Purification and kinetic characterization of polyphenol oxidase from Barbados cherry (Malpighia glabra L.)

Polyphenol oxidase (PPO) of Barbados cherry was extracted and purified through ammonium sulfate precipitation, gel filtration, and affinity chromatography. The purification factor for PPO was 60% with 8.3% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of PPO was approximately the sum of 52 and 38 kDa estimated by SDS–PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 7.2. The enzyme had a temperature optimum at 40 °C and was relatively stable at 60 °C, with 55% loss of activity. Sodium diethyl dithiocarbamate (SDDC), l-cysteine and ascorbate significantly inhibited PPO activity. 4-Methyl catechol and catechol were found to be efficient diphenolic substrates for cherry PPO, considering the _Vmax/_Km$V_{\text{max}}/K_{\mathrm{m}}$ ratio. The data obtained in this study may help to understand cherry fruit browning.     

Heat acclimation and the role of RpoS in prolonged heat shock of Escherichia coli O157

Escherichia coli, a commensal mesophile that primarily inhabits the gastro-intestinal tract, responds to temperature up-shifts with transient expression of stress-response proteins. The goal of this study was to identify adaptive proteins of E. coli O157 crucial for growth resumption of this human pathogen after heat shock, with specific focus on the role of the RpoS sigma factor. Using the comparative proteomic analysis of hyper-thermally acclimatized wild-type strain B-1 and rpoS-mutant strain SV521, we identified 39 proteins that underwent significantly-different induction upon temperature shock at 45°C or rpoS mutation. All identified proteins of the heat post-acclimation stimulon fell into two large sub-groups: (i) stress proteins, including molecular chaperons, proteases, DNA/RNA stabilizing enzymes, and anti-oxidant proteins, and (ii) housekeeping proteins. It was found that in the heat stress stimulon RpoS has significantly (P=0.012) limited control over the key stress proteins involved in translation, translational elongation, protein folding and refolding. However, RpoS showed a significant (P=0.035) control over the cellular metabolic processes that included NADPH regeneration, pentose-phosphate shunt, nicotinamide nucleotide and NADP metabolic processes, reflecting its specific importance in promoting resource utilization (energy, protein synthesis etc.) during proliferation of hyperthermally-adapted cells. Pathogenic strains, like E. coli O157, have the ability to survive a variety of harsh stress conditions, leading to their entry into the food chain, and subsequent pathogenesis. This research offers insights into the physiological response of this pathogen during the critical period following adaptation to thermal stress and subsequent resumption of growth.     

Natural plant enzyme inhibitors. Isolation and characterisation of a trypsin/chymotrypsin inhibitor from indian red wood (Adenanthera pavonia) seeds

A trypsin/chymotrypsin inhibitor was isolated from Indian red wood seeds by extraction with 0.01m HCl, chromatography on diethyl amino ethyl-cellulose, ammonium sulphate fractionation and gel chromatography on Sephadex G-100. The homogeneity of the final product was ascertained by affinity chromatography on trypsin sepharose and chromatography on phenyl sepharose CL-4B columns. During all stages of purification and characterisation the ratio of activities against trypsin and chymotrypsin remained constant at about 1.1:1 indicating that the same factor is responsible for both activities. The size of the inhibitor was found to be 24 000 daltons based on gel chromatographic studies on Sephadex G-100 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molar ratio of interaction between the inhibitor and bovine trypsin for complete inactivation of the enzyme was found to be 1.04:1. Electrophoretic and gel chromatographic studies indicated that the purified inhibitor is capable of undergoing aggregation to form dimers and trimers. Even in the presence of sodium dodecyl sulphate and sodium dodecyl sulphateurea, this phenomenon was discernible. The binding sites on the inhibitor for trypsin and chymotrypsin were not mutually exclusive, based on the data from mixed enzyme studies and on analysis of the inhibitor-enzyme complexes by gel chromatography on Sephadex G-100. Modification of the arginyl residues of the inhibitor resulted in the loss of more of the antitryptic activity than of antichymotryptic activity. Conversely, modification of amino groups resulted in the loss of more of the antichymotryptic activity. The inhibitor was stable to exposure to a wide range of pH (1.0–12.0), but it was completely inactivated on heat-treatment at 100°C for 15 min. The mode of inhibition of trypsin as well as chymotrypsin was non-competitive and K_i values for the inhibitor were 2.92 × 10-¹⁰M and 4.46 × 10-¹⁰M , respectively, for the two enzymes.     

Preparation and characterization of porous polyester fibres by salt leaching method

Porous polyester fibres with cotton-like dull characteristics were produced by salt leaching method. Polyester was synthesized in the presence of common salt. Fibres containing submicron size salt particles (0.5–1%) were prepared by melt spinning. The fibres were washed in hot water to remove the salt particles creating small craters of size 50–1000?nm. Partially oriented yarn (POY) and fully drawn yarn (FDY) were produced. POY samples were textured to produce polyester textured yarn containing salt particles. The influence of salt particles on polymer crystallization was investigated and found that the presence of salt significantly influences the rate of crystallization. The gloss values of porous FDYs were compared with standard semi-dull polyester yarns of same denier. The light scattering behaviour of the yarn has been enhanced due to porosity. This resulted in a fibre with better dullness characteristics. The light reflectance of porous yarn is lower in the 400–700?nm region compared to control FDY samples. The dyeability of the porous fibre was better than non-porous counterpart.     

Synergistic antifungal effects of thyme essential oil and Lactobacillus plantarum cell‐free supernatant against Penicillium spp. and in situ effects

This study evaluates the synergistic antifungal effects between thyme essential oils and Lactobacillus plantarum cell‐free supernatant (LCFS) against Penicillium spp. and in situ antifungal activity in rice grains. Thyme essential oil and LCFS showed remarkable antifungal activities against Penicillium spp. with the minimum inhibitory concentration (MIC) of 40 and 80 µL/mL, respectively. The analysis of fractional inhibitory concentration (FIC) index showed the antifungal synergism between thyme essential oil and LCFS against Penicillium spp. with FIC index of 0.5. This synergism also resulted in fourfold reduction in their MICs when applied in combination. The antifungal modes of action were characterized by observing the changes in cell membrane permeability and degradation of fungal cell wall. The combination of thyme essential oil and LCFS (2 × MIC of each) showed remarkable in situ antifungal effect and completely inhibit the growth of Penicillium in rice seeds. The results suggested the possible applications of the observed synergism on actual crops.

Practical applications

Essential oils are used as preservative in food industry and high concentration of essential oil is associated with negative organoleptic characteristics. This study presented a novel approach for synergistic antifungal effects by using the combination of thyme essential oil and Lactobacillus plantarum cell‐free supernatant (LCFS) against Penicillium spp. and systematic evaluation of the antifungal effect by using fractional inhibitory concentration (FIC) index method. This approach will be a role model for future research on synergism and overcome the major drawbacks of using live bacteria and the negative effects arising from antimicrobial activities of essential oils.     

A suboptimal controller for minimun sensitivity of closed loop eigenvalues to parameter variations

The design of a suboptimal controller for linear time invariant multivariable systems, which assigns the closed loop eigenvalues at desired locations and minimizes their sensitivity with respect to plant parameters, is outlined. The dominant open loop poles are shifted in groups employing a dyadic structure of state feedback for each group. Freedom available in choosing the constants of proportionality of the different rows of the dyadic matrices is used for eigenvalue sensitivity minimization. Since the final controller matrix is the sum of different dyadic matrices, the sensitivity of the final closed loop poles can be made less than that obtained by shifting all the dominant eigenvalues in one stage using unity rank feedback. An example illustrating the design method is given.     

Photoelastic Studies on Rubber-to-Rubber Joints

Photoelasticity is a method which yields information on the principal stress difference and orientation in a composite structure. Various problems associated with this technique, especially those concerning the fundamental relationship between the fringe order and the stress, have yet to be investigated. A few studies of this relationship in a universal stress state have been presented, particularly in the field of rubber-to-metal and rubber-to-fabric composites, but no evaluations have so far been made in the field of rubber-to-rubber joints. Applying the photoelastic method, we report our observations on the stress distribution in a natural rubber to natural rubber joint subjected to uniaxial tension. A comparison between the results of experimental photoelastic studies and the corresponding computer modelling has been illustrated. The theoretical displacement pattern of the angular joints of bi-rubber part has also been highlighted.     

Viscosity-molecular weight concentration relationships beyond the critical region in polymer solutions and melts

A multivariate regression analysis of viscosity-molecular weight-concentration data is presented which is superior to the methods currently used. Four sets of data from the literature are analysed to show the efficacy of the method.