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Virgin olive oil (VOO), characterized by its unique aroma, flavor, and health benefits, is subject to adulteration with the addition of oils obtained from other edible species. The consumption of adulterated olive oil with nut species, such as hazelnut or almond, leads to health and safety issues for consumers, due to their high allergenic potential. To detect almond and hazelnut in olive oil, several amplification systems have been analyzed by qPCR assay with a SYBR Green post-PCR melting curve analysis. The systems selected were Cora1F2/R2 and Madl, targeting the genes coding the allergenic protein Cor a 1 (hazelnut) and Pru av 1 (almond), respectively. These primers revealed adequate specificity for each of the targeted species. In addition, the result obtained demonstrated that this methodology can be used to detect olive oil adulteration with up to 5% of hazelnut or almond oil by a single qPCR assay, and with a level as low as 2.5% by a nested-qPCR assay. Thus, the present research has shown that the SYBR-based qPCR assay can be a rapid, precise, and accurate method to detect adulteration in olive oil.

The aim of the work was to use an effluent with high chemical oxygen demand (COD) from small brewery as substrate for the production of Chlorella vulgaris biomass. A non-axenic strain from a Patagonian river was employed, and the effects of COD and pH value on the microalga growth were studied through a central composite design. A medium with COD 18300 mg O2 mL−1 and initial pH: 6.5 optimized microalga growth. With the optimal condition, C. vulgaris adapted rapidly to medium and stationary phase was attained at 75 h. Moreover, lack of illumination did not affect μmax, neither final biomass concentration, while supplementation with BG11 enhanced the biomass productivity at 0.47 ± 0.07 g L−1 h−1, and pigment contents at least four times respect to the heterotrophic mode. For the first time, a brewery wastewater with high COD could be successfully used as substrate for C. vulgaris production, without presenting inhibitions, which represents a significant advance contributing toward more sustainable promising perspectives. Fil: Lois Milevicich, Julieta. Universidad Tecnológica Nacional. Facultad Regional Buenos Aires; Argentina Fil: Casá, Nahuel Esteban. Universidad Tecnológica Nacional. Facultad Regional Buenos Aires; Argentina Fil: Alvarez, Paola. Universidad Tecnológica Nacional. Facultad Regional Buenos Aires; Argentina Fil: Mateucci, Ricardo Rafael. Universidad Tecnológica Nacional. Facultad Regional Buenos Aires; Argentina Fil: Busto, Víctor Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina. Universidad Tecnológica Nacional. Facultad Regional Buenos Aires; Argentina Fil: de Escalada Pla, Marina Francisca. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias. Instituto de Tecnología de Alimentos y Procesos Quimicos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Tecnología de Alimentos y Procesos Quimicos.; Argentina. Universidad Tecnológica Nacional. Facultad Regional Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina

The behavior against temperature and thermal stability of enzymes is a topic of importance for industrial biocatalysis. This study focuses on the kinetics and thermodynamics of the thermal inactivation of Lipase PS from B. cepacia and Palatase from R. miehei. Thermal inactivation was investigated using eight inactivation models at a temperature range of 40–70 °C. Kinetic modeling showed that the first-order model and Weibull distribution were the best equations to describe the residual activity of Lipase PS and Palatase, respectively. The results obtained from the kinetic parameters, decimal reduction time (D and tR), and temperature required (z and z’) indicated a higher thermal stability of Lipase PS compared to Palatase. The activation energy values (Ea) also indicated that higher energy was required to denature bacterial (34.8 kJ mol−1) than fungal (23.3 kJ mol−1) lipase. The thermodynamic inactivation parameters, Gibbs free energy (ΔG#), entropy (ΔS#), and enthalpy (ΔH#) were also determined. The results showed a ΔG# for Palatase (86.0–92.1 kJ mol−1) lower than for Lipase PS (98.6–104.9 kJ mol−1), and a negative entropic and positive enthalpic contribution for both lipases. A comparative molecular dynamics simulation and structural analysis at 40 °C and 70 °C were also performed.

The behavior against temperature and thermal stability of enzymes is a topic of importance for industrial biocatalysis. This study focuses on the kinetics and thermodynamics of the thermal inactivation of Lipase PS from

Monoacylglycerols (MAG) containing polyunsaturated fatty acids (PUFA) have been synthesized by glycerolysis of anchovy oil using Lipozyme RM-IM in a solvent-free system. The experimental conditions, reaction temperature, substrate molar ratio (glycerol/triacylglycerol, Gly/TAG) and enzyme concentration were studied. A response surface methodology was employed to analyze the effect of the individual variables on MAG production and oxidative stability with respect to induction time. The operating conditions that simultaneously optimized MAG yields and oxidative stability were a reaction temperature of 40 °C, a Gly/TAG molar ratio of 2:1 and 6% enzyme load. The yield obtained was 20.34% of MAG (with 12.12% of 2-MAG) and an induction time of 1.88 h.

Resumen: El objetivo de esta investigación fue optimizar la inmovilización de naringinasa de Penicillium decumbens mediante atrapamiento en criogeles de alcohol polivinílico y polietilenglicol. En primer lugar, se determinó el efecto de las características de los componentes de la matriz sobre la eficacia de inmovilización y la estabilidad mecánica de los biocatalizadores obtenidos. Se empleó la herramienta de diseño de experimentos y superficie de respuesta para analizar el efecto del pH (3,0-8,0), de las concentraciones de alcohol polivinílico (5,3-8,7%) y polietilenglicol (7,0-12,0%) sobre la eficacia de inmovilización, y para optimizar estos parámetros. Adicionalmente, se estudió la capacidad de reutilización de los inmovilizados en zumos sintéticos. Se consiguieron biocatalizadores inmovilizados que conservaban un 45-47% de la enzima activa y que, tras tres ciclos de reutilización en zumo sintético, mantenían un 54,3% de la actividad inicial. Los resultados obtenidos mostraron que las condiciones de inmovilización afectaban a la eficacia de inmovilización y a la estabilidad operativa de la naringinasa atrapada.

In this study, the use of spray-drying technology for encapsulating Flavourzyme® (protease–peptidase complex) was evaluated to overcome the limitations (low encapsulation efficiency and no large-scale production) of other encapsulation processes. To the best of our knowledge, spray drying has not been applied previously for the immobilization of this enzyme. Firstly, bovine serum albumin (BSA), as a model protein, was encapsulated by spray drying in chitosan and tripolyphoshate (TPP) cross-linked-chitosan shell matrices. The results showed that the chitosan–TPP microcapsules provided a high encapsulation efficiency and better protein stability compared to the non-crosslinked chitosan microcapsules. The effect of enzyme concentration and drying temperature were tested during the spray drying of Flavourzyme®. In this regard, an activity yield of 88.0% and encapsulation efficiency of 78.6% were obtained with a concentration of 0.1% (v/v) and an inlet temperature of 130 °C. Flavourzyme®-loaded chitosan microcapsules were also characterized in terms of their size and morphology using scanning electron microscopy and laser diffractometry.

The photoionization of xenon atoms in the 70–100 eV range reveals several fascinating physical phenomena such as a giant resonance induced by the dynamic rearrangement of the electron cloud after photon absorption, an anomalous branching ratio between intermediate Xe+ states separated by the spin-orbit interaction and multiple Auger decay processes. These phenomena have been studied in the past, using in particular synchrotron radiation, but without access to real-time dynamics. Here, we study the dynamics of Xe 4d photoionization on its natural time scale combining attosecond interferometry and coincidence spectroscopy. A time-frequency analysis of the involved transitions allows us to identify two interfering ionization mechanisms: the broad giant dipole resonance with a fast decay time less than 50 as, and a narrow resonance at threshold induced by spin-flip transitions, with much longer decay times of several hundred as. Our results provide insight into the complex electron-spin dynamics of photo-induced phenomena., Here the authors report experiment and theory study of the photoionization of xenon inner shell 4d electron using attosecond pulses. They have identified two ionization paths - one corresponding to broad giant dipole resonance with short decay time and the other involving spin-flip transitions.

Shape resonances in physics and chemistry arise from the spatial confinement of a particle by a potential barrier. In molecular photoionization, these barriers prevent the electron from escaping instantaneously, so that nuclei may move and modify the potential, thereby affecting the ionization process. By using an attosecond two-color interferometric approach in combination with high spectral resolution, we have captured the changes induced by the nuclear motion on the centrifugal barrier that sustains the well-known shape resonance in valence-ionized N$_2$. We show that despite the nuclear motion altering the bond length by only $2\%$, which leads to tiny changes in the potential barrier, the corresponding change in the ionization time can be as large as $200$ attoseconds. This result poses limits to the concept of instantaneous electronic transitions in molecules, which is at the basis of the Franck-Condon principle of molecular spectroscopy., 24 pages, 5 figures

The production of mono- and diacylglycerols rich in polyunsaturated fatty acids is achieved in this study, by solvent-free glycerolysis of anchovy oil with lipase PS-DI from Burkholderia cepacia. Attention is focused on the oxidative stability of the reaction products, determined in terms of induction time (It). The effects of glycerol/triacylglycerol molar ratio, enzyme concentration, and reaction temperature on mono- and diacylglycerol production and It are all assessed. The operating conditions that optimized monoacylglycerol yields and oxidative stability were a glycerol/triacylglycerol ratio of 3/1, 9.0% (w/w) Lipase PS-DI, a stirring rate of 200 rpm, and a reaction time of 4 h, at 45.8 °C, producing a content of 24.8% and 51.9% of mono- and diacylglycerols, respectively, over an It of 1.41 h. The glycerolysis conditions determined by simultaneous optimization strategy increased the oxidative stability of the glycerolysis products by 68%, which rose from 0.84 h (individual optimization) to 1.41 h.

The treatment of must with glucose oxidase (GOX) is a biotechnological alternative to the production of reduced-alcohol wines. Nevertheless, the low pH of must is a limiting factor for the enzyme's activity. This study reports on a simple immobilization method that uses enzyme encapsulation to overcome that limiting factor. The encapsulation of GOX was acceptably optimized using response surface methodology (RSM). An immobilization yield of 73% was obtained with 22 g L−1 Na-alginate, 20 g L−1 carboxymethylcellulose, and 12 g L−1 CaCl2. The apparent Km value of the immobilized GOX (4.55 mM) was slightly higher than that of the soluble enzyme (3.99 mM) and the activation energy was similar (~ 35 kJ mol−1). The encapsulated enzyme maintained about 68% and 92% of its initial activity at pH 3.0 and 4.0, respectively. Moreover, GOX-loaded alginate hollow beads could be re-used with 7 reaction cycles in model must, with a final reaction efficacy of 37%. GOX was successfully encapsulated in Ca-alginate hollow beads, and their catalytic potential remained unaffected by the immobilization process. In view of these results and the activity of the immobilized enzyme at the acidic pH of the must, this encapsulated biocatalyst is a promising alternative for industrial use in the production of reduced-alcohol wines.

Electron correlation and multielectron effects are fundamental interactions that govern many physical and chemical processes in atomic, molecular and solid state systems. The process of autoionization, induced by resonant excitation of electrons into discrete states present in the spectral continuum of atomic and molecular targets, is mediated by electron correlation. Here we investigate the attosecond photoemission dynamics in argon in the 20–40 eV spectral range, in the vicinity of the 3s−1np autoionizing resonances. We present measurements of the differential photoionization cross section and extract energy and angle-dependent atomic time delays with an attosecond interferometric method. With the support of a theoretical model, we are able to attribute a large part of the measured time delay anisotropy to the presence of autoionizing resonances, which not only distort the phase of the emitted photoelectron wave packet but also introduce an angular dependence., Nature Communications, 9, ISSN:2041-1723

Light pulses of sub-100 as (1 as=10-18 s) duration, with photon energies in the extreme-ultraviolet (XUV) spectral domain, represent the shortest event in time ever made and controlled by human beings. Their first experimental observation in 2001 has opened the door to investigating the fundamental dynamics of the quantum world on the natural time scale for electrons in atoms, molecules and solids and marks the beginning of the scientific field now called attosecond science.

An enzymatic membrane reactor (EMR) is assembled for the immobilization of naringinase on a polyethersulfone ultrafiltration membrane, based on fouling-induced method. The effects of molecular weight cut-off, membrane configuration, applied pressure, enzyme concentration and pH are studied in terms of permeate rate, immobilization efficiency, and biocatalytic conversion. The 10 kDa membrane operating in reverse mode, 0.2 MPa, 0.3 gL−1 of enzyme in acetate buffer at pH 5 and cross-linking with 0.25% glutaraldehyde showed the highest naringin conversion (73%). It was determined that the intermediate pore blocking model was the predominant fouling mechanism for the enzymatic immobilization. The EMR was applied for debittering of grapefruit juice, achieving a conversion of naringin below bitterness threshold and maintaining the antioxidant capacity of the juice. Furthermore, the biocatalytic activity of immobilized enzyme was retained at a high level at least during three consecutive reaction runs, with overnight storage at 4 °C after each run. Industrial relevance The potential of membrane technologies in the juice industries is widely recognized today. The development of EMR with naringinase activity is an attractive option for reducing bitterness that could replace current techniques, due to its high specificity and effectiveness, possibility of repeated and continuous use, and in order to retain the properties of juice as much as possible. The research therefore represents an advance in the application of biocatalytic membranes as technological alternative for juice debittering.

The applicability of qPCR in olive-oil authentication depends on the DNA obtained from the oils and the amplification primers. Therefore, four olive-specific amplification systems based on the trnL gene were designed (A-, B-, C- and D-trnL systems). The qPCR conditions, primer concentration and annealing temperature, were optimized. The systems were tested for efficiency and sensitivity to select the most suitable for olive oil authentication. The selected system (D-trnL) demonstrated specificity toward olive in contrast to other oleaginous species (canola, soybean, sunflower, maize, peanut and coconut) and showed high sensitivity in a broad linear dynamic range (LOD and LOQ: 500 ng – 0.0625 pg). This qPCR system enabled detection, with high sensitivity and specificity, of olive DNA isolated from oils processed in different ways, establishing it as an efficient method for the authentication of olive oil regardless of its category.

Plant-derived drugs represent a significant proportion of the pharmaceutical market. Plant in vitro culture has emerged as a useful platform for producing secondary metabolites due to its multiple advantages: production under controlled culture conditions; independence from geographic, seasonal or weather conditions; and since it avoids the use of land and the extraction from the natural source, it has a low environmental impact. Since the production of secondary metabolites in plant in vitro cultures can be low, there are several strategies that can be applied to enhance the production of these compounds. Elicitation has been widely used to increase secondary metabolite production in plant in vitro cultures. This phenomenon involves the treatment of plant cultures with different agents (physical, chemical or biological) that triggers a defence response, which generally involves the production of secondary metabolites. Anthraquinones are secondary metabolites traditionally used as dyes that exhibit interesting therapeutic applications, such as antiviral (against hepatitis C virus), and as photosensitizers of cancer cells. In this chapter, we will present an approach for the establishment of hairy root cultures of Rubia tinctorum and a protocol for elicitation with methyl jasmonate. We will also discuss the possibility of combining elicitation with other strategies, such as in situ removal with Miglyol® 812.

International audience; We apply a spectrally-resolved electron interferometry technique to the measurement of the spectral phase in the vicinity of the 3s13p64p Fano resonance of argon. We show that it allows disentangling the phases of the two nearly-overlapping electron wavepackets corresponding to different spin–orbit final states. Using simple assumptions, it is possible to process the experimental data and numerically isolate each component in a self-consistent manner. This in turn allows reconstructing the autoionization dynamics of the dominant channel.

In this work, barley seed coating with urease and phosphatase, free and immobilized in humic acids, was studied as a tool to enhance the use efficiency of N and P by plants. The seeds were encapsulated in gels of Ca-alginate by a method that allows gelation of the polymer, containing enzymes, from the surface of seeds outward. The coating process did not affect seed germination. A pot experiment with the coated seeds was carried out. The seed coating with free enzymes increased soil ammonium-N (NH4+-N) and inorganic P up to 39 and 54 %, respectively, and up to 62 and 64 %, with the immobilized enzymes, in comparison with control pots. Seed coating improved N (47-137 %) and P (29-61 %) uptake by plants. Moreover, a synergistic response in N and P uptake with both urease and phosphatase was observed. It can be concluded that the seed coating with urease and phosphatase exerted a positive effect enhancing the use of N and P by plants, which could lead to the development of natural fertilizers, minimizing the application of chemical fertilizers.

In a seminal article, Fano predicts that absorption of light occurs preferably with increase of angular momentum. Here we generalize Fano's propensity rule to laser-assisted photoionization, consisting of absorption of an extreme-ultraviolet photon followed by absorption or emission of an infrared photon. The predicted asymmetry between absorption and emission leads to incomplete quantum interference in attosecond photoelectron interferometry. It explains both the angular-dependence of the photoionization time delays and the delay-dependence of the photoelectron angular distributions. Our theory is verified by experimental results in Ar in the 20-40 eV range.

One of the most ubiquitous techniques within attosecond science is the so-called reconstruction of attosecond beating by interference of two-photon transitions (RABBIT). Originally proposed for the characterization of attosecond pulses, it has been successfully applied to the accurate determination of time delays in photoemission. Here, we examine in detail, using numerical simulations, the effect of the spatial and temporal properties of the light fields and of the experimental procedure on the accuracy of the method. This allows us to identify the necessary conditions to achieve the best temporal precision in RABBIT measurements. This article is part of the theme issue ‘Measurement of ultrafast electronic and structural dynamics with X-rays’.

We investigate the angle-resolved photoelectron spectra from laser-assisted photoionization for helium and neon atoms using an ab initio method based on time-dependent surface flux and configuration interaction singles. We find that the shape of the distributions can be interpreted using a propensity rule, an intrinsic difference in the absorption and the emission processes, as well as interference effects between multiple paths to the final angular momentum state. The difference between absorption and emission is hidden in the first sideband in neon due to the multiple competing m channels, while in the second sideband a qualitative difference between absorption and emission is observed. In the m-resolved case, a clear difference is observed already in the first sideband, as exemplified in helium and m-resolved neon.

BACKGROUND Most N fertilizers added to soil are not efficiently used by plants and are lost to the atmosphere or leached from the soil, causing environmental pollution and increasing cost. Barley seed encapsulation in calcium alginate gels containing free or immobilized urease to enhance plant utilization of soil N was investigated. RESULTS Urease was immobilized with soil humic acids (HA). A central composite face-centered design was applied to optimize the immobilization process, reaching an immobilization yield of 127%. Soil stability of urease was enhanced after the immobilization. Seed encapsulation with free urease (FU) and humic-urease complex (HUC) resulted in a urease activity retention in the coating layer of 46% and 24%, and in germination rates of 87% and 92%, respectively. Under pot culture conditions, the pots planted with seeds encapsulated with FU and HUC showed higher ammonium N (NH4 (+) -N) (26% and 64%, respectively) than the control soil at 28 days after planting (DAP). Moreover, the seed encapsulation with FU and HUC increased the N uptake 83% and 97%, respectively, at 35 DAP. CONCLUSION Seed encapsulation with urease could substantially contribute to enhancing plant N nutrition in the early stages of seedling establishment. © 2015 Society of Chemical Industry.

International audience; Autoionization, which results from the interference between direct photoionization and photoexcitation to a discrete state decaying to the continuum by configuration interaction, is a well known example of the important role of electron correlation in light–matter interaction. Information on this process can be obtained by studying the spectral, or equivalently, temporal complex amplitude of the ionized electron wave packet. Using an energy-resolved interferometric technique, we measure the spectral amplitude and phase of autoionized wave packets emitted via the sp2+ and sp3+ resonances in helium. These measurements allow us to reconstruct the corresponding temporal profiles by Fourier transform. In addition, applying various time–frequency representations, we observe the build-up of the wave packets in the continuum, monitor the instantaneous frequencies emitted at any time and disentangle the dynamics of the direct and resonant ionization channels.

Photoionization time delays are measured with two-color (XUV+IR) interferometric techniques. The combination of attosecond temporal resolution and high spectral resolution from narrowband harmonics allows the study of ultrafast dynamics in both time and frequency domain.

In the present chapter, we review some aspects of the biosynthesis of sesquiterpene lactones and its regulation in different medicinal and aromatic plants used in the pharmaceutical industry. In this sense, we describe the mevalonate and the 2-C-methyl-D-erythritol 4-phosphate pathways, which generate the corresponding isoprenoid precursors (isopentenyl diphosphate and dimethylallyl diphosphate), as well as the late pathways that lead to sesquiterpene lactone biosynthesis. This chapter also analyses the role of the transcription factors involved in the regulation of sesquiterpene lactone biosynthesis and the different biotechnological approaches that have been developed for sesquiterpene lactone production. In vitro plant cell cultures (comprising micropropagation and plant cell suspension, shoot and root cultures) have emerged as a production platform for many plant secondary metabolites, since they allow their production under controlled conditions and shorter production cycles. The characterisation and isolation of genes involved in the regulation of sesquiterpene lactone biosynthetic pathways have allowed the design of metabolic engineering strategies to increase the production of these metabolites. Moreover, we discuss different strategies to increase sesquiterpene lactone production through genetic engineering. We also focus on the metabolic engineering of the artemisinin biosynthetic pathway in Artemisia annua. This metabolic pathway has become a model system not only for the biotechnological production of sesquiterpene lactones but also for the improvement of other plant secondary metabolic pathways. Finally, we analyse the successful expression of the complete artemisinin biosynthetic pathway in Escherichia coli and Saccharomyces cerevisiae, which has led to the efficient accumulation of artemisinic acid in these microorganisms.

Ultrafast processes in matter, such as the electron emission following light absorption, can now be studied using ultrashort light pulses of attosecond duration ($10^{-18}$s) in the extreme ultraviolet spectral range. The lack of spectral resolution due to the use of short light pulses may raise serious issues in the interpretation of the experimental results and the comparison with detailed theoretical calculations. Here, we determine photoionization time delays in neon atoms over a 40 eV energy range with an interferometric technique combining high temporal and spectral resolution. We spectrally disentangle direct ionization from ionization with shake up, where a second electron is left in an excited state, thus obtaining excellent agreement with theoretical calculations and thereby solving a puzzle raised by seven-year-old measurements. Our experimental approach does not have conceptual limits, allowing us to foresee, with the help of upcoming laser technology, ultra-high resolution time-frequency studies from the visible to the x-ray range., 5 pages, 4 figures

Synopsis Above-threshold ionization is a corner stone of attsecond science. In this work we extend Fano’s propensity rule to two-photon above-threshold ionization and show that the asymmetry between absorption and emission of the second photon predicted by this propensity rule has strong implications for angle-resolved pump-probe experiments and in particular for attosecond photoelectron interferometry.

A new spectrophotometric end-point method to evaluate lipase activity in aqueous media is described. This procedure is based on blocking the enzyme reaction by adding chloroform:isoamyl alcohol (24:1) as a denaturating agent, and removing the precipitate by centrifugation. Emulsifier screening showed that the chemical component with the least effect on lipase activity from Thermomyces lanuginosus, Mucor javanicus, Aspergillus niger, Rhizomucor miehei, Penicillium camemberti and Burkholderia cepacia, in the proposed method, was 0.1% (w/w) gum arabic. In contrast, Triton X-100, SDS, sodium cholate and Tween 80 reduced lipase activity, depending on the microbial source of the latter. A comparative study with other end-point methods based on the addition of chemical reagents (NaOH, Na2CO3, THAM or acetone–ethanol) or in thermal treatments (chilling or heating) was performed. The proposed procedure was shown to be more accurate in comparison with other methods that were tested. Furthermore, the effectiveness of the method was demonstrated in a study of the specificity of six commercial lipases toward p-nitrophenyl decanoate (C10) and p-nitrophenyl palmitate (C16). The study also analyzed the kinetic behavior and catalytic efficiency of lipase from T. lanuginosus and B. cepacia toward p-nitrophenyl decanoate and p-nitrophenyl palmitate.

A large portion of the soluble P fertilizers introduced into soil reacts with soil components forming insoluble P products, which are not efficiently taken up by plants. Barley seed (Hordeum vulgare L. 'Volley') encapsulation in Ca-alginate gels containing phosphatase free or immobilized with soil humates (humate-phosphatase complexes, HPC) was investigated as a tool to enhance the utilization by plants of accumulated soil P. The effect of the enzyme/carboxymethylcellulose solution ratio on the coating phosphatase activity and on seed germination was studied. A pot experiment with encapsulated seeds was conducted. Dry weight, length and shoot P content were estimated at different days after planting (DAP). Under optimal conditions (0.20 mL mL-1 free phosphatase and 0.13 g mL-1 HPC), the seed encapsulation with free phosphatase and HPC resulted in a coating phosphatase activity of 20 and 17% and in germination percentages of 92 and 85%, respectively. Increased phosphatase activity was observed in the rhizosphere of encapsulated seeds in comparison with non-treated ones. Under pot culture conditions, at 35 DAP, the inorganic P in soil planted with encapsulated seeds was higher (14-16%) than in control soil. In contrast, soil planted with treated seeds showed lower content of organic P (20-29%) than the control soil. Furthermore, the seed encapsulation significantly increased the shoot P content and the P uptake between 24 and 28% and 15 to 20%, respectively, at 35 DAP. In conclusion, seeds treated with phosphatase could substantially contribute to enhance the plant P nutrition in the early stages of seedling establishment. © 2013 by the American Society of Agronomy, 5585 Guilford Road, Madison, WI 53711. All rights reserved.

Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl- d -erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.

Antioxidant capacity, radical scavenging activity, as well as protective effect on lipoperoxidation, glutathione oxidation and DNA damage, were evaluated in grapefruit juice subjected to bitterness removal by naringinase or by physical adsorption with Amberlite®IRA-400. The results showed a reduction in naringin content for the naringinase-treated juice (N-PJ) and those processed with the exchange resin (R-PJ), which made both juices acceptable to consumers. Total antioxidant capacity, measured by ABTS and FRAP assays, was lower in R-PJ samples. The highest superoxide and hydroxyl radical scavenger activity was observed in N-PJ. With regard to inhibitory effect of juice samples on lipoperoxidation, N-PJ also provided the greatest effectiveness. In addition, R-PJ showed the lowest levels of GSH. The results showed a protective effect on DNA oxidative damage for all juice samples tested. In summary, enzymatic technology was more effective than physical adsorption in preserving the antioxidant and biomolecule protection capacity of fresh grapefruit juice.

Hyoscyamine, anisodamine (6β-hydroxyhyoscyamine) and scopolamine are tropane alkaloids produced by plants belonging to the Solanaceae family such as Brugmansia candida. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Further therapeutical properties for anisodamine were recently described renewing the interest in these alkaloids. The scaling-up of hairy root cultures is an interesting strategy for the pharmaceutical production of these compounds instead of the isolation from plants. In this work, B. candida hairy roots were cultured in a modified 1.5 L stirred tank in order to analyze an alternative production system of scopolamine and anisodamine. It was found that these cultures produced an increased biomass and alkaloids concentration compared to the processes carried out in Erlenmeyer flasks. Anisodamine was the predominant alkaloid reaching a maximum concentration of 10.05 ± 0.76 mg/g DW in modified bioreactor culture system. The results obtained in this work are potentially applicable for the rational scale-up of the process.

BACKGROUND: This study was designed to evaluate and compare antioxidant capacity and radical scavenging activity of naringin and its aglycone by different in vitro assays. The effects of flavanones on lipid peroxidation, glutathione (GSH) oxidation and DNA cleavage were also assessed. RESULTS: The results showed that naringenin exhibited higher antioxidant capacity and hydroxyl and superoxide radical scavenger efficiency than naringin. Our results evidenced that glycosylation attenuated the efficiency in inhibiting the enzyme xanthine oxidase and the aglycone could act like a more active chelator of metallic ions than the glycoside. Additionally, naringenin showed a greater effectiveness in the protection against oxidative damage to lipids in a dose-dependent manner. Both flavanones were equally effective in reducing DNA damage. However, they show no protective effect on oxidation of GSH. CONCLUSION: The data obtained support the importance of characterizing the ratio naringin/naringenin in foods when they are evaluated for their health benefits. Copyright © 2010 Society of Chemical Industry

An alkaline phosphatase (EC 3.1.3.1) from Escherichia coli ATCC27257 was immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retained about 74% of the original enzymatic activity. The pH and temperature profile of the immobilized and free enzyme revealed a similar behavior. Kinetic parameters were determined: K(m) and K(i) values were 2.44 and 0.423 mM, respectively, for the phosphatase-polyresorcinol complex and 1.07 and 0.069 mM, respectively, for free phosphatase. The thermal and storage stabilities of the immobilized phosphatase were higher than those of the native one. On addition to soil, free enzyme was completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex was comparatively stable. Barley seed coated with the immobilized enzyme exhibited higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus was detected when the seed was encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot was observed.

Neutrase, a commercial preparation of Bacillus subtilis , was covalently immobilized on alginate-glutaraldehyde beads. Immobilization conditions and characterization of the immobilized enzyme were investigated. Central composite design and response surface methods were employed to evaluate the effects of immobilization parameters, such as glutaraldehyde concentration, enzyme loading, immobilization pH, and immobilization time. Under optimized working conditions (2% alginate, 6.2% glutaraldehyde, 61.84 U mL(-1) Neutrase, pH 6.2, and 60 min) the immobilization yield was about 50%. The immobilized enzyme exhibited higher K(m) compared to the soluble enzyme. The pH-activity profile was widened upon immobilization. The optimum temperature was shifted from 50 to 60 degrees C, and the apparent activation energy was decreased from 47.7 to 22.0 kJ mol(-1) by immobilization. The immobilized enzyme also showed significantly enhanced thermal stability.

Phenolic compounds present in the drainage from several industries are harmful pollutants and represent a potential danger to human health. In this work we have studied the removal of phenol from water using Brassica napus hairy roots as a source of enzymes, such as peroxidases, which were able to oxidise phenol. These hairy roots were investigated for their tolerance to highly toxic concentrations of phenol and for the involvement of their peroxidase isoenzymes in the removal of phenol. Roots grew normally in medium containing phenol in concentrations not exceeding 100 mg l −1 , without the addition of H 2 O 2 . However, roots were able to remove phenol concentrations up to 500 mg l −1 , in the presence of H 2 O 2 , reaching high removal efficiency, within 1 h of treatment and over a wide range of pH (4–9). Hairy roots could be re-used, at least, for three to four consecutive cycles. Peroxidase activity gradually decreased to approximately 20% of the control, at the fifth cycle. Basic and near neutral isoenzymes (BNP) decreased along time of recycling while acidic isoenzymes (AP) remained without changes. Although both group of isoenzymes would be involved in phenol removal, AP showed higher affinity and catalytic efficiency for phenol as substrate than BNP. In addition, AP retained more activity than BNP after phenol treatment. Thus, AP appears to be a promising isoenzyme for phenol removal and for application in continuous treatments. Furthermore, enzyme isolation might not be necessary and the entire hairy roots, might constitute less expensive enzymatic systems for decontamination processes.

In this paper, a long-term investigation of the enzyme hydrolysis of pectin, using a free enzyme membrane reactor (FEMR), is presented. Production of crude enzymes (polygalacturonase and pectin lyase), in Aspergillus niger CECT 2088 cultures grown on an agricultural subproduct (apple pomace) as a low cost inducer, was also studied. Observing the retention of induced pectinases by the ultrafiltration membranes, the best FEMR configuration was achieved with the membrane of 10,000 nominal molecular weight limit (NMWL), since the biocatalyst was retained with no loss of enzyme activity. The monitoring of pectin hydrolysis in the FEMR was carried out by determining the viscosity and amount of reducing groups in both reaction mixture and permeate. A viscosity reduction of 88%, below the initial value, was reached in the reaction mixture only after 0.25 h of operation, while a gradual release of reduced sugars, in both reaction mixture and permeate, was detected. The FEMR was maintained in operation for 15 days, achieving an excellent catalytic efficiency.

Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon.

Capillary electrophoresis using an untreated fused-silica capillary and a low-pH buffer containing urea and hydroxypropyl methyl cellulose was used to follow the proteolytic action of neutral protease of Bacillus subtilis on isolated casein fractions and whole milk casein (CN) samples. Electrophoretic analysis showed that this protease caused intense hydrolysis of α -CN, β -CN and κ -CN. The breakdown products due to the attack of the neutral protease on β -CN, γ 2 -CN A, γ 1 -CN B, γ 1 -CN A 1 , γ 3 -CN B, γ 1 -CN A 2 , γ 3 -CN A, β -CN-I A 1 , β -CN-I A 2 and β -CN-I B, and on α s -CN, α s1 -CN-I, α s0 -CN-I and α s1 -CN f(1–23) were separated.

Anthraquinones (AQs) are compounds with a wide variety of interesting properties, which could be obtained by Rubia tinctorum suspension cultures. The present paper reports the effect of proline and aminoindan-2-phosphonic acid (AIP) on AQs production. The addition of proline (0.25 mM) or AIP (100 μM) enhance, up to 50% the AQ biosynthesis in R. tinctorum suspension culture. This increase is similar to those obtained by elicitation. Phenylalanine ammonia liase (PAL) activity was higher in presence of proline but no change in phenolic acid contents was found. Glucose-6-phosphate dehydrogenase (G6PDH) activity was not affected in cell cultures treated with proline. Addition of AIP produced PAL inhibition and lower levels of phenolic acid contents. These results show that it is possible to increase the AQs level in R. tinctorum suspension culture by metabolic manipulation and by inhibition of the key enzymes of the metabolic pathways that compete with AQs for a common substrate.

Optimisation of pectin hydrolysis using pectolytic enzymes produced by Aspergillus niger CECT 2088 was carried out. The effect of enzyme concentration (0.018–0.072%, w/v), substrate concentration (0.004–0.896%, w/v), pH (4.49–5.91), temperature (32.2–67.8 °C) and reaction time (18.2–71.8 min) on the enzymatic process was studied. The experiments were arranged according to a central composite statistical design. Response surface methodology (RSM) was used to assess factor interactions and empirical models regarding four product response variables (i.e., reactor conversion, reducing sugar concentration, endopectolytic productivity and enzymatic depolymerization productivity). Multi-response optimisation was also performed on the reactor and endopectolytic productivity data set of the factorial design. The highest reactor conversion (61%) and endopectolytic productivity (7.2 cSt mg −1 ml −1 ) were achieved with 1 h reaction time at 46 °C and pH 4.8 at a substrate/enzyme ratio of 11.6.