The influence of water activity on thermal stability of horseradish peroxidase

The thermal stability of horseradish peroxidase in the solid state was studied as a function of water activity, from 0.11 to 0.88. At all activities the enzyme was found to be much more stable in the solid state than in solution. Inactivation temperatures were in the range of 140–160°C. Inactivation curves show a biphasic behaviour which can be described by a model assuming two fractions (heat labile and heat stable) with independent first order inactivation kinetics. The labile fraction represents approximately 30% of the total activity. The z-value for both stable and labile fractions depends on water activity (moisture content) and has a maximum at a_w= 0.76 (44.4°C and 43.8°C, respectively).     

Analysis of the kinetic patterns of horseradish peroxidase thermal inactivation in sodium phosphate buffer solutions of different ionic strength

The thermal inactivation of horseradish peroxidase was studied in sodium phosphate buffer solutions and in pure water at pH 7 in the temperature range of 70–95°C. The sodium phosphate ions concentration affected both the thermostability and the kinetic patterns and had a stabilizing effect. The gradual change observed at low concentrations made a series-type mechanism theoretically more coherent with the experimental observations than the conventionally applied two-fraction model. In water the kinetics is apparently First order at high temperatures, while the results obtained at 25°C support the occurrence of a series-type inactivation mechanism. The pH and enzyme concentration also affect the inactivation proFile, supporting the conclusion that the thermal inactivation is not a monomolecular process with respect to protein concentration.     

Stability of immobilized thermolysin in organic solvents

Various factors affecting the stability of thermolysin immobilized by cross-linking with glutaraldehyde were elucidated, particularly in the water-immiscible organic solvents such as ethyl acetate and tert-amyl alcohol. The main reason for enzyme inactivation in water-immiscible organic solvents was found to be autolysis in the water phase, which may surround the enzyme immobilized inside the support. By contrast, in water-miscible organic solvents thermal denaturation was the predominant cause of enzyme inactivation. Courses of inactivation were expressed by second-order kinetics in the initial stage, after which inactivation proceeded at a slower rate. The extent of autolysis was found to strongly depend on the kind of organic solvent, the water content, and type of support and these dependencies were explained by the difference in the amount and state of water inside the support. Thermolysin was immobilized onto Amberlite XAD-7 as a compact aggregate inside the support which may increase the stability of the enzyme. Finally, it was shown that the stability of the immobilized enzyme could be correlated with the logP value for water-miscible organic solvents and with the solubility of water for water-immiscible organic solvents.     

Comparison of high pressure treatment and thermal pasteurization effects on the quality and shelf life of guava puree

The effects of high pressures and thermal pasteurization on the survival of microorganisms, enzyme inactivation and quality changes of guava puree during storage at 4°C were investigated and compared with untreated samples. After treatment at a pressure of 600MPa and 25°C for 15 min, the microorganisms in guava puree were inactivated to less than 10 cfu mL⁻¹ and the product exhibited no change in colour, pectin, cloud and ascorbic acid content as compared with fresh samples. The inactivation of enzymes in guava puree by thermal pasteurization was greater than by high pressures. The microbial count in guava puree reduced to 200 cfu mL⁻¹ and the product showed marked changes in viscosity, turbidity and colour when heated at 88–90°C for 24s. The content of pectin, cloud and ascorbic acid as well as colour in untreated and high pressurized (400MPa) guava puree gradually decreased, whereas these changes were not observed in pasteurized (88–90°C) and high pressurized (6000MPa) puree during storage at 4°C for 60 days. The guava puree treated at 600MPa and 25°C for 15 min retained good quality similar to the freshly extracted puree after storage at 4°C for 40 days.     

Inactivation of Pectin Methyl Esterase in Orange Juice by Pulsed Electric Fields

ABSTRACT: Pulsed electric fields (PEF) treatments were applied to nonpasteurized orange juice using a bench top PEF system to study effects of PEF on the activity of pectin methyl esterase (PME). Effects of electric strength on PME activity at a constant water bath temperature were studied using electric field strengths up to 35 kV/cm at 30 °C. Increase of electric field strength caused a significant inactivation of PME with increase in orange juice temperature ( p < 0.05). A thermal inactivation study showed that heating of orange juice at the same temperature as orange juice during PEF treatment was not effective as PEF treatment in inactivating PME. Effects of electric field strength at different water bath temperatures were studied using electric field strengths up to 25 kV/cm and water bath temperatures of 10–50 °C. Higher electric field strengths at higher water bath temperature were the more effective to inactivate PME. A combination of PEF treatment at 25 kV/cm and a water bath temperature of 50 °C caused 90% inactivation of PME.     

The proteolytic activity of the crude enzyme extracts of Baltic cod (Gadus morhua) alimentary tract

The crude enzyme extract from Baltic cod alimentary tract had maximum activity of aspartic proteinases towards haemoglobin at pH 2.0 and 3.4, and of serine proteinases with casein as the substrate at pH 8.3 and 10.4. With bovine myofibrils as the substrate the proteolysis at pH 5.0-8.0 was maximum at the lowest and highest pH values. the optimum temperature for the proteolytic activity at pH 3.4 and 10.4 was 30–45°C and at pH 8.3 it was 40–50°C. Heating the crude extract for 10 min at pH 3.4,8.3, and 10.4 in the absence of the substrate had no effect on the activity of the acid and alkaline proteinases up to about 35–40°C. the stability at higher temperature decreased gradually and total inactivation occurred at 55–60°C. In the pH range 5.5–7.5 the proteolytic activity against bovine myofibrils was low at 0°C but brought about significant loss of myosin heavy chain at 20°C.     

Development of an Enzymic Time Temperature Integrator for Sterilization Processes Based on Bacillus licheniformis α-amylase at Reduced Water Content

ABSTRACT: The thermal stability of Bacillus licheniformis α-amylase at low moisture content was studied, based on isothermal experiments performed in a temperature range 113 to 125 °C. The thermal inactivation was monitored by measuring the decrease in thermal denaturation enthalpy and/or the decrease in enzymic activity on pnitrophenyl-α-D-maltoheptaoside, or on starch as a substrate. Based on enthalpy readings, an enzymic system with a z-value of 10.4 °C was observed when using a relative humidity of 81% at 4 °C. A theoretical study showed that this system could be used as a Time Temperature Integrator (TTI) to monitor the safety of sterilization processes of numerous food products.     

Amorphous state and delayed ice formation in sucrose solutions

Phase transitions of amorphous sucrose and sucrose solutions (20–100% sucrose) were studied using differential scanning calorimetry, and related to viscosity and delayed ice formation. Glass transition temperature (Tg) was decreased by increasing water content. Ice formation and concurrent freeze concentration of the unfrozen solution increased apparent Tg. Tg could be predicted weight fractions and Tg values of components. Williams–Landel-Ferry (WLF) relation could be used to characterize temperature dependence of viscosity above Tg. Crystallization of water above Tg was time dependent, and annealing of solutions with less than 80% sucrose at –35°C led to a maximally freeze-concentrated state with onset of glass transition at –46°C, and onset of ice melting at -34°C. The state diagram established with experimental and predicted Tg values is useful for characterization of thermal phenomena and physical state at various water contents.     

A circular dichroism study of the effect of some organic solvents on the actomyosin of sardine (Sardinops melanosticta)

Circular dichroism (CD) was used to study changes in conformation of sardine ( Sardinops melanosticta ) actomyosin after treatment with some organic solvents. Untreated sardine actomyosin showed two negative bands at 208 and 222 nm, typical of proteins possessing α-helix configurations. The water-miscible alcohols methanol, ethanol, isopropanol and n-butanol, and the water-immiscible solvents n-hexane, n-octanol, and dichloromethane altered the native conformation of sardine actomyosin. The degree of alteration of native sardine actomyosin was principally dependent on the alcohol concentration and temperature used for dewatering. The least damage occurred with isopropanol, or with 10 or 90% aqueous ethanol; most damage occurred with 40–50% aqueous ethanol. Dichloromethane and n-butanol increased the α-helical content of the native protein. Treatment with hydrophilic and hydrophobic solvents at 70 and 20°C unfolded the protein to form a random coil, but defatting treatment at 2°C caused little damage.     

The aqueous extraction of glucosinolates from rapeseed

The losses of seed constituents from Brassica campestris var. Toria for various conditions of the leaching process developed at the Overseas Development Natural Resources Institute (ODNRI) were studied. Boiling seeds for 3 min at a seed/water ratio of 1:3 was sufficient to allow inactivation of the enzyme myrosinase; higher ratios did not increase losses in any of the constituents studied.
Heat treatment of the seeds (5 min in boiling water) reduced the nitrogen solubility at the native pH (6.5) from 28 to 8%. The pH had little effect on the extent of glucosinolate leaching from coarsely ground seeds and the minimum protein loss occurred close to the native pH. Increased water temperatures (40, 50 and 80°C) did not lead to an increased leaching efficiency over ambient temperature (20°C). Seed/water ratio was found to be the most important factor during leaching. Cross-current extraction over three stages at a seed/water ratio of 1:10 reduced the glucosinolate content by 98% while the crude protein loss was about 8.6%.     

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.     

Kinetics of ascorbic acid degradation in potato blanching

Degradation kinetics of ascorbic acid was investigated on potato strips wrapped in aluminium foil and blanched for 5–60 min at different temperatures between 65°C and 100°C. The results confirmed that first order kinetics is adequate in describing the degradation reaction. The value of the rate constant was found to pass through a maximum at 85–90°C, indicating the inactivation of oxidative enzymes around these temperatures. Comparison of the degradation losses based on the present investigation with results on total loss of ascorbic acid during blanching reported in the literature showed that thermal degradation accounts for a considerable portion of the total loss.     

Effect of pH on Thermal and/or Pressure Inactivation of Victoria Grape (Vitis vinifera sativa) Polyphenol Oxidase: A Kinetic Study

ABSTRACT: Thermal and/or high pressure inactivation of polyphenol oxidase (PPO), isolated from grapes (var. Victoria, South Africa) was studied as a function of pH. Temperature as well as pressure stability increased with increasing pH from 3 to 6. Both thermal (pH range, 3 to 6) and thermal-high pressure inactivation (pH = 3) of the enzyme followed 1st order kinetics. The z_t value and activation energy, both reflecting the temperature dependency of the inactivation rate constant, respectively, slightly decreased and increased with increasing pH from 3 to 6. The thermal-high pressure inactivation of Victoria grape PPO was kinetically investigated in a model system at pH 3.0. A3rd-degree polynomial model was successfully applied to describe the temperature/pressure dependence of the inactivation rate constants. Pressure and temperature were found to act synergistically, except in the high temperature (≥45°C)-low pressure (≤300 MPa) region where an antagonistic effect was observed.     

Efficacy of Pressure-Assisted Thermal Processing, in Combination with Organic Acids, against Bacillus amyloliquefaciens Spores Suspended in Deionized Water and Carrot Puree

ABSTRACT:  Effect of organic acids (acetic, citric, and lactic; 100 mM, pH 5) on spore inactivation by pressure-assisted thermal processing (PATP; 700 MPa and 105 °C), high pressure processing (HPP; 700 MPa, 35 °C), and thermal processing (TP; 105 °C, 0.1 MPa) was investigated.  Bacillus amyloliquefaciens  spores were inoculated into sterile organic acid solutions to obtain a final concentration of approximately 1.3 × 10⁸ CFU/mL.  B. amyloliquefaciens  spores were inactivated to undetectable levels with or without organic acids after 3 min PATP holding time. At a shorter PATP treatment time (approximately 2 min), the inactivation was greater when spores were suspended in citric and acetic acids than in lactic acid or deionized water. Presence of organic acids during PATP resulted in 33% to 80% germination in the population of spores that survived the treatment. In contrast to PATP, neither HPP nor TP, for up to 5 min holding time with or without addition of organic acids, was sporicidal. In a separate set of experiments, carrot puree was tested, as a low-acid food matrix, to study spore recovery during extended storage following PATP. Results showed that organic acids were effective in inhibiting spore recovery in treated carrot puree during extended storage (up to 28 d) at 32 °C. In conclusion, addition of some organic acids provided significant lethality enhancement ( P  < 0.05) during PATP treatments and suppressed spore recovery in the treated carrot puree.     

INTERRELATIONSHIP BETWEEN CERTAIN PHYSICOCHEMICAL PROPERTIES OF RICE

Water uptake at 80°C and starch-iodine blue value at 65°C, both expressed as percentage of the respective parameter at 96°C gave good inverse estimates of the gelatinization temperature of rice. The equilibrium moisture content on soaking in water was 28–29s in indica , and 30–31% in dwarf indica and japonica rice. Similarly the fraction of total amylose soluble in water (96° C) was around 50% in indica and japonica , but 35% in dwarf indica. Dwarf indica rice viscogram showed a rather high setback value. The blue value of the excess cooking water of rice gave no useful information. Varieties with less than 25% amylose gave C-type alkali reaction.     

THE EFFECT OF DEMETHYLATION PROCEDURES ON THE QUALITY OF LOW-ESTER PECTINS USED IN DESSERT GELS

Water uptake at 80°C and starch-iodine blue value at 65°C, both expressed as percentage of the respective parameter at 96°C gave good inverse estimates of the gelatinization temperature of rice. The equilibrium moisture content on soaking in water was 28–29s in indica , and 30–31% in dwarf indica and japonica rice. Similarly the fraction of total amylose soluble in water (96° C) was around 50% in indica and japonica , but 35% in dwarf indica. Dwarf indica rice viscogram showed a rather high setback value. The blue value of the excess cooking water of rice gave no useful information. Varieties with less than 25% amylose gave C-type alkali reaction.     

Rennin-like milk coagulant enzyme produced by a local isolate of Mucor

Among 20 isolates of Mucor isolated from various environments in Jordan and found to produce a rennin-like acid protease, known as Mucor rennin-like enzyme (MRE), Mucor J20 was found to produce the highest level of MRE. The optimum incubation conditions for enzyme production in a fortified wheat bran mixture using solid-state fermentation were 3–4 days at 30°C. The highest MRE activity (185–200 rennin units or RU) was produced in a medium containing wheat bran and lentil straw (1 : 1 w/w) moistened with whey, and incubated in clay pots at 30°C for 4 days. A slightly lower activity value (178 RU) was found when using a mineral salt solution or distilled water instead of whey, or when using wheat bran alone with whey. At pH 4, the MRE retained its complete activity (100%) for 6 weeks at 5°C and 10°C, and for 3 and 2 weeks at 20°C and 30°C, respectively. After heating at 60°C for 10 min, the enzyme lost its activity at all pH levels used (pH 2–8). The crude extract of MRE was successfully applied in the manufacture of a cheese curd.     

Extraction of ginger rhizome: kinetic studies with supercritical carbon dioxide

The rates of extraction of [6]-gingerol from ground dried Jamaican ginger rhizomes were determined in supercritical carbon dioxide over the pressure range 128–197 bar (1850–2850 psi) and the temperature range 50–65°C. The CO₂ densities varied from 0.415 to 0.775 g cm⁻³. First-order plots showed two approximately linear sections with an initial intercept. The first linear section corresponded to a relatively fast initial extraction stage while the last 20% or so of the gingerol was extracted in the much slower subsequent stage. Reducing the ginger particle size by a factor of 3 increased the rate constant of the fast stage nine-fold. Despite the low viscosity of the supercritical fluid, the rate constants of the fast stage were much smaller than in extractions with organic solvents. Only when the CO₂ density was increased to 0.775 g cm⁻³ did the rate constant of the fast stage rise to a value comparable with, but still smaller than, those in organic solvents of similar density. The rate constants of the later slow extraction stage were also somewhat smaller than the corresponding values in organic solvents at 30°C.     

Inactivation of Shigella boydii 18 IDPH and Listeria monocytogenes Scott A with power ultrasound at different acoustic energy densities and temperatures

ABSTRACT:  The effect of acoustic energy density (AED) on inactivation of Shigella boydii 18 IDPH and Listeria monocytogenes Scott A in a cell suspension was studied at sublethal temperatures and at AEDs of 0.49, 0.85, and 1.43 W/mL. The effect of temperature on ultrasonic inactivation of L. monocytogenes Scott A at 35, 50, and 65 °C was examined at an AED of 1.43 W/mL. Increasing AED increased the rate of inactivation for both S. boydii and L. monocytogenes . The destruction of S. boydii and L. monocytogenes followed 1st order kinetics in a 20-min treatment, except for S. boydii inactivation at 1.43 W/mL where a tailing effect was observed after 15 min. At sublethal temperatures, the D-values of S. boydii were 8.8, 4.3, and 2.5 min for AEDs of 0.49, 0.85, and 1.43 W/mL, whereas those for L. monocytogenes at the 3 AED levels were 31.5, 13.5, and 7.3 min, respectively. Ultrasonic treatment of L. monocytogenes at 35 and 50 °C enhanced inactivation. However, at 65 °C, application of ultrasound did not result in additional inactivation compared to thermal treatment alone at the same temperature. With the experimental conditions and the ultrasound system used in this study, an upper temperature limit for thermosonication was evident above which no added killing due to ultrasound was observed.     

Enhanced deactivation of bacterial lipases by a modified UHT treatment

Lipase was produced by three non-fluorescent pseudomonads during culturing in resterilized UHT whole milk at 10°C. The enzymes exhibited pronounced thermostability in milk with 85–88 and 82–89% of the original activities retained after heat treatments at 140°C for 5 sec and 80°C for 10 min, respectively. Also, after a single treatment at 60°C for 10 min approximately 95% of the untreated activities remained.
Double heat treatments consisting of either 130 or 140°C for 5 sec followed by 60–80°C for 3 min enhanced lipase deactivation by as much as 40% compared with the combined effect of both higher and lower temperature treatments performed on separate enzyme samples. No significant enhancement of deactivation was noted when samples were heated at 60°C for 3 min followed by 140°C for 5 sec compared with the latter treatment alone.
Lipase deactivation was non-linear at 60°C following treatment at 140°C for 5 sec; no substantial additional loss of activity occurred at the lower temperature between 5 and 10 min.