Thermolabile Fraction Research Articles

Thermal inactivation kinetics of partially purified mango pectin methylesterase

Kinetic parameters of thermal inactivation of pectin methylesterase (PME) in a partially purified mango enzyme extract were determined. The PME of mango partially purified by salting out showed different patterns of thermal inactivation, indicating the presence of a thermostable fraction at 70 °C and a thermolabile fraction at lower temperatures. The inactivation of the thermostable fraction exhibited a linear behavior that yielded a z-value of 9.44 °C and an activation energy (Ea) of 245.6 kJ mol-1 K-1 using the Arrhenius model. The thermostable mango PME fraction represented 17% of total crude enzyme extract, which emphasizes the importance of residual enzyme activity after heat treatment.

Effect of ohmic heating of soymilk on urease inactivation and kinetic analysis in holding time.

To verify the effect of the ohmic heating on the urease activity in the soymilk, the ohmic heating methods with the different electrical field conditions (the frequency and the voltage ranging from 50 to 10 kHz and from 160 to 220 V, respectively) were employed. The results showed that if the value of the urease activity measured with the quantitative spectrophotometry method was lower than 16.8 IU, the urease activity measured with the qualitative method was negative. The urease activity of the sample ohmically heated was significantly lower than that of the sample conventionally heated (P < 0.01) at the same target temperature. It was concluded that the electrical field enhanced the urease inactivation. In addition, the inactivation kinetics of the urease in the soymilk could be described with a biphasic model during holding time at a target temperature. Thus, it was concluded that the urease in the soymilk would contain 2 isoenzymes, one is the thermolabile fraction, the other the thermostable fraction, and that the thermostable isoenzyme could not be completely inactivated when the holding time increased, whether the soymilk was cooked with the conventional method or with the ohmic heating method. Therefore, the electric field had no effect on the inactivation of the thermostable isoenzyme of the urease.

Perennially and annually frozen soil carbon differ in their susceptibility to decomposition: Analysis of Subarctic earth hummocks by bioassay, XANES and pyrolysis

Earth hummocks are the most common patterned ground feature in the Subarctic region and subduction of organic matter by cryoturbation (i.e., mixing of soil layers by repeated freezing–thawing) physically protects soil organic carbon (SOC) from decomposition. Climate models predict that Subarctic regions will experience disproportionate rates of warming, which may accelerate rates of decomposition of stored SOC and thus cause increased release of CO2. Our objectives in this study were to characterize and develop relationships between the chemistry and bioavailability of SOC in the horizons of Subarctic earth hummocks. Biodegradability was assessed in a controlled mineralization study in the laboratory. The chemical composition of soil organic matter (SOM) was characterized by X-ray absorption near-edge structure (XANES) spectroscopy at the carbon (C) K-edge and its thermal stability was determined by Rock-Eval pyrolysis.The mineralization bioassay showed that buried organic horizons were less susceptible than surface SOM to biodegradation, and not significantly more susceptible than the adjacent mineral soil. Analysis by XANES showed the accumulation of ketones in buried organic horizons, and the loss of carbohydrate, phenolic and carboxylic compounds. This suggests that ketones can be used as biomarkers for microbially transformed SOM. In contrast, SOM in perennially frozen mineral soils (i.e., below the permafrost table), was more susceptible to biodegradation than that in buried mineral and organic soils in the annually frozen active layer. The SOM in these horizons did not show ketone signals but instead showed strong phenolic content. Analysis by pyrolysis indicated that the thermolabile fraction was related to the bioavailability of C, and that in perennially frozen soils, this fraction contained proportionately higher oxygen-containing functional groups. These results point to a pool of labile SOC, relatively rich in phenolic compounds, in perennially frozen soils which may be susceptible to decomposition in a warming climate. Future warming-induced C losses, therefore, may mostly occur not from annually-frozen SOM buried by cryoturbation, but from perennially-frozen C made accessible by falling permafrost table.

Development of enzymic time-temperature integrators with rapid detection for evaluation of continuous HTST pasteurization processes

The assessment of the thermal process impact in terms of food safety and quality is of great importance for process evaluation and design. This can be accomplished from the analysis of the residence time and temperature distributions coupled with the kinetics of thermal change, or from the use of a proper time-temperature integrator (TTI) as indicator of safety and quality. The objective of this work was to develop and test enzymic TTIs with rapid detection for the evaluation of continuous HTST pasteurization processes (70–85 °C, 10–60 s) of low-viscosity liquid foods, such as milk and juices. Enzymes peroxidase, lactoperoxidase and alkaline phosphatase in phosphate buffer were tested and activity was determined with commercial reflectometric strips. Discontinuous thermal treatments at various time-temperature combinations were performed in order to adjust a first order kinetic model of a two-component system. The measured time-temperature history was considered instead of assuming isothermal conditions. Experiments with slow heating and cooling were used to validate the adjusted model. Only the alkaline phosphatase TTI showed potential to be used for the evaluation of pasteurization processes. The choice was based on the obtained z-values of the thermostable and thermolabile fractions, on the cost and on the validation tests.

Contribution to characterisation of biochar to estimate the labile fraction of carbon

Different analytical techniques were used to find the most reliable and economic method for determining the labile fraction of C in biochar. Biochar was produced from pine, poplar and willow (PI, PO and WI, respectively) at two temperatures (400 and 550 °C) and characterised using spectroscopic techniques [solid state 13C nuclear magnetic resonance spectroscopy (NMR)], molecular markers [pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS)], thermogravimetry (TG), elemental composition and wet oxidation (potassium permanganate and potassium dichromate). Short term incubation (110 h) of an A horizon from an Umbrisol amended with the biochar samples at two doses (7.5 and 15 t ha −1) was also carried out to provide supplementary information on the influence of biochar–soil interaction on CO 2 evolution. Spectroscopic analysis demonstrated that the degree of biochar carbonisation was influenced by the type of feedstock and heating conditions and followed the order WI-400 < PI-400 ∼ WI-550 ∼ PO-400 < PO-550 < PI-550. The thermo-labile fraction of the biochar samples, estimated from TG, ranged between 21% and 49%. The fraction of total C oxidised with potassium permanganate (C per/C total) was <50 g kg −1 in all cases, whereas potassium dichromate (C dichro/C total) oxidation efficiency ranged between 180 and 545 g kg −1. For each type of feedstock, the highest values of either chemically or thermally degradable C corresponded to the biochar produced at low temperature. Results indicate that low cost methodologies, such as dichromate oxidation and TG, reflected the degree of biochar carbonisation, and could therefore be used to estimate the labile fraction of C in biochar.

Calorimetric characterization of grass during its decomposition

Grass is cultivated for bioenergy purposes yet and expected to play a more prominent role as a source of renewable energy in the future. Understanding its burning characteristics is thus crucial to optimize the energetic efficiency. The aim of this study was to reveal the effect of i) microbial decomposition and ii) enzymatic depolymerisation of grass on its thermal behaviour. Thermal characteristics of grass during the course of aerobic decomposition and of fresh samples after treatment by enzymatic hydrolysis were measured by differential scanning calorimetry (DSC). Heat of reaction, 50% burnoff values and positions and areas of peaks were taken as indicators for the thermal behaviour. An increase in biochemical stability of the grass during 140 days of decomposition was indicated by an exponential mass loss and decreasing specific CO2 respiration rates. Parameters representing the thermal stability of the materials coincided with this pattern. The relative contribution of the thermolabile fraction decreased, and 50% burnoff values increased almost steadily during decomposition from 366 to 407°C. These thermal stability parameters are thus indicators for biochemical degradation. However, peak temperatures of the individual peaks continuously decreased during decomposition. A comparison of composted and fresh, cellulase-treated samples without long-term microbial decomposition indicated that enzymatic depolymerisation rather than microbial consumption of the plant tissue led to the observed decrease in peak temperatures.

Improved kinetic modeling of the early generation of CO 2 from the Boom Clay kerogen. Implications for simulation of CO 2 production upon disposal of high activity nuclear waste

A new kinetic model accounting for the generation of “early” CO 2 from kerogen within the Boom Clay Formation (Oligocene, Belgium) was developed and calibrated using a large set of experimental pyrolysis data. The model also was constrained by the present day temperature of the clay where no CO 2 generation is assumed. The kinetic scheme involved three competitive reactions characterized by relatively low activation energies, i.e., E a < 35 kcal/mol (146.5 kJ/mol) with an associated gas potential of ca. 49 mg of CO 2 per gram of organic carbon. Focusing on the fastest reaction specifically, this study showed that the earliest stage of CO 2 formation is associated with the thermal decomposition of moieties containing activated carboxylic acid functions present in the thermolabile fraction of the kerogen. Considering the temperature elevation within the Boom Clay Formation that would be imposed by the presence of a nuclear waste repository, the kinetic model predicts that (i) CO 2 release would start about 1 year after disposal, and might reach the limits of the clay layer after about 100 years and (ii) for each meter length of gallery, the absolute cumulative mass of CO 2 generated in a radius of 50 m around the gallery might reach ca. 3 tons.

Changes in litter properties during decomposition: A study by differential thermogravimetry and scanning calorimetry

To verify the paradigm that organic matter (OM) quality ( q) decreases with decomposition it is necessary to define q in strictly chemical, operational terms. We suggest defining q as the result of a balance between the energy stored in OM and the external supply of energy needed to release it. We apply this concept to the study of litter decomposition in four European pine forests: boreal, cool Atlantic, Mediterranean and warm Atlantic. Intact litter cores were taken and transported to the laboratory, where needles were sorted into six classes that summarize the main facts of the decomposition: melanisation, fragmentation and perforation by mesofauna. Each class was analyzed by both differential thermogravimetry and differential scanning calorimetry to obtain its spectra of weight loss and energy release. In the non-decomposed needles, two peaks of weight loss and energy release appear: a labile peak at about 350 °C, and a recalcitrant peak at about 450 °C. During decomposition, both peaks (but especially the recalcitrant one) move to lower temperatures, and their shapes change from well defined to flattened. In Mediterranean litters, a third peak appears at about 500 °C, due probably to refractory products of neoformation. There is a continuous increase in the energy stored in the remaining litter (in Joules per unit OM): this increase is concentrated in both the most thermolabile fractions (lost at temperatures <350 °C) and the most thermostable ones (>450 °C). With decomposition OM becomes more recalcitrant (i.e., it is lost at higher temperatures), but its stored energy becomes more available (i.e., it is released at lower temperatures). Overall, the energetic benefit/cost ratio increases. Thus, our results to date do not agree with the current paradigm that q decreases with decomposition; rather, they suggest that, at least in the first phases we studied, q is maintained or even increases.

Association of metals (mercury, cadmium and zinc) with metallothionein-like proteins in storage organs of stranded dolphins from the Mediterranean sea (Southern Italy).

Selected toxic and essential metals (mercury, Hg; cadmium, Cd; and zinc, Zn) were determined in the liver and in the kidney collected from 13 dolphins (Stenella coeruleoalba and Tursiops truncatus) stranded along the southern coasts of Italy from 1991 to 1999. Liver samples were also analysed for their methyl mercury (MeHg) and selenium (Se) contents. For subcellular fractionation, liver and kidney samples were homogenized in Tris-HCl buffer; after centrifugation, the supernatant (cytosol) was separated from pellets (insoluble fraction), heated at 80 degrees C for 10 min and centrifuged in order to separate the precipitate containing the thermolabile high molecular weight proteins. The cytosol heat-stable fraction, including metallothionein-like proteins (MTLPs), was then purified by gel filtration chromatography on Sephadex G-75 column. The three subcellular fractions collected (insoluble fraction, thermolabile fraction and purified heat-stable fraction) were analysed for their Hg, Cd and Zn contents. The analytical results confirm previous similar studies on toothed whales in showing that: (1) in the liver, as well as in the kidney, Hg was mainly found in the insoluble fraction, therefore, metallothioneins seem to have no role in the Hg detoxification; (2) in the liver, the molar ratio between Se and inorganic Hg was very close to 1; this suggests that the final compound of MeHg detoxification could be HgSe (tiemannite); (3) in almost all the samples, Cd and Zn were detected in the purified heat-stable fraction (including MTLPs). The mechanism of Cd detoxification and Zn homeostasis is also discussed.

Occurrence and nature of thermolabile compounds in the Boom Clay kerogen (Oligocene, underground Mol Laboratory, Belgium)

The Boom Clay Formation has been selected as a model for studying the long term disposal of high activity nuclear waste in clay. During the resulting storage, the immature kerogen in the clay would be subjected to thermal stress and some of the products which could be generated might affect the efficiency of the geological barrier. The kerogen isolated from a representative clay sample, collected in the underground laboratory of the Nuclear Energy Research Centre at Mol (Belgium), was subjected to short heating at 300 °C to examine the thermolabile fraction released. The fraction contains a wide variety of components including hydrocarbons, ketones, alkanoic acids, phenols, furans and pyrroles. Information about the origin of these compounds was obtained through comparison with the bitumen and with the previously examined pyrolysis products generated from the kerogen at higher temperature (400 °C). The thermolabile fraction appears to correspond to “free” compounds trapped in the macromolecular structure of the kerogen and “labile” moieties linked by covalent bonds with a relatively low thermal stability. The study affords additional information on the sources and composition of the kerogen. Furthermore, the thermolabile components in the Boom Clay kerogen might affect the effectiveness of the clay barrier when subjected to thermal stress, especially through the release of oxygen-containing products, like carboxylic acids and phenols, that might complex released radioactive elements.

High Temperature Stabilization of DNA in Complexes with Cationic Lipids

The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100°C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T m(labile), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T m(stable), at 105 to 115°C. The parameter T m(stable) did not depend on the charge ratio, R(±). Instead, the amount of thermostable DNA and the enthalpy ratio Δ H (stable)/Δ H (labile) depended upon R(±) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(±) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(±) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130°C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.

Humic Marine Matter and Insoluble Materials in Antarctic Snow

The aim of the present work is to show that the insoluble components of marine origin are always present, together with the soluble ones, in both the coarse fraction and in the fine fraction of the marine aerosol and that they make up a non negligible part of the insoluble material in Antarctic snow. The results obtained show that a large proportion of the insoluble matter present in high altitude Antarctic snow consists of marine organic matter involved in the aerosolization process and that the thermolabile fraction (4000C), largely consisting of fulvic acids, increases with increasing altitude. The effect of distance from the sea coast is harder to demonstrate for particulate fulvic acids at least over the short distances investigated herein (150 km) and owing to the complex orography of the sample areas.

Pharmacological aspects of mouse hind-paw oedema induced by Lachesis muta rhombeata venom

Pharmacological aspects of mouse hind-paw oedema induced by subplantar injections of Lachesis muta rhombeata (LMR) venom were investigated. The oedema induced by subplantar injections of 10 to 50 ng/g of LMR venom is dose dependent, with onset, peak and duration at 30, 60 and 180 min, respectively. Subplantar injection of 30 ng/g of Bothrops jararaca (BJ) venom induced oedema that has the same intensity as 30 ng/g of LMR venom but lasts for more than 4 h suggesting different time course. Systemic effects or haemorrhage were not observed with doses less than 50 ng/g. Oedema is not due to the presence of oedematogenic amines since dialysis did not change the oedema induced by 30 ng/g of LMR venom. Part of the oedema induced by LMR venom is due to a thermolabile fraction since pre-heating the venom at 100°C for 15 min induced a significant reduction (56.19±6.8%) of the oedematogenic activity. The oedema induced by LMR venom is possibly induced by release of a pharmacological active substance at the site of injection. Histamine, arachidonate metabolites, nitric oxide and serotonin may play important roles in the oedematogenic effect of LMR venom since pre-treatment of mice with pyrilamine, indomethacin, dexamethasone, L-NAME and methysergide induced a significant reduction (49.86±10%; 51.06±5.9%; 77.66±3.6%; 73.30±6.1% and 93.77±2.8%, respectively) of the oedema formation. The present results demonstrate that the oedema induced by LMR and BJ venoms may be triggered and maintained by different pharmacological mechanisms. Since methysergide and L-NAME were the most active inhibitors of the oedema we can suggest that a link between serotonin release by the venom and a NO synthase activation may be an important step in the oedema formation induced by LMR venom.

Physico-chemical forms of storage of metals (Cd, Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from the Tunisian coast: ecotoxicological consequences

Three fish species ( Diplodus annularis, Scorpaena porcus and Scorpaena scrofa) were sampled off the coast of the industrialized area of Sfax. The distribution of Cd, Cu and Zn was examined in different fractions of both gills and liver: insoluble fraction and cytosol divided into thermolabile compounds and heat-stable compounds. In the latest fraction, quantitative analysis of metallothionein-like proteins (MTLP) was achieved by using differential pulse polarography. In the gills, the insoluble fraction was the major compartment for the storage of all three metals. In the liver, 50% Cd and Cu at least were also associated with this fraction whereas Zn was bound primarily with heat-stable compounds, a pattern which is probably due to the role of MTLP in Zn homeostasis and to the fact that Zn is a constituent element of metallothionein. According to the species, one eighth to a little more than one quarter of Cd, Cu or Zn was bound to the thermolabile fraction in the liver, probably due to the presence of numerous enzymes involved in the metabolic functions of this organ. In both gills and liver, Cd and Cu concentrations in the thermolabile fraction were increased with increasing organ burden of these metals. This observation suggests an enhancement of the toxic hazard since this fraction includes metabolic targets of metals. Statistically significant relationships have been shown between metals and MTLP concentrations in both organs. However, the use of MTLP as a biomarker has to be revised in the light of natural changes (according to age, sex, etc.) which could conceal variations due to metabolic pollution.

The value of the Emit-dau system in the examination of cadaveric urine

Randomly taken postmortem urine samples (170) were analyzed by the Emit-dau system for their barbiturate and benzodiazepine content. Of the samples, 23% and 25% were found positive for barbiturate and benzodiazepine, respectively. The percentages of the positive samples were reduced by a heating process to 9% and 11%, respectively. TLC and Emit-st were used for reference procedures. The relative high percentage (above 30%) of the urine samples analyzed exhibited elevated lysozyme activity and protein value. It was found that the disturbing proteins in the Emit-dau system contained not only endogene lysozyme but other thermolabile fractions with higher molecular weight.

Can muscle lysosomal enzymes affect muscle collagen post-mortem?

Incubation of collagen fibres with muscle lysosomal enzymes at 37°C, pH 5·5, affects significantly the thermal stability of muscle collagen. The thermolabile fraction increases and the strength of the fibres measured through thermal contraction decreases by/nearly 50% after treatment. It is concluded that the muscle lysosomal fraction contains an enzyme system which can reduce the strength of muscle collagen at pH 5·5. The reason why this potential is not exhibited post-mortem could be due to a limited release of the enzymes or the presence of enzyme inhibitors.

Specific features of the structural organization of the mitochondrial genome in RAT liver

The nature of intramolecular heterogeneity of mtDNA in the liver of white rats has been studied. The peculiarities of the melting curve, and the possibility of DNA fractionation of nucleotide compounds with hydroxylapatite (HA) column chromatography has shown the presence of sequences differing in the mean nucleotide content. A section of about 350 pairs in size repeated four times was found in the reassociation of most thermolabile fraction with a mean composition of 28% GC. These sections are well seen on the denaturation map of the recorded molecules formed in the range of temperature transition 'helix-coil'. The distance between the centers of fusible sections (in percentage of total length) is 32.5, 32, 14.0 and 21.5.

Serum Enzyme Changes in Lames with Experimentally Induced Acute Muscular Dystrophy

An experiment was performed to study some serum enzyme changes taking place in artificially fed lambs made dystrophic on a skim milk ration supplemented with α-tocopherolextracted cod liver oil. In the course of two to three weeks the lambs showed highly increased serum values of aspartate- and alanine aminotransferase (AspAT = GOT and A1AT = GPT), total lactate dehydrogenase (LDH), and creatine phosphokinase (GPK). The latter enzyme proved to be the most sensitive test of myopathies, with serum values above 1000 times the normal level. A high-grade hyaline skeletal muscle degeneration was confirmed by histology. Myocardial changes were also present. Contrary to expectations, electrophoretic separation of LDH isoenzymes in the serum of the dystrophic lambs did not reflect any increase of the prevalent muscular cathodic thermolabile fraction LDH5. The use of a so-called LDH heat stability test as an aid in clinical diagnostic work, as previously suggested, therefore does not seem to be valid in this disease. The results are discussed.

Hereditary haemolytic anaemia due to an abnormal haemoglobin (haemoglobin Kings County).

Two patients in an American Negro family with a hereditary non‐spherocytic haemolytic Heinz‐body anaemia, mesobilifuscinuria, and an abnormal haemoglobin have been studied. The absence of any abnormality in the red cell metabolism and the presence of a thermolabile fraction of the haemoglobin constituting 10 per cent of the patients’total haemoglobin make us believe that the cause of the haemolysis is the abnormal haemoglobin. Since this haemoglobin appears to differ from all the other abnormal haemoglobins described in patients having congenital non‐spherocytic haemolytic anaemia, a new name (haemoglobin Kings County) is proposed for it.

The mineralogical composition of the calcareous corpuscles of Taenia taeniaeformis

1. 1. The mineral content of calcareous corpuscles of cestodes is amorphous in vivo and consists primarily of Ca, Mg, P, and CO 2. 2. 2. The results of a series of heat and chemical transformations of isolated Taenia taeniaeformis corpuscles and synthetic mixtures suggest that the major inorganic components of the native corpuscles are in the form of an amorphous calcium phosphate and an amorphous MgCaCO 2 complex. 3. 3. The fact that still other inorganic constituents must be present is demonstrable not only from the analytical data but also from the isolation of a thermolabile fraction.