Effect of added μ-calpain and post-mortem storage on the mechanical properties of bovine single muscle fibres extended to fracture

The effects of μ-calpain and post-mortem storage on the strength of single muscle fibres were investigated. During the 10 min of incubation at pH 7.5, μ-calpain became evenly distributed throughout the fibre. μ-Calpain-incubation resulted in thinner (P <0.001) Z-lines and reduced (P <0.001) the strength of the fibres compared to controls. These results demonstrate that μ-calpain is capable of mechanically weakening the muscle fibres. Post-mortem storage of meat for 10 days at 2?°C weakened (P <0.001) the muscle fibres compared to 24-h fibres. The presence or absence of Ca(2+) affected fibre stiffness. Fibres incubated at pH 7.5 in 100 μM Ca(2+) were less stiff than fibres incubated in 200 μM EGTA. Breaking stress and strain were not affected by Ca(2+). We hypothesise that Ca(2+) causes conformational changes in some of the load-bearing proteins, which alters their initial resistance to extension, but does not affect the breaking strength of the fibres.     

Optimizing Assay and Extraction of Lipoxygenase in Wheat Germ

Using three-level seven-factor response surface methodology, wheat germ lipoxygenase (LPO) assay conditions were standardized. The important parameters were concentration of the substrate (linoleic acid), and surfactant (Tween 20), pH and temperature. The standardized LPO assay conditions for the 1 mL reaction volume were : 450 μM linoleic acid, 129 μM Tween 20, 175 mM ethanol, 1 mM EDTA, pH 6.2 (phosphate buffer), ionic strength 100 mM and 40°C. LPO extraction conditions were standardized by sequential variation of parameters. Optimum conditions were a single extraction of defatted wheat germ flour at 2–5°C with magnetic stirring of an extractant acetate buffer pH 4.5, ionic strength 100 mM, at buffer-to-solid ratio 10:l.     

Effect of gelation factors on the formation and characteristics of protease‐induced whey protein gel

Whey protein gel formed at 10% (w/v) whey protein concentration, 0.5% E/S, pH 7.0, 55°C and 2.5 mM CaCl₂ concentration had an average particle size of 23.46 μm, hardness of 0.46, cohesiveness of 0.13 and adhesiveness of 1.40, and the gel showed semisolid, smooth and creamy texture. There were no distinct changes in gel textural properties after heating at 80 and 90°C for 5 min, respectively, or being kept at 4°C for 1 month. The textural properties of the gel showed no significant difference after its pH was adjusted to 4.5, 5.5 and 7.5 compared with that of pH 6.5 (control gel). However, the average particle size significantly increased after being adjusted to pH 4.5 and pH 5.5. Transmission electron micrographs showed that protease‐induced gel possessed much looser aggregate structure compared with heat‐induced compact gel, which may give support to its potential application in low‐fat foods that no need of extensive heating.     

Zinc binding in bovine milk

About 90% of the Zn in bovine skim milk was sedimented by ultracentrifugation at 100,000 g for 1 h. About half of the non-sedimentable Zn was non-dialysable, indicating that it was associated with protein, probably non-sedimented casein micelles. Casein micelles incorporated considerable amounts of Zn added to skim milk as ZnCl2, and at Zn concentrations greater than or equal to 16 mM coagulation of casein micelles occurred. Ca was displaced from casein micelles by increasing ZnCl2 concentration and approximately 40% of micellar Ca was displaced by 16 mM-ZnCl2. Micellar Zn, Ca and Pi were gradually rendered soluble as the pH of milk was lowered and at pH 4.6 greater than 95% of the Zn, Ca and Pi were non-sedimentable. These changes were largely reversible by readjustment of the pH to 6.7. About 40% of the total Zn in skim milk was non-sedimentable at 0.2 mM-EDTA and most of the remainder was gradually rendered soluble by EDTA over the concentration range 1-50 mM. This indicates that there are two distinct micellar Zn fractions. No micellar Ca or Pi was solubilized at EDTA concentrations up to 1.0 mM, indicating that both colloidal calcium phosphate (CCP) and casein micelles remained intact under conditions where the more loosely bound micellar Zn fraction dissolved. Depletion of casein micelles of colloidal Ca and Pi by acidification and equilibrium dialysis resulted in removal of Zn, and in colloidal Pi-free milk non-dialysable Zn was reduced to 1.2 mg/l (approximately 32% of the original Zn). Thus, approximately 32% of the Zn in skim milk is directly bound to caseins, while approximately 63% is associated with CCP. Over 80% of the Zn in colloidal Pi-free milk was rendered soluble by 0.2 mM-EDTA, indicating that the casein-bound Zn is the loosely bound Zn fraction in casein micelles. A considerable fraction of the Zn in acid whey (pH 4.6) co-precipitated with Ca and Pi on raising the pH to 6.7 and heating for 2 h at 40 degrees C, indicating that insoluble Zn phosphate complexes form readily under these conditions. Studies on dialysis of milk against water, or dilution of milk or casein micelles with water, showed that CCP and its associated Zn is very stable and dissolves only very slowly at pH 6.6. The nature of Zn binding in casein micelles may help to explain the lower nutritional bioavailability of Zn in bovine milk and infant formulae compared with human milk.     

Ionic strength and composition affect the mobility of surface-modified Fe0 nanoparticles in water-saturated sand columns

The surfaces of nanoscale zerovalent iron (NZVI) used for groundwater remediation must be modified to be mobile in the subsurface for emplacement. Adsorbed polymers and surfactants can electrostatically, sterically, or electrosterically stabilize nanoparticle suspensions in water, but their efficacy will depend on groundwater ionic strength and cation type as well as physical and chemical heterogeneities of the aquifer material. Here, the effect of ionic strength and cation type on the mobility of bare, polymer-, and surfactant-modified NZVI is evaluated in water-saturated sand columns at low particle concentrations where filtration theory is applicable. NZVI surface modifiers include a high molecular weight (MW) (125 kg/mol) poly(methacrylic acid)-b-(methyl methacrylate)-b-(styrene sulfonate) triblock copolymer (PMAA-PMMA-PSS), polyaspartate which is a low MW (2-3 kg/mol) biopolymer, and the surfactant sodium dodecyl benzene sulfonate (SDBS, MW = 348.5 g/mol). Bare NZVI with an apparent zeta-potential of -30 +/- 3 mV was immobile. Polyaspartate-modified nanoiron (MRNIP) with an apparent zeta-potential of -39 +/- 1 mV was mobile at low ionic strengths (< 40 mM for Na+ and < 0.5 mM for Ca2+), and had a critical deposition concentration (CDC) of approximately 770 mM Na+ and approximately 4 mM for Ca2+. SDBS-modified NZVI with a similar apparent zeta-potential (-38.3 +/- 0.9 mV) showed similar behavior (CDC approximately 350 mM for Na+ and approximately 3.5 mM for Ca2+). Triblock copolymer-modified NZVI had the highest apparent zeta-potential (-50 +/- 1.2 mV), the greatest mobility in porous media, and a CDC of approximately 4 M for Na+ and approximately 100s of mM for Ca2+. The high mobility and CDC is attributed to the electrosteric stabilization afforded by the triblock copolymer but not the other modifiers which provide primarily electrostatic stabilization. Thus, electrosteric stabilization provides the best resistance to changing electrolyte conditions likely to be encountered in real groundwater aquifers, and may provide transport distances of 10s to 100s of meters in unconsolidated sandy aquifers at injection velocities used for emplacement.     

Studies on the pathogenesis and survival of different culture forms of Listeria monocytogenes to pulsed UV-light irradiation after exposure to mild-food processing stresses

The effects of mild conventional food-processing conditions on Listeria monocytogenes survival to pulsed UV (PUV) irradiation and virulence-associated characteristics were investigated. Specifically, this study describes the inability of 10 strains representative of 3 different culture forms or morphotypes of L. monocytogenes to adapt to normally lethal levels of PUV-irradiation after exposure to sub-lethal concentrations of salt (7.5% (w/v) NaCl for 1 h), acid (pH 5.5 for 1 h), heating (48 °C for 1 h) or PUV (UV dose 0.08 μJ/cm(2)). Findings showed that the order of increasing sensitivity of L. monocytogenes of non-adapted and stressed morphotypes to low pH (pH 3.5 for 5 h, adjusted with lactic), high salt (17.5% w/v NaCl for 5 h), heating (60 °C for 1 h) and PUV-irradiation (100 pulses at 7.2 J and 12.8 J, equivalent to UV doses of 2.7 and 8.4 μJ/cm(2) respectively) was typical wild-type smooth (S/WT), atypical filamentous rough (FR) and atypical multiple-cell-chain (MCR) variants. Exposure of L. monocytogenes cells to sub-lethal acid, salt or heating conditions resulted in similar or increased susceptibility to PUV treatments. Only prior exposure to mild heat stressing significantly enhanced invasion of Caco-2 cells, whereas subjection of L. monocytogenes cells to combined sub-lethal salt, acid and heating conditions produced the greatest reduction in invasiveness. Implications of these findings are discussed. This constitutes the first study to show that pre-exposure to mild conventional food-processing stresses enhances sensitivity of different culture morphotypes of L. monocytogenes to PUV, which is growing in popularity as an alternative or complementary approach for decontamination in the food environment.     

Purification and characterization of polyphenol oxidase from Trametes sp. MS39401

Two polyphenol oxidases (EC 1.14.18.1), P-1 and P-2, were purified as electrophoretically homogeneous proteins from the culture filtrate of Trametes sp. MS39401 by acetone precipitation and column chromatographies on DEAE-Sephadex A-50, Sephadex G-150 and hydroxylapatite. P-1 was purified 34-fold with a yield of 4.2%, while P-2 was purified 37-fold with a yield of 20.7%. The molecular masses of P-1 and P-2 were estimated to be 61 kDa and 90 kDa, respectively, by gel filtration. The isoelectric points of P-1 and P-2 were 3.4 and 2.7, respectively. The optimum pH range of both enzymes was 4.5-5.0 at 45 degrees C. The optimum temperature of both enzymes was 55 degrees C at pH 5.0. P-1 was stable at pH 5.0-7.5 and temperatures up to 60 degrees C. P-2 was stable at pH 3.0-7.5 and temperatures up to 50 degrees C. The thermostability of P-1 was comparable to that of the PM1 laccase of basidiomycetes, which was reported to be the most stable among basidiomycete laccases. Both enzymes were active toward various phenolic compounds and aminophenols. However, they lacked activity toward l-tyrosine. The K(m) values for (+)-catechin were 0.19 mM for P-1 and 0.67 mM for P-2. Both enzymes were appreciably inactivated by Hg(2+) and Sn(2+). Significant activation of neither enzyme was observed in the presence of metal ions and reagents. Both enzymes were significantly inhibited by copper-chelating agents, reducing agents and N-bromosuccinimide. Carbon monoxide caused appreciable inactivation of neither enzyme, so it is suggested that P-1 and P-2 belong to the group of laccases.     

Heat-induced gelation of actomyosin

Rheological properties of actomyosin gels were markedly affected by protein concentration, pH and heating temperature. Gel strength increased with increasing protein concentration (30-60 mg ml(-1)) and heating temperature (55-75°C), but decreased with increasing pH (5·5-9·0). Low heating temperatures (50-55°C) favoured the formation of more cohesive actomyosin gels than the higher heating temperatures (60-75°C). Gels formed at low pH (5·5 and 6·0) were less cohesive than those formed at high pH (7·5-9·0). Addition of ATP and pyrophosphate (10 mm) prior to heating decreased gel strength and cohesiveness, whereas EDTA (1-5 mM) reduced gel strength but did not affect gel cohesiveness.     

Myosin heavy chain isoforms influence myofibrillar ATPase activity under simulated postmortem pH, calcium, and temperature conditions

The pH and Ca(2+) sensitivity of myofibrillar ATPase activity plays an integral role in regulating postmortem muscle ATP utilization and likely paces postmortem glycolysis. The objective of this study was to determine the influence of pH and Ca(2+) concentration on the ATPase activity of myofibrils from red semitendinosus (RST) and white semitendinosus (WST) porcine muscles. Myofibrillar ATPase was measured at 39 °C over a pH range 5-7.5 and a [Ca(2+)] range pCa 4-9 (10(-4)-10(-9)M). At maximum Ca(2+)-dependent activation (pCa 4), RST myofibrils had lower (p<0.0001) ATPase activity than WST myofibrils. This maximum activity of myofibrils from both muscle regions was not influenced from pH 7.5 to 6.5, declined between pH 6.5 and 5.75 (Hill coefficient, n(H)=2.7-3.4; pH at half maximum activity, pH(50)=5.97) and was near zero at pH 5.5. At pH 7, pCa-activity relationships showed that RST required less Ca(2+) for half-maximum activation (higher pCa(50); 6.50) than WST myofibrils (pCa(50)=6.35) but had no difference in n(H). At pH 7, both RST and WST myofibrils had maximum Ca(2+)-dependent, actin-activated ATPase activity at pCa ?6 and Ca(2+)-independent myosin ATPase activity at pCa ?6.75. pCa-activity relationships at different pH levels indicated that pCa(50) decreased with pH from pH 6.5 to 6.125 in both RST and WST myofibrils. At pH <5.75, [Ca(2+)] did not influence ATPase activity in RST or WST myofibrils. These data show that myofibrils with predominantly fast MyHC (WST) have a higher actin-activated myosin ATPase activity than myofibrils with primarily slow MyHC isoforms (RST) at Ca(2+) concentrations and pH values characteristic of postmortem muscle.     

Interactive Effects of Factors Affecting Gelation of Whey Proteins

The individual effects of heating time (15–120 min), pH (3–9) and NaCl (0–2M), sucrose (0–30% w/v) and protein (10–30% w/v) concentrations on the strength, turbidity and water holding capacity were investigated on a commercial whey protein concentrate (WPC, 75% protein) when heated at temperatures ranging from 65 to 90°C. Interactive effects were investigated using a four-variable, five-level central composite rotatable design (CCRD) analyzed by response surface methodology (RSM). Gel strength (GS) and water holding capacity (WHC) increased with protein concentration, heating temperature and time. Increasing sucrose concentration decreased GS but increased WHC. Increasing NaCl concentration increased GS and WHC below pH 5 but resulted in weaker gels at high pH (>7).     

Proteolytic activity of isolated lamb calpains on myofibrils under the conditions of pH, Ca2+ concentration and temperature existing in postmortem muscle.

The two calcium-activated neutral proteinases (calpains I and II) and their specific inhibitor were isolated by ion exchange chromatography in DEAE-Sephacel from lamb skeletal muscle (longissimus dorsi). Their proteolytic activities were then determined using myofibrils as substrate. The Ca2+ requirements were different for each form of the enzyme: calpain I needed only 50 mumol Ca2+ for half-maximal activity, while the other isoenzyme, calpain II, needed 1,000 mumol Ca2+ for reaching 50% of its maximum activity. Both calpains showed a relevant activity in the pH range 5.5-6.5 (over 40% of maximum activity found at pH 7.5). With regard to the effect of temperature, both isoenzymes retained about 25% of their activity at 25 degrees C with a temperature reduction down to 4 degrees C. It is concluded that calpain I is an active protease under conditions similar to that prevalent in lamb meat during postmortem storage.     

Simultaneous application of microbial transglutaminase and high hydrostatic pressure to improve heat induced gelation of pork plasma

The effects of treating porcine plasma with microbial tranglutaminase (MTGase) under high hydrostatic pressure (HHP) were studied as a means of improving its gel-forming properties when subsequently heated at pH 5.5, near the pH of meats. Plasma containing varying levels of commercial MTGase was pressurized (400MPa, room temperature, pH 7) for different times, and adjusted to pH 5.5 prior to heating to induce gelation. MTGase-treatment under HHP led to greater enhancement of heat-induced plasma gel properties as compared to control samples. The greatest improvements were achieved by pressurising plasma with 43.3U MTGase/g protein for 30min, thereby achieving recoveries of 49% and 63% in fracture force (gel strength) and fracture distance (gel deformability) of the subsequently heat-induced gels, respectively, relative to gel properties obtained by heating untreated plasma at physiological conditions (pH 7.5).     

Mass spectrometric investigations on lactate adduction to equine myoglobin

Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15 mM) were incubated with sodium lactate (200 mM) at 4 °C, pH 5.6 in 50 mM sodium citrate buffer or at 37 °C, pH 7.4 in 50 mM sodium phosphate buffer, simulating typical meat storage and physiological conditions, respectively. Controls consisted of myoglobin plus a volume of deionized water equivalent to that used to deliver the lactate treatments. No peaks corresponding to lactate-Mb adducts could be detected in the mass spectra of samples incubated up to 360 min at pH 7.4, 37 °C or 8 days at pH 5.6 and 4 °C. Our results suggest that lactate did not form covalent adducts with equine oxy- and carboxy-myoglobin.     

The effect of pH,sodium chloride,sodium nitrite and storage temperature on the growth of Clostridium perfringens and faecal streptococci in laboratory media

The effects of salt concentration (1–12% w/v) in combination with unheated sodium nitrite (0–400 μg/ml) on growth of mixed strains of Clostridium perfringens and of faecal streptococci at three pH values (5.6, 6.2, 6.8) and storage temperatures ranging from 10°C to 35°C is reported. At pH 6.2, following storage at 15°C, 1% salt and 50 μg/ml nitrite inhibited growth of C. perfringens. At 20°C and pH 6.2, 200 μg/ml nitrite plus 3% salt, or 50 μg/ml plus 4% salt were required to inhibit growth. Growth of C. perfringens was prevented by levels of curing salts used commercially providing the pH was 6.2 or below. At pH 6.8 or above at least 4% salt and 50 μg/ml nitrite was required to prevent growth at 20°C. The faecal streptococci grew in medium containing 6% salt and 400 μg/ml nitrite irrespective of pH or storage temperature. In 8% salt growth was prevented by storing at or below 17.5°C or, if pH was 6.2 or lower, by adding 200 μg/ml nitrite irrespective of storage temperature. Growth of faecal streptococci was not controlled by concentrations of curing salts which would be acceptable in meat products.     

THERMAL BROWNING OF TOMATO SOLIDS AS AFFECTED BY CONCENTRATION AND INHIBITORS

SUMMARY– The amount of brown color developed was always determined by reconstituting the sample to 5.6% total solids and measuring optical density (O.D.) of an acetone extract at 420 mμ. Tomato solids at concentrations of 5.6 to 98% were heated for 15 min at 95°C. The O.D. increased slowly with increasing concentration to about 50% solids. Thereafter, the O.D. increased rapidly to a peak at 95% and decreased to half this value at 98%. The effect of heating time at 55, 75 and 95°C and at 5.6 and 45% solids was studied. The browning rate was slight at the lower 2 temperatures relative to that at 95°C, and was much more rapid at the higher concentration than at single strength. Under each temperature and solids condition, the rate of browning was highest at the earlier times. The inhibition effects of pH and SO₂ were studied. Browning was at a minimum at pH 2.5. Browning could be further reduced at each pH by increasing levels of SO₂. The protective effect of acidification and SO₂ addition increased with increasing solids concentration.     

Purification and Characterization of Peroxidase Isoenzymes from Green Peas (Pisum sativum)

Peroxidase isoenzymes were purified from green peas with ion-exchange chromatography on DEAE- and S-Sepharose. Three isoenzymes were identified, one neutral (N) and two cationic (C1, C2). N was extremely heat labile, with 50% original activity lost after heating 1.5 min at 25°C. N had Km values (pH 5.0) of 10.2 mM and 2.6 mM for guaiacol and H₂O₂, respectively. C1 and C2 retained activity on heating at 30–70°C. C1 was able to reactivate after thermal inactivation. Km values for guaiacol/H₂O₂ were 10.8 mM/7.2 mM (pH 5.0) and 10.8 mM/4.3 mM (pH 6.0) for C1 and C2, respectively. The three isoenzymes exhibited different peroxidase activities with different H-donors, different sensitivities to cyanide and different abilities to catalyze oxidation of indoleacetic acid.     

CHARACTERIZATION OF THE PROTEASES INVOLVED IN HYDROLYZING PADDLEFISH (POLYODON SPATHULA) MYOSIN

An extract from paddlefish surimi possessed activities of B, L, and H‐like cathepsins. The optimal pH was around 5.0 for cathepsins B and L, and was between 6.0–6.5 for the H‐like cathepsin. The enzyme activities were not impaired by heating at 40Cfor 20 min. However, the protease extract lost about 20% of its cathepsin B, 50% B+L, and 90% H‐like cathepsin activities after heating at 50C for 20 min. The activity of H‐like cathepsin was not inhibited by E‐64, suggesting that it did not belong to ike known cysteme protease group. The protease extract was capable ofhydrolyzing myosin heavy chain, producing a major fragments) around 140 kDa. Degradation of myosin by the protease extract was substantially reduced by protease inhibitors including E‐64, a protease inhibitor mixture, and bovine plasma powder.     

Core-Shell Biopolymer Nanoparticles Produced by Electrostatic Deposition of Beet Pectin onto Heat-Denatured β-Lactoglobulin Aggregates

ABSTRACT:  The purpose of this study was to produce and characterize core-shell biopolymer particles based on electrostatic deposition of an anionic polysaccharide (beet pectin) onto amphoteric protein aggregates (heat-denatured β-lactoglobulin [β-lg]). Initially, the optimum conditions for forming stable protein particles were established by thermal treatment (80 °C for 15 min) of 0.5 wt%β-lg solutions at different pH values (3 to 7). After heating, stable submicron-sized ( d = 100 to 300 nm) protein aggregates could be formed in the pH range from 5.6 to 6. Core-shell biopolymer particles were formed by mixing a suspension of protein aggregates (formed by heating at pH 5.8) with a beet pectin solution at pH 7 and then adjusting the pH to values where the beet pectin is adsorbed (< pH 6). The impact of pH (3 to 7) and salt concentration (0 to 250 mM NaCl) on the properties of the core-shell biopolymer particles formed was then established. The biopolymer particles were stable to aggregation from pH 4 to 6, but aggregated at lower pH values because they had a relatively small ζ-potential. The biopolymer particles remained intact and stable to aggregation up to 250 mM NaCl at pH 4, indicating that they had good salt stability. The core-shell biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components or as substitutes for lipid droplets.     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Sodium lactate influences myoglobin redox stability in vitro

Injection-enhancement of beef with lactate improves color stability; however, the mechanism is unclear. Thus, our objectives were to assess the effects of sodium lactate on equine myoglobin redox stability in vitro. Oxymyoglobin at pH 5.6 (50 mM sodium citrate) and pH 7.4 (50 mM sodium phosphate) was incubated at 4 °C with lactate (0, 5, 10, 100, or 200 mM) and myoglobin redox form was determined using absorbance spectra. Metmyoglobin formation at pH 5.6 and 7.4 was significantly (P < 0.05) decreased by lactate at concentrations of 100 and 200 mM. In general, increasing lactate concentration from 100 to 200 mM increased (P < 0.05) oxymyoglobin redox stability. This effect of lactate on myoglobin redox stability could be partially responsible for the improved color stability associated with lactate injection-enhanced beef products. Further work should determine the effect of lactate on beef myoglobin.