Effects of different calcium salts on properties of milk related to heat stability

Insoluble calcium salts were added to milk to increase total calcium by 30 mM, without changing properties influencing heat stability, such as pH and ionic calcium. There were no major signs of instability associated with coagulation, sediment formation or fouling when subjected to ultra high temperature (UHT) and in-container sterilisation. The buffering capacity was also unaltered. On the other hand, addition of soluble calcium salts reduced pH, increased ionic calcium and caused coagulation to occur. Calcium chloride showed the largest destabilising effect, followed by calcium lactate and calcium gluconate. Milk became unstable to UHT processing at lower calcium additions compared to in-container sterilisation.

The effect of free Ca2+ on the heat stability and other characteristics of low-heat skim milk powder

Low-heat skim milk powder (SMP), reconstituted to 25% total solids, was found to have poor heat stability. This could be improved by reducing the free Ca2+ concentration to 1.14 mm, or lower, by the addition of either Amberlite IR-120 ion-exchange resin in its sodium form or tri-sodium citrate in skim milk prior to evaporation and spray drying. Reduction in Ca2+ concentration was accompanied by increases in pH, particle size, and kinematic viscosity, and by a reduction in zeta-potential and changes in colour. In-container sterilisation of the reconstituted powder increased particle size, zeta-potential, kinematic viscosity and a* and b* values. However. Ca2+ concentration, pH and whiteness decreased. This study elucidated the importance of Ca2+ concentration and pH on heat stability of low-heat SMP, suggesting that Ca2+ concentration and pH in bulk milk are useful indicators for ensuring that spray dried milk powder has good heat stability. (C) 2009 Elsevier Ltd. All rights reserved.

Comparison of heat stability of goat milk subjected to ultra-high temperature and in-container sterilisation

Goatmilk with and without stabilizing salt was subjected to in-container and UHTsterilization. Heatstability was assessed by measuring the amount of sediment in the milk. Without stabilizing salts, goatmilk usually produced less sediment when subjected to in-containersterilization compared with UHT processing. Addition of stabilizing salts up to 12.8 mM resulted in a progressive increase in sediment for in-containersterilization. In contrast, adding stabilizing salts at 6.4 mM initially reduced sediment formation in UHT-treated milk but addition of stabilizing salts at 12.8 mM increased sediment formation. Adding stabilizing salts to goatmilk increased pH, decreased ionic calcium, and increased ethanol stability. Adding up to 2 mM calcium chloride increased sediment formation more after UHT treatment than after in-containersterilization. These results suggest that no single mechanism or set of reactions causes milk to produce sediment during heating and that the favored pathway is different for UHT and in-containersterilization processes. Poor heatstability could be induced both by increasing ionic calcium and by decreasing it. Ethanol stability is not a good indicator of heatstability for in-containersterilization, but it may be for UHTsterilization, if milk does not enter the region of poor heatstability found at low concentrations of ionic calcium.

Effect of seasonal variation on some physical properties and heat stability of milk subjected to ultra-high temperature and in-container sterilisation

Heat stability was evaluated in bulk raw milk, collected throughout the year and subjected to ultra-high temperature (UHT) or in-container sterilisation, with and without added calcium chloride (2 mM), disodium hydrogen phosphate (DSHP, 10 mM) and trisodium citrate (TSC, 10 mM). More sediment was observed following in-container sterilisation (0.24%) compared with UHT (0.19%). Adding CaCl2 made the milk more unstable to UHT than to in-container sterilisation, while adding DSHP and TSC made the milk more unstable during in-container sterilisation than to UHT processing, although TSC addition increased the sediment formed by UHT processing. Better heat stability was observed in autumn and winter than in spring and summer following UHT. However, following in-container sterilisation, samples with added stabilising salts showed significantly improved heat stability in autumn, whereas with added CaCl2, the best heat stability was observed in spring. No correlation was found between urea and heat stability. DSHP and TSC made the milk more unstable during in-container sterilisation than to UHT processing, although TSC addition increased the sediment formed by UHT processing. Better heat stability was observed in autumn and winter than in spring and summer following UHT. However, following in-container sterilisation, samples with added stabilising salts showed significantly improved heat stability in autumn, whereas with added CaCl2, the best heat stability was observed in spring. No correlation was found between urea and heat stability.

Partial purification, heat stability and kinetic characterization of the pectinmethylesterase from Brazilian guava, Paluma cultivars

Pectinmethylesterase (PME) was extracted from guava fruit (Psidium guajava L.), cultivar Paluma, by 70% ammonium sulphate saturation and partially purified by gel filtration on Sephadex G100. Gel filtration showed PME isoenzymes with different values of molecular mass. Two samples were examined: concPME (70% saturation by ammonium sulphate) and Iso4 PME (one of the isoforms from gel filtration with the greatest specific activity). Optimum pH of the enzyme (for both samples) was 8.5 and optimum temperature ranged from 75 and 85 degrees C. The optimum sodium chloride concentration was 0.15 M. The K-M and V-max ranged from 0.32 to 0.23 mg m1(-1) and 244 to 53.2 mu mol/min, respectively, for concPME and Iso4PME. The activation energies (E-a) were 64.5 and 103 kJ/mol, respectively, for concPME and Iso4PME. Guava PME, cv Paluma, is a very thermostable enzyme, showing great heat stability at all temperatures studied. (c) 2005 Elsevier Ltd. All rights reserved.

Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids. To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral changes indicated an alteration in the apparent T, of sheep liver and E. coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 degrees C to 72 +/- 1 degrees C at 20 mM serine, respectively. Using stopped flow spectrophotometry k values of (49 +/- 5)(.)10(-3) s(-1) and (69 +/- 7).10(-3) s(-1) for sheep liver and E. coli enzymes were determined at 50 mM serine. The binding of serine monitored by intrinsic fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins. However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact structure leading to increased thermal stability of the enzyme.

Effects of stabiliser addition and in-container sterilisation on selected properties of milk related to casein micelle stability

Different stabilising salts and calcium chloride were added to raw milk to evaluate changes in pH, ionic calcium, ethanol stability, casein micelle size and zeta potential. These milk samples were then sterilised at 121 °C for 15 min and stored for 6 months to determine how these properties changed. Addition of tri-sodium citrate (TSC) and di-sodium hydrogen phosphate (DSHP) to milk reduced ionic calcium, increased pH and increased ethanol stability in a concentration-dependent fashion. There was relatively little change in casein micelle size and a slight decrease in zeta potential. Sodium hexametaphosphate (SHMP) also reduced ionic calcium considerably, but its effect on pH was less noticeable. In contrast, sodium dihydrogen phosphate (SDHP) reduced pH but had little effect on ionic calcium. In-container sterilisation of these samples reduced pH, increased ethanol stability and increased casein micelle size, but had variable effects on ionic calcium; for DSHP and SDHP, ionic calcium decreased after sterilisation but, for SHMP, it remained little changed or increased. Milk containing 3.2 mM SHMP and more than 4.5 mM CaCl2 coagulated upon sterilisation. All other samples were stable but there were differences in browning, which increased in intensity as milk pH increased. Heat-induced sediment was not directly related to ionic calcium concentration, so reducing ionic calcium was not the only consideration in terms of improving heat stability. After 6 months of storage, the most acceptable product, in appearance, was that containing SDHP, as this minimised browning during sterilisation and further development of browning during storage.

Ionic calcium and pH as predictors of stability of milk to UHT processing

This paper describes the use of pH and calcium ion electrodes for investigating factors affecting the heat stability of UHT milk with added calcium chloride. Calcium chloride was added to raw milk to manipulate ionic calcium and pH to within the range that may be typically encountered in raw milk of different compositions and microbial quality. Addition of only 5 mM calcium chloride was sufficient to induce considerable changes in pH, ionic calcium and ethanol stability and alter its stability to UHT treatment. There was a strong relationship between pH decrease and increase in ionic calcium when pH was reduced, whether by addition of calcium chloride or by acidification. Calcium chloride addition was found to increase sediment formation in UHT treated milk. However, sediment could be reduced by addition of stabilizers. Those most effective were ones which decreased ionic calcium and increased pH, such as trisodium citrate and disodium hydrogen phosphate. Sediment formation following UHT treatment was only slight for milk samples whose ethanol stability was greater than 80%.

Peroxidase from peach fruit: Thermal stability

Peroxidase from peach fruit was purified 28.9-fold by DEAE-cellulose, Sephadex G-100 and hydroxylapatite chromatography. The purified enzyme showed only one peak of activity with an optimum pH of 5.0 and temperature of 40 degreesC. The calculated activation energy (Ea) for the reaction was 7.97 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 80 degreesC with a fast inactivation at 80 degreesC. PAGE of the inactivation course at 70 degreesC showed only one band of activity. Different sugars increased the heat stability of the activity in the following order: sucrose>lactose>glucose>fructose. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (10 to 40%, w/w) with the Ea for inactivation increasing with sucrose concentration from 0 to 20% (w/w). After inactivation at 70 degreesC and 75 degreesC the enzyme was able to be reactivated by up to 40% of the initial activity when stared at 30 degreesC.

Identification of optimal epitopes for Plasmodium falciparum rapid diagnostic tests that target histidine-rich proteins 2 and 3

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

Occurrence and properties of steryl ferulates and glycosides in wheat and rye

Cereal kernels are known to contain a number of minor components that possess beneficial health attributes. In this thesis rye and wheat were studied as sources of steryl ferulates and steryl glycosides and their behaviour in processing were evaluated. Further, enzymatic hydrolysis of these conjugates was studied, as well as the capacity of steryl ferulates to inhibit lipid oxidation at different temperatures. Steryl ferulates were shown to have a strong positive correlation with dietary fibre contents in milling fractions from the outer parts of the kernels obtained from a commercial scale mill. Highest contents of steryl ferulates were found in the bran in both cereals, with the content decreasing once moving towards the inner parts of the kernel. Variation in the contents of steryl ferulates was higher in wheat fractions than rye fractions. Steryl glycosides, on the other hand, had either negative or no correlation with dietary fibre, and the range of the steryl glycoside contents was much narrower than that of steryl ferulates in both cereals. There were significant differences in the sterol compositions of these steryl conjugates when compared with each other or with the total plant sterols in the corresponding fractions. Properties of steryl ferulates and steryl glycosides were evaluated after common processing methods and in enzymatic hydrolysis. Thermal and mechanical processing had only minor or no effects on the contents of steryl conjugates from rye and wheat bran. Enzymatic treatments on the other hand caused some changes, especially in the contents of glycosylated sterols. When steryl ferulates extracted from rye or wheat bran were subjected to enzymatic treatments by steryl esterase, significant differences in the rates of hydrolysis were observed between steryl ferulates from different sources with differing sterol compositions. Further, differences were also observed between enzymes from different sources. Steryl glycosides were shown to be hydrolysed by β-glucosidase (cellobiase) from A. niger, but less with β-glucosidases from other sources. Steryl ferulates showed good antioxidant activity at both moderate and high temperatures. In bulk and emulsion systems of methyl linoleate at 40°C steryl ferulates extracted from rye and wheat bran inhibited hydroperoxide formation much more effectively than synthetic steryl ferulates or those extracted from rice (γ-oryzanol), demonstrating that the sterol composition has an effect on the activity. At cooking (100°C) and frying temperatures (180°C) sitostanyl ferulate was shown to inhibit polymer formation significantly and, especially at 100°C, comparably to α-tocopherol. The rate of antioxidant degradation was slower for sitostanyl ferulate, showing higher heat stability than α-tocopherol. When evaluated as a mixture, no synergistic effect was observed between these two antioxidants. The data presented in this thesis provides information that may henceforth be applied when evaluating the intakes of steryl conjugates from cereal sources, as well as their possible influences as minor bioactive components. Wheat and rye both are good sources of steryl ferulates and steryl glycosides and, especially with steryl ferulates, what may be lost out to some other cereals on quantity is compensated with quality of the sterol composition.

Mechanical and thermal properties of high-density polyethylene toughened with glass beads

Glass beads were used to improve the mechanical and thermal properties of high-density polyethylene (HDPE). HDPE/glass-bead blends were prepared in a Brabender-like apparatus, and this was followed by press molding. Static tensile measurements showed that the modulus of the HDPE/glass-bead blends increased considerably with increasing glass-bead content, whereas the yield stress remained roughly unchanged at first and then decreased slowly with increasing glass-bead content. Izod impact tests at room temperature revealed that the impact strength changed very slowly with increasing glass-bead content up to a critical value; thereafter, it increased sharply with increasing glass-bead content. That is, the lzod impact strength of the blends underwent a sharp transition with increasing glass-bead content. It was calculated that the critical interparticle distance for the HDPE/glass-bead blends at room temperature (25degreesC) was 2.5 mum. Scanning electron microscopy observations indicated that the high impact strength of the HDPE/glass-bead blends resulted from the deformation of the HDPE matrix. Dynamic mechanical analyses and thermogravimetric measurements implied that the heat resistance and heat stability of the blends tended to increase considerably with increasing glass-bead content.