbit and bovine muscle during rigor and post-rigor shortening. Muscle was stored at four different temperatures (2°, 16°, 25° and 37°), corresponding to three types of post-mortem muscle shortening: cold, minimal and high temperature. These three types of shortening are presumably related to different states of the actin-myosin interaction in post-mortem muscle. Post-mortem tenderization may be the result of either actin-myosin dissociation or F-actin depolymerization.
To detect the occurrence of either of these possible changes, two salt solutions, differing widely in their myofibrillar protein extracting abilities, were used to compare post-mortem myofibrillar protein solubility after different times of post-mortem storage and to provide information about the actin-myosin complex. Myofibrillar protein solubility of both rabbit and beef muscle in 0.5M KCl, 0.1M phosphate, pH 7.4, increased markedly with increasing post-mortem storage at temperatures up to 25deg;. Similar solubility changes were obtained with 1.1M Kl, 0.1M K phosphate, pH 7.4, but these changes were much smaller in magnitude. Solubility in both salt solutions, in general, decreased for muscle stored at 37°.
Although time and temperature of post-mortem storage caused appreciable alterations in protein solubility, these alterations could not be directly related to changes in tenderness or sarcomere length or to species differences in the effects of temperature on post-mortem shortening. Viscosity, analytical ultracentrifugation, and ATPase assays all indicated the absence of "normal" actomyosin in all myofibrillar protein extracts in this study. It was suggested that the 1.1 M KI extracts contained G-actomyosin, but the available evidence indicated the presence of only myosin in 3-hr, 0.5 M KCI extracts.     

Conditioning of bovine muscle. II. Changes in the composition of extracts of myofibrils after conditioning

Myofibrils from longissimus dorsi muscles conditioned at 4° for 8 days and 15 days, were extracted by Hasselbach-Schneider solution and 5 mM Tris-HCl, pH 8·2. Both extractants removed increasing amounts of protein from the myofibrils as conditioning proceeded. More myosin, actin, tropomyosin and troponin was extracted by the Hasselbach-Schneider solution and 5 mM Tris, pH 8·2, extracted increasing amounts of a complex mixture containing actin and α-actinin. There was no evidence that any particular protein had been degraded or had disappeared and it is suggested that one of the effects of conditioning may have been an alteration in the binding of some of the proteins to each other in the myofibril.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats III. The effect of potassium sorbate

The growth of Clostridium botulinum types A and B spores, at 10¹ or 10³ per container, was studied in a pork slurry system containing nitrite (40 μg/g), sodium chloride (2.5, 3.5, 4.5% w/v) sodium isoascorbate (550 μg/g) at varying pH levels, with or without potassium sorbate (0.26% w/v), without heating and after two heat treatments (80°C for 7 min, and 80°C for 7 min + 70°C for 1 hr) followed by storage at 15, 17.5, 20 or 35°C for up to 6 months. At a given spore inoculum, potassium sorbate significantly decreased toxin production, as did increasing NaCl, decreasing pH or decreasing storage temperature. Heat treatment did not significantly affect spoilage or toxin production overall, but interacted significantly with some factors. The effect of sorbate was greater at 3.5% NaCl than at 2.5%, at pH values below 6.0, and at low storage temperature.     

INSTABILITY OF SODIUM NITRITE IN A CHEMICALLY DEFINED MICROBIOLOGICAL MEDIUM

Mixtures of sodium nitrite, amino acids and ascorbic acid at pH 6.3 or 7.2 were filter sterilized or heated for various times at 121°C. Samples were analyzed for nitrite concentration immediately after treatment and after storage at 37 or 43°C. Heating the complete medium for 15 min at 121°C reduced the nitrite concentration by 30–50% independent of the initial nitrite concentration. Storage of complete filter sterilized medium at 43°C for 18–24 hr resulted in 50–65% loss of nitrite while in heat sterilized medium the loss was as great as 90%. None of the 19 amino acids stored individually with sodium nitrite at 37°C for up to 4 hr affected the nitrite concentration, whereas ascorbic acid resulted in total disappearance of nitrite. During heating at 121°C of the 19 amino acids only cystine resulted in any significant nitrite loss after 15 min of treatment while ascorbic acid effected total nitrite disappearance after 5 min. The defined medium containing nitrite demonstrated inhibitory activity against seven strains of Clostridium perfringens.     

Studies on Bovine Natural Actomyosin 2. Physico-Chemical Properties and Tenderness of Muscle

SUMMARY: Studies were made of physicochemical characteristics of natural actomyosin from bovine longissimus of different post-mortem ages and tenderness classifications. Reduced viscosity, ATP sensitivity, and "actin" content (polyethylene sulfonate treatment) were higher for natural actomyosin prepared from muscle 12-24 hr post-mortem than from pre-rigor muscle, which confirms previous reports for rabbit natural actomyosin. A higher actin to myosin ratio in actomyosin from muscle 12-24 hr was therefore postulated. A stronger interaction of actin and myosin in actomyosin from muscle 12-24 hr post-mortem than from pre-rigor or aged muscle was also suggested by reduced viscosity and ultracentrifugation data. Reduced viscosity differences between actomyosins from tough and tender muscle suggested a higher gel character in actomyosin from tough muscle. This possibly indicated a higher content of α-actinin. No consistent differences in ATP sensitivity, myosin and actin content of natural actomyosin of tough and tender muscle were found. Natural actomyosin from muscle aged post-mortem showed the appearance during analytical ultracentrifugation of an additional component which sedimented at about 11S to 12S. This component appeared in the actomyosin prepared from tender muscle after 24 hr but did not appear until 10 days in the actomyosin from tough muscle.     

Stability of β-glucosidase Activity Produced by Bifidobacterium and Lactobacillus spp. in Fermented Soymilk During Processing and Storage

ABSTRACT: Microorganisms possess endogenous enzymes, however the stability of these enzymes during storage in soymilk has not been studied. β-glucosidase is an important enzyme that could be used in the bioconversion of the predominant soy isoflavone glucosides to their bioactive aglycone forms. Fifteen probiotic microorganisms including bifidobacterium, Lactobacillus acidophilus , and Lactobacillus casei were screened for β-glucosidase activity using p-nitrophenyl-β-d-glucopyranoside as a substrate. Six strains were selected on the basis of β-glucosidase activity produced during fermentation of soymilk. The stability of the enzyme activity was assessed during incubation for up to 48 h and storage for 8 wk at frozen (-80°C), refrigerated (4°C), room (24.8°C), and incubation (37°C) temperatures. L. casei strains showed the highest β-glucosidase activity after 24 h of incubation followed by L. acidophilus strains, whereas bifidobacterium strains showedleast activity. However, p-glucosidase from Bifidobacterium animalis BB12 showed the best stability during the 48 h fermentation. Lower storage temperatures (-80°C and 4°C) showed significantly higher ( P < 0.05) β-glucosidase activity and better stability than that at higher temperatures (24.8°C and 37°C). The stability of β-glucosidase from these microorganisms should be considered for enzymic biotransformation during storage of isoflavone β-glucosides to bioactive isoflavone aglycone forms with potential health benefits.     

Mackerel Cathepsins B and L Effects on Thermal Degradation of Surimi

During surimi processing, cathepsins B and L activities in minced, leached and NaCl-ground meats were 6.02, 5.23, and 4.07 units/g, respectively. About 80% activity remained in surimi after 8 wk storage at ?40°C suggesting that these proteinases were stable and difficult to remove. At 40°～ 55°C, pH 6.5 ～ 7.5, cathepsins B and L and purified cathepsin B had high hydrolytic activity on myosin heavy chain (MHC). The strength of surimi gel with cathepsins B and L or with purified B decreased (p < 0.05) after 2 hr incubation at 55°C. This suggested that the residual cathepsins B and L had MHC-degrading activity and consequently caused gel softening.     

Effect of molecular weight, type of chitosan, and chitosan solution pH on the shelf-life and quality of coated eggs

ABSTRACT:  Effects of the molecular weight and type of chitosans and pH of chitosan solution on antibacterial activity against Salmonella enterica Enteritidis and on internal quality of chitosan-coated eggs were evaluated during 4 wk of storage at 25 °C. Two types of chitosans were studied: α-chitosans with 4 different molecular weights (Mw = 282, 440, 746, and 1110 kDa) and β-chitosan (Mw = 577 kDa). The α-chitosan with 282 kDa exhibited stronger bactericidal effects than did other α- and β-chitosans. The weight loss, Haugh unit, and yolk index values suggested that coating of eggs with α-chitosan with 282 kDa increased the shelf-life of eggs by almost 3 wk at 25 °C compared with noncoated eggs. The pH (4.5, 5.0, and 5.5) of the α-chitosan (282 kDa) solution did not affect the internal quality of chitosan-coated eggs. Therefore, coating of eggs with 282 kDa α-chitosan without pH adjustment (initial pH of 4.5) may offer a protective barrier against contamination of S. Enteritidis while simultaneously preserving the internal quality of eggs.     

Studies on genetic variants of α-lactalbumin and β-lactoglobulin from milk of native Portuguese ovine and caprine breeds

Summary α-Lactalbumin (α-La) and β-lactoglobulin (β-Lg) fractions were obtained from Portuguese native breeds of ewes and goats by preparative gel filtration and further purified by ion exchange; their genetic variants were characterized by isolectric focusing, and β-Lg isolated was further characterized by differential scanning calorimetry. Separation of β-Lg and α-La by molecular exclusion from native whey was relatively easy, whereas β-Lg from both breeds accounted for a single peak via ion exchange under various gradients of NaCl. Isoelectric focusing has indicated that α-La from ovine and caprine wheys appears as a single variant in each case, as well as β-Lg from caprine whey; however, β-Lg from ovine whey appears as two peaks, tentatively denoted as β-Lg A and B. Further comparison with bovine whey made it possible to rank whey proteins by increasing value of pI as follows: bovine β-Lg A, bovine α-La, bovine β-Lg B, ovine and caprine α-La, ovine β-Lg A, and finally ovine β-Lg B and caprine β-Lg. β-Lg from goat's whey showed the highest onset temperature of denaturation in the presence (78–97 °C) or absence (90–100 °C) of NaCl for every pH tested; when NaCl was present, a good correlation between pI and onset temperature of denaturation was obtained for pH values in the range 3.5–7.0.     

Effects of Dense Phase Carbon Dioxide Pasteurization on the Physical and Quality Attributes of a Red Grapefruit Juice

ABSTRACT:  Red grapefruit juice was treated with continuous dense phase carbon dioxide (DPCD) equipment to inactivate yeasts and molds and total aerobic microorganisms. A central composite design was used with pressure (13.8, 24.1, and 34.5 MPa) and residence time (5, 7, and 9 min) as variables at constant temperature (40 °C), and CO₂ level (5.7%) after experimentally measuring CO₂ solubility in the juice. Five log reduction for yeasts and molds and total aerobic microorganisms occurred at 34.5 MPa and 7 min of treatment. A storage study was performed on the fresh juice DPCD treated at these conditions. °Brix, pH, titratable acidity (TA), pectinesterase (PE) inactivation, cloud, color, hue tint and color density, total phenolics, antioxidant capacity, and ascorbic acid were measured after the treatment and during 6 wk storage at 4 °C. During storage, the DPCD-treated juice showed no growth of total aerobic microorganisms and yeasts and molds. Cloud increased (91%) while percent PE inactivation was partial (69.17%). No significant (α= 0.05) differences were detected between treated and untreated samples for °Brix, pH, and TA. Treated juice had higher lightness and redness and lower yellowness. No significant differences (α= 0.05) were detected for the hue tint values while the color density value was higher for the treated samples compared to the untreated. The treatment and the storage did not affect the total phenolic content of the juice. Slight differences were detected for the ascorbic acid content and the antioxidant capacity. The experimental results showed evidence that the treatment can maintain the physical and quality attributes of the juice, extending its shelf life and safety.     

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.