Do ultrasonic field effects upon the polyphenolics profile of propolis extracts improve their antioxidant and antimicrobial activity?

Ultrasound-assisted extraction (UAE) was applied for polyphenols extraction from Romanian propolis, followed by comparison with previous maceration work. The effects consisted not only in time reduction and extraction yield increase, but also in polyphenolics profile modification in terms of flavonoids / polyphenolic acids ratio. The operating parameters were ultrasounds (US) field exposure time (10-100 min), solvent composition (water, 25 % and 50 % ethanolic solutions, w/w), and liquid:solid ratio (2:1, 4:1 and 6:1, w:w), while keeping temperature constant. 24 polyphenolic derivatives were quantified by UHPLC-HRMS. UAE favored the extraction of pinocembrin, isorhamnetin and chrysin in water and 25 % ethanol, leading to different profiles than maceration, and further influences upon the antioxidant and antimicrobial activity. All extracts demonstrated increased antibacterial and antifungal activity compared to maceration, particularly the 50 % ethanolic extracts, which presented a three-times larger antioxidant capacity. Chemometric methods (Principal Component Analysis - PCA and Partial Least Squares Regression - PLS) and a saturation type model were used to correlate the polyphenolics profiles and antioxidant capacity. Experimental and modelling results concluded that 50 % ethanolic solutions and UAE represent the favorable operating conditions in terms of yield and extracts quality.

Polyphenolics profile effects upon the antioxidant and antimicrobial activity of propolis extracts.

Propolis, a complex bee product, is a source of numerous bioactive principles, beneficial for human health, therefore it is intensively studied. In the present work, extracts of propolis from Bihor Romanian County were studied to identify the relationship between the polyphenolic derivatives profile and their antioxidant and antimicrobial activity. Extracts were obtained using water and 25%, 50%, and 70% ethanolic solutions (w/w), at 2:1, 4:1, and 6:1 liquid: solid ratios (w/w). 21 polyphenolic derivatives were quantified by UHPLC-MS, proving that the extracts composition strongly depends on the solvent. The sum of quantified polyphenolics extracted varied between 1.5 and 91.2 mg/g propolis. The antioxidant capacity was evaluated using the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6 sulfonic acid) diammonium salt (ABTS) and 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging methods. Antimicrobial efficiency was tested against Gram-positive (B. subtilis), Gram-negative bacteria (E. coli), and fungi (C. albicans) by disc-diffusion method. All extracts, even the aqueous ones, demonstrated antibacterial and antifungal activity. Chemometric methods (partial least squares) and a saturation-type model were used to evaluate the contribution of various bioactive principles in building the antioxidant capacity of extracts. Both experimental and modelling results show that 50% ethanolic extracts provide a rich polyphenolics profile and ensure a good antioxidant capacity.

A new reactor for process intensification involving the simultaneous application of adjustable ultrasound and microwave radiation.

Ultrasound (US) and Microwaves (MW) are effective methods for processes intensification. Their combined use in the same reactor can lead to remarkable results. Recently there has been a resurgence of interest in this field for new synthetic applications using reactors based upon existing technologies. We describe here a new type of apparatus in which the thermal energy is continuously removed from the system making possible the use of high power and adjustable ultrasonic and microwave densities throughout the process. The installation consists of a glass reactor located in a monomode applicator which is immersed at the same time in an ultrasonic device which can be operated at different frequencies and powers. A liquid, transparent to microwaves, was used to couple ultrasonic energy to the reactor and to remove the heat generated. Comsol software was used to get information about the distribution of ultrasonic and microwave energy between the reactor liquid and the coupling fluid. The performance was assessed using the conversion of p-nitrophenol into 4-nitrocatechol as a chemical dosimeter and a transesterification.

Preclinical Evaluation of NHS-Activated Gold Nanoparticles Functionalized with Bombesin or Neurotensin-Like Peptides for Targeting Colon and Prostate Tumours.

Recent advances and large-scale use of hybrid imaging modalities like PET-CT have led to the necessity of improving nano-drug carriers that can facilitate both functional and metabolic screening in nuclear medicine applications. In this study, we focused on the evaluation of four potential imaging nanoparticle structures labelled with the 68Ga positron emitter. For this purpose, we functionalized NHS-activated PEG-gold nanoparticles with 68Ga-DOTA-Neuromedin B, 68Ga-DOTA-PEG(4)-BBN(7-14), 68Ga-DOTA-NT and 68Ga-DOTA-Neuromedin N. In vitro binding kinetics and specific binding to human HT-29 colon carcinoma cells and DU-145 prostate carcinoma cells respectively were assessed, over 75% retention being obtained in the case of 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP in prostate tumour cells and over 50% in colon carcinoma cells. Biodistribution in NU/J mice highlighted a three-fold uptake increase in tumours at 30 min post-injection of 68Ga-DOTA-NT-AuNP and 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP compared to 68Ga-DOTA-NT and 68Ga-DOTA-PEG(4)-BBN(7-14) respectively, therewith fast distribution in prostate and colon tumours and minimum accumulation in non-targeted tissues.

Ultrasound assisted preparation of calcium alginate beads to improve absorption of Pb+2 from water.

Calcium alginate (CaAlg) beads were prepared using ultrasound for use in the removal of lead from natural and wastewaters by ion exchange. Ultrasound was applied in a batch mode with an ultrasonic bath or in a flow mode using an ultrasonic clamp-on device. For comparison purposes the synthesis was performed in batch mode in the absence of the ultrasound. The beads prepared using ultrasound showed a greater ion exchange capability which could be ascribed to a larger specific surface area as a result of surface roughening induced by cavitation. Scanning Electron Microscopy (SEM) images revealed that the roughening was in the form of corrugation for the product with the best ion exchange capability obtained in the flow process where preformed CaAlg droplets were subjected to ultrasound during the setting process. These beads performed 11% better for lead removal than those synthesized in the absence of ultrasound.

Simultaneous production of oil enriched in ω-3 polyunsaturated fatty acids and biodiesel from fish wastes.

The waste resulted from fish processing industries are discarded into the environment around the world, causing environmental pollution. The main problem of fish oil extracted from waste is the high content in free fatty acids (FFA) which decrease the yield in fatty acids esters during transesterification reactions. Therefore, to correct the fish-oil properties, a new environmentally friendly heterogeneous superacid catalyst (SO42-/SnO2-ZrO2) was tested in the esterification reaction of FFA with ethanol. The catalyst was characterized by different techniques (XRD, FT-IR, FT-IR of adsorbed pyridine, BET, SEM-EDX, TGA and acidity measurements). The reaction was found to follow a Langmuir-Hinshelwood (L-H) dual-site mechanism with the novelty that both Brönsted and Lewis acid centers participate equally in the esterification reaction. The pre-treated oil was subjected to transesterification reaction with ethanol over a heterogeneous base catalyst and then, the saturated and unsaturated fractions of fatty acid ethyl esters (FAEE) were separated using a vacuum rectification unit with falling film. The saturated content can be used as biofuel, while the unsaturated FAEE are further transesterified with glycerol in order to obtain oil with high content in polyunsaturated fatty acids (PUFA). A detailed study of the intrinsic kinetic process at the surface of the superacid catalyst and a thorough mathematical model of the fixed bed reactor were written and validated by an experimental program, designed according to the D-optimal methodology.

Multi-scale modelling of bioreactor-separator system for wastewater treatment with two-dimensional activated sludge floc dynamics.

A simple "first generation" multi-scale computational model of the formation of activated sludge flocs at micro-scale and reactor performance at macro-scale is proposed. The model couples mass balances for substrates and biomass at reactor scale with an individual-based approach for the floc morphology, shape and micro-colony development. Among the novel model processes included are the group attachment/detachment of micro-flocs to the core structure and the clustering of nitrifiers. Simulation results qualitatively describe the formation of micro-colonies of ammonia and nitrite oxidizers and the extracellular polymeric substance produced by heterotrophic microorganisms, as typically observed in fluorescence in situ hybridization images. These results are the first step towards realistic multi-scale multispecies models of the activated sludge wastewater treatment systems and a generic modelling strategy that could be extended to other engineered biological systems.

Determination of bioactive, free isothiocyanates from a glucosinolate-containing phytotherapeutic agent: a pilot study with in vitro models and human intervention.

Isothiocyanates (ITCs) derived from plants of the order Brassicales are known for their antibacterial, anti-inflammatory or anticarcinogenic potential. Although only the free ITCs exert bioactivity, quantification in vivo is almost exclusively performed on total ITC/metabolite content. We therefore investigated in a pilot study the amount of free ITC at different steps critical for therapeutic efficacy. A sensitive and specific GC-MS/MS method for the simultaneous quantification of individual free ITC after solid-phase extraction (SPE) was developed. We show here that release of biologically active ITC from plants occurs at not only alkaline but also acidic pH. Furthermore, in human urine conversion of the ultimate, inactive mercapturic acid conjugate back into its corresponding bioactive form is increased at alkaline as compared to neutral pH. This was also observed in the urine of human volunteers, where - in correlation with the pH value - a mean of 0.16 to 1.03 µmol ITC was detected after oral application of a phytotherapeutic agent containing 30.4 µmol of the initial pro-drugs. The amounts of free ITC being necessary for bioactivity in vitro were found to be indeed achieved in vivo. These data might be helpful to better understand the beneficial effects of ITC observed in vivo.

Conditional confined oscillatory dynamics of Escherichia coli strain K12-MG1655 in chemostat systems.

A series of continuous- and sequencing-batch reactor experiments were performed to assess the growth dynamics of Escherichia coli strain K12-MG1655 in chemostat systems. Previous mathematical predictions and early experimental results had shown that confined oscillatory dynamics ensue in bioreactor populations, which relates to "group birth and death" events within the population. New results are reported here that generally verify the predictions of the model and show that confined oscillations occur under different initial conditions, but the characteristics of the oscillatory dynamics vary as a function of the hydraulic retention time (HRT). Bioreactors were operated at HRTs ranging from 2.7 to 35 h and, regardless of initial conditions or the imposition of transient operational instabilities, highly patterned oscillations developed when HRT was between ∼3 and 8 h. However, outside of this range, bioreactor populations tended to form biofilms on the reactor walls (although the majority of the cells remained suspended in the bulk solution) and stable oscillations were not seen in the bulk phase. This suggests that alternate operating "states" might exist in chemostat populations with biofilm formation and non-homogenous spatial growth influencing "system" dynamics at very low and high HRTs. Although the model accurately predicts a confined dynamic equilibrium for mid-range HRT operations, experimental data show that model predictions do not extend outside of this range, when an alternate stable-state seems to be attained.

Non-linear population dynamics in chemostats associated with live-dead cell cycling in Escherichia coli strain K12-MG1655.

Bacterial populations conditionally display non-linear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live-dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD600 and live-dead cell oscillations (within ~33-38 min cycles) only became statistically significant (p < 0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD600 maxima and lowest at local OD600 minima, showing that oscillations followed live-dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.

Birth, growth and death as structuring operators in bacterial population dynamics.

A new model is presented that describes microbial population dynamics that emerge from complex interactions among birth, growth and death as oriented, discrete events. Specifically, birth and death act as structuring operators for individual organisms within the population, which become synchronised as age clusters (called cell generations that are structured in age classes) that are born at the same time and die in concert; a pattern very consistent with recent experimental data that show bacterial group death correlates with temporal population dynamics in chemostats operating at carrying capacity. Although the model only assumes "natural death" (i.e., no death from predation or antimicrobial exposure), it indicates that short-term non-linear dynamic behaviour can exist in a bacterial population growing under longer term pseudo-steady-state conditions (a confined dynamic equilibrium). After summarizing traditional assumptions about bacterial aging, simulations of batch, continuous-flow, and bioreactors with recycle are used to show how population dynamics vary as function of hydraulic retention time, microbial kinetics, substrate level, and other factors that cause differential changes in the distribution of living and dead cells within the system. In summary, we show that population structures induced by birth and death (as discrete and delayed events) intrinsically create a non-linear dynamic system, implying that a true steady state can never exist in growing bacterial populations. This conclusion is discussed within the context of process stability in biotechnology.

Continuous hybridoma bioreactor: sensitivity analysis and optimal control.

Animal cell culture has already established itself as a mature technology able to make a wide range of valuable products, the actual focus being to find the competitive bioreactor design and operating conditions for increasing production. A complex analysis, implying sensitivity calculus and optimal control computation, is done for a system composed of a continuous perfectly mixed bioreactor, with cell recirculation, a cell separator, a mixer and a purge. The bioreactor's sensitivity to the control parameters is measured by a new concept, entropic density, developed from the notion of Shannon entropy. An optimization procedure based on a genetic-algorithms approach is applied for the computation of the inlet flow profile in time, which guarantees optimum monoclonal-antibody production. Our studies, including the present one, proved that the best approach to obtain high production is to use a hybrid operating sequence: fed-batch mode followed by the continuous mode.

A sensitivity analysis of the fed-batch animal-cell bioreactor with respect to some control parameters.

Animal cell culture is widely used in the manufacture of valuable products, and this process is nowadays seeing a rapid expansion. The growth of animal cells is a complex process, because the cells are very sensitive to environmental changes (in, for example, nutrients, pH, temperature, oxygen and osmolarity) during this phase and to the toxic compounds produced by the cell itself. Ammonia and lactate are the two major waste materials of cell culture. They can have inhibitory effects on cell growth and product (monoclonal antibodies among others) formation. In order to model the behaviour of a fed-batch animal cell bioreactor producing monoclonal antibodies, it is necessary to use a complex kinetic model with optimal operating patterns ensuring high productivities. Good knowledge of such domains of operating parameters, together with the understanding of the response of this rather complex system to small modifications in the working conditions, are essential for on-line control to improve the quality of product and the yield of an animal cell culture. The present study focuses on the sensitivity analysis of a fed-batch animal cell bioreactor with respect to some candidate control parameters (substrate set-point concentrations, feeding time-step patterns and concentration of feeding solutions), emphasizing the influence of these on the overall performance of the system.