Investigation of Mirabegron-loaded Nanostructured Lipid Carriers for Improved Bioabsorption: Formulation, Statistical Optimization, and In-Vivo Evaluation.

Overactive bladder (OAB) is a usual medical syndrome that affects the bladder, and Mirabegron (MBG) is preferred medicine for its control. Currently, available marketed formulations (MYRBETRIQ® granules and MYRBETRIQ® ER tablets) suffer from low bioavailability (29-35%) hampering their therapeutic effectiveness and compromising patient compliance. By creating MBG nanostructured lipid carriers (MBG-NLCs) for improved systemic availability and drug release, specifically in oral administration of OAB treatment, this study aimed to address these issues. MBG-NLCs were fabricated using a hot-melt ultrasonication technique. MBG-GMS; MBG-oleic acid interaction was assessed by in silico molecular docking. QbD relied on the concentration of Span 80 (X1) and homogenizer speed (X2) as critical material attribute (CMA) and critical process parameter (CPP) respectively, while critical quality attributes (CQA) such as particle size (Y1) and cumulative drug release at 24 h (Y2) were estimated as dependent variables. 32 factorial design was utilized to investigate the interconnection in variables that are dependent and independents. Optimized MBG-NLCs with a particle size of 194.4 ± 2.25 nm were suitable for lymphatic uptake. A PDI score of 0.275 ± 0.02 and zeta potential of -36.2 ± 0.721 mV indicated a uniform monodisperse system with stable dispersion properties. MBG-NLCs exhibited entrapment efficiency of 77.3 ± 1.17% and a sustained release in SIF of 94.75 ± 1.60% for 24 h. MBG-NLCs exhibited the Higuchi model with diffusion as a release mechanism. A pharmacokinetic study in Wistar rats exhibited a 1.67-fold higher bioavailability as compared to MBG suspension. Hence, MBG-NLCs hold promise for treating OAB by improving MBG's oral bio absorption.

Step-by-step full factorial design to optimize a quantitative sandwich ELISA.

In this work, a quantitative sandwich ELISA was optimized, through a full factorial design of experiments (DOE) in successive steps of a preliminary protocol obtained by the method of one factor at a time (OFAT). The specificity of the optimized ELISA, the lower limit of quantification, the quantification range and the analytical sensitivity of the antigen quantification curve were evaluated, in comparison with the curve obtained from the preliminary protocol. The full factorial DOE was linked to a simple statistical processing, which facilitates the interpretation of the results in those laboratories where there is no trained statistician. The step-by-step optimization of the ELISA and the successive incorporation into the protocol of the best combination of factors and levels, allowed obtaining a specific immunoassay, with an analytical sensitivity 20 times greater and with a lower limit of antigen quantification that decreased from 156.25 at 9.766 ng/mL. As far as we know, there are no reports of optimization of an ELISA following the step-by-step scheme used in this work. The optimized ELISA will be used for the quantification of the TT-P0 protein, the active principle of a vaccine candidate against sea lice.

Phyto-assisted synthesis of CuO/industrial waste derived biochar composite for adsorptive removal of doxycycline hydrochloride and recycling of spent biochar as green energy storage device.

The highest exposure of Endocrine disrupting compounds (EDC) into the water bodies as a result of extensive production and application of Covid-19 related drugs is a growing concern now a days. Herein, a novel nanocomposite material was developed by impregnating green synthesized copper oxide nanoparticles on the porous surface of fabric waste derived biochar to eliminate the concerned EDCs along with a sustainable disposal strategy for the spent adsorbent. Morphological characterizations by Field emission scanning electron microscopy confirmed the formation of hierarchical porous structured material. X-ray analysis revealed presence of both amorphous nature of biochar matrix as well as the crystalline nature attributed from monodispersion of copper oxide nanoparticles onto biochar surface. Batch sorption study showed removal of doxycycline hydrochloride (DOX) of >97% after 2 h at pH 7, 30 mg L-1 initial concentration of DOX and 2 g L-1 of adsorbent dose at room temperature after a two-step optimization process. Spectroscopic study and Raman shift suggested that pore filling, strong complexation and electrostatic interactions maximise the adsorption of DOX in the CuO/biochar composite as compared to the pristine biochar. However disposal of spent adsorbent is a crucial aspect for the environment and therefore, a sustainable recycling strategy for DOX loaded adsorbent as electrode material has been proposed for the first time in this study. Maximum specific capacitance value was observed in the range of 221.9-297.3 F g-1 for the DOX loaded nanocomposite at 1 mV s-1 comparable with other reported heteroatom-doped carbonaceous material as electrode. Therefore the excellent adsorption capacity of green synthesized CuO/biochar composite and its recycling after DOX adsorption can be recommended as a sustainable solution for mitigation of pharmaceuticals from wastewater. A detail study on degradation of DOX into eco-friendly products and its cost-effectiveness would be beneficial to suggest appropriate mitigation strategy for such compounds.

Development, stability-indicating assessment, and evaluation of influential method conditions using a full factorial design for the determination of Nintedanib esylate-related impurities.

The design of an appropriate analytical method for assessing the quality of pharmaceuticals requires a deep understanding of science, and risk evaluation approaches are appreciated. The current study discusses how a related substance method was developed for Nintedanib esylate. The best possible separation between the critical peak pairs was achieved using an X-Select charged surface hybrid Phenyl Hexyl (150 × 4.6) mm, 3.5 µm column. A mixture of water, acetonitrile, and methanol in mobile phase-A (70:20:10) and mobile phase-B (20:70:10), with 0.1% trifluoroacetic acid and 0.05% formic acid in both eluents. The set flow rate, wavelength, and injection volumes were 1.0 ml/min, 285 nm, and 5 µl, respectively, with gradient elution. The method conditions were validated as per regulatory requirements and United States Pharmacopeia general chapter < 1225 >. The correlation coefficient for all impurities from the linearity experiment was found to be > 0.999. The % relative standard deviation from the precision experiments ranged from 0.4 to 3.6. The mean %recovery from the accuracy study ranged from 92.5 to 106.5. Demonstrated the power of the stability-indicating method through degradation studies; the active drug component is more vulnerable to oxidation than other conditions. Final method conditions were further evaluated using a full-factorial design. The robust method conditions were identified using the graphical optimization from the design space.

Potential of Antarctic lipase from Acinetobacter johnsonii Ant12 for treatment of lipid-rich wastewater: screening, production, properties and applications.

The present study aimed to screen and optimize lipase production by the Antarctic strain Acinetobacter johnsonii Ant12 for lipid-rich wastewater treatment. Lipase production was successfully enhanced threefold through optimization of culture conditions. The optimum crude lipase activity was observed at 50 °C with high stability in a wide temperature range. The lipase also exhibited high activity and stability in the presence of solvents, metal ions, and surfactants. The crude lipase was used for the treatment of lipid-rich wastewater, which poses a significant challenge, as traditional removal methods are often inefficient or non-eco-friendly. In this study, bioaugmentation with Ant12 resulted in substantial lipid reduction in synthetic as well as real-world wastewater. Multiple linear regression analysis showed that lipid concentration and time were the most significant factors influencing lipid degradation. Bioaugmentation of real-world wastewater with Ant12 cells resulted in 84% removal of lipids in 72 h, while its crude lipase degraded 73.7% of lipids after 24 h. Thus, the specific rate of lipid degradation was higher for crude lipase (0.095/h) than the whole cell treatment (0.031/h). Economic analysis revealed that crude lipase production was much cheaper, faster and more eco-friendly than purified or partially purified lipase production, which justifies its use in wastewater treatment. The high activity of enzyme also implicates its application as a detergent additive. In our knowledge, it is the first study to establish A. johnsonii isolate from Antarctica for lipid-rich wastewater treatment.

Development of Photoprotective Formulations: Influence of Formulation Composition on the SPF and Mechanical Properties.

The evaluation of the vehicle formulation is important during the development of sunscreens, as it influences their efficacy. In this context, the aim of the present study was to develop photoprotective formulations and evaluate the influence of the formulation components in the sun protection factor (SPF) and physical-mechanical and sensory properties of the formulations. We evaluated four sunscreens through a 22 full factorial design in terms of concentration and emulsifier type. The design of experiments (DOE) parameters were SPF, thixotropy, and work of shear. After the screening of the formulations by DOE, the SPF values, mechanical and sensory properties, and stability were evaluated. All study formulations showed non-Newtonian pseudoplastic behavior, compatible with sunscreens, and presented SPF values above 30. The factors evaluated in DOE had significant interactions for all the analyzed parameters. The concentration of the phosphate-based emulsifier influenced the SPF parameter. The work of shear was influenced by the concentration of polyglyceryl-based emulsifier. The concentration and the type of emulsifier influenced the thixotropy. Finally, effective sunscreens were developed, and the type and concentration of emulsifiers had an influence on the SPF of the formulations. In addition, the formulations chosen by DOE were stable and showed good sensory properties.

In Vivo Evaluation of Almotriptan malate Formulation through Intranasal Route for the Treatment of Migraine: Systematic Development and Pharmacokinetic Assessment.

Migraine headaches are usually intolerable, and a quick-relief treatment remains an unmet medical need. Almotriptan malate is a serotonin (5-HT1B/1D) receptor agonist approved for the treatment of acute migraine in adults. It is currently available in an oral tablet dosage form and has a Tmax of 1-3 h, and therefore, there is a medical need to develop a non-invasive rapidly acting formulation. We have developed an intranasal formulation of almotriptan malate using the quality-by-design (QbD) approach. A 2-factor 3-level full factorial design was selected to build up the experimental setting. The developed formulation was characterized for pH, viscosity, in vitro permeation, ex vivo permeation, and histopathological tolerance. To assess the potential of the developed formulation to produce a rapid onset of action following intranasal delivery, a pharmacokinetic study was performed in the Sprague-Dawley rat model and compared to the currently available marketed oral tablet formulation. For this, the LC-MS/MS bioanalytical method was developed and used for the determination of plasma almotriptan malate concentrations. Results of a pharmacokinetic study revealed that intranasal administration of optimized almotriptan malate formulation enabled an almost five-fold reduction in Tmax and about seven-fold increase in bioavailability in comparison to the currently available oral tablet formulation, suggesting the potential of developed almotriptan malate intranasal formulation in producing a rapid onset of action as well as enhanced bioavailability.

Extracellular lipase production by Yarrowia lipolytica under magnetic fields.

This study aimed at estimating cultivation conditions to enable Yarrowia lipolytica NNRL Y-1095 to produce extracellular lipase and at evaluating the influence of magnetic fields (MF) on the lipase production and on its catalytic conditions. Culture conditions of carbon sources and surfactant defined to produce extracellular lipase were 10 g L-1 glucose, 15 g L-1 olive oil and 2 g L-1 Triton X-100. The highest lipase activity (34.8 U mL-1) was reached after 144 h when MFs were applied from 72 to 144 h of culture. It corresponds to an increase of 287.5% by comparison with the highest lipase activity in the control culture. MF application from 72 to 144 h did not change the optimal temperature of lipase, which was 37 °C, by comparison with the control. However, the optimal pH of the control was 7.0 while the one of lipase produced with MF was 8.0. Findings highlighted that the presence of MFs led to increase in synthesis of lipase by Y. lipolytica, with changes in the catalytic profile. This is one of the first studies of MF application to Y. lipolytica NRRL Y-1095 cultures to produce lipase.

Gefitinib-loaded starch nanoparticles for battling lung cancer: Optimization by full factorial design and in vitro cytotoxicity evaluation.

Lung cancer is the number one killer among all cancer types. For decades, clinicians have been using conventional chemotherapeutics, but they can't rely on them alone anymore, because they poison bad cells and good cells as well. Researchers exploited nanotechnology as a potential tool to develop a platform for drug delivery to improve therapeutic efficiency. A quality by design synthesis of gefitinib-loaded starch nanoparticles (Gef-StNPs) has emerged as an essential tool to study and optimize the factors included in their synthesis. Therefore, we applied design of experiment (DOE) tools to attain the essential knowledge for the synthesis of high-quality Gef-StNPs that can deliver and concentrate the gefitinib (Gef) at A549 cells, thereby improving therapeutic efficacy and minimizing adverse effects. The in vitro cytotoxicity after exposing the A549 human lung cancer cells to the optimized Gef-StNPs was found to be much higher than that of the pure Gef (IC50 = 6.037 ± 0.24 and 21.65 ± 0.32 µg/mL, respectively). The optimized Gef-StNPs formula showed superiority over the pure Gef regarding the cellular uptake in A549 human cell line (3.976 ± 0.14 and 1.777 ± 0.1 µg/mL) and apoptotic population (77.14 ± 1.43 and 29.38 ± 1.11 %), respectively. The results elucidate why researchers have a voracious appetite for using natural biopolymers to combat lung cancer and paint an optimistic picture of their potential to be a promising tool in battling lung cancer.

A computer vision chemometric-assisted approach to access pH and glucose influence on susceptibility of Candida pathogenic strains.

Microorganisms adapt to environmental conditions as a survival strategy for different interactions with the environment. The adaptive capacity of fungi allows them to cause disease at various sites of infection in humans. In this study, we propose digital images as responses of a complete factorial 23. Furthermore, we compared two experimental approaches: the experimental design (3D) and the checkerboard assay (2D) to know the influence of pH, glucose, and fluconazole concentration on different strains of the genus Candida. The digital images obtained from the factorial 23 were used as input in the PCA-ANOVA to analyze the results of this experimental design. pH modification in the culture medium modifies the susceptibility in some species less adapted to this type of modification. For the first time, to the best of our knowledge, digital images were used as input to PCA-ANOVA to obtain information on Candida spp.. Therefore, a higher concentration of antifungals is needed to inhibit the same strain at a lower pH. In short, we present an alternative with less use of reagents and time. In addition, the use of digital images allows obtaining information about fungal susceptibility with three or more factors.

HPLC-PDA identification and resolution of rufinamide forced degradation impurities: A congregated chemometric expedite optimization coupled with factorials and desirability.

Rufinamide is used presently to treat Lenaux-Gastaut syndrome. A full factorial design and desirability approach was investigated for the optimization of hydrolytic stress via response surface curves (RSCs). The degradation impurities were identified and resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) on the Qualisil® BDS C8 column. Acetonitrile-water (29:71, v/v) was optimized for the mobile phase and used at a flow rate of 1.0 ml/min with detection at a wavelength of 230 nm. Rufinamide showed appreciable susceptibility to hydrolysis under acidic and alkaline stress, and substantial degradation in the neutral condition. It degraded much less under oxidative stress. Exposure towards thermal and photolytic stress conditions indicated appreciable stability. The developed method was subjected to validation as per the recommendations of the International Conference on Harmonization. The proposed method showed no influence from the excipients and the degradation products. As well as good precision and accuracy in determination, the method showed a linear response between 2 and 12 µg ml-1 . The method was extended for determination in a human plasma sample, which resulted in excellent recovery without interference from matrix effects. The combined use of desirability and design for the optimization of acidic and alkaline hydrolytic stress led to simple and rapid analysis.

Development of eplerenone nano sono-crystals using factorial design: enhanced solubility and dissolution rate via anti solvent crystallization technique.

OBJECTIVE: The purpose of this work was to improve EP solubility by using a sono-crystalization approach to reduce particle size and hence, increase the dissolution rate. Significance Eplerenone (EP) is an antagonist of the aldosterone receptor and is used for the treatment of hypertension and chronic heart failure. EP was classed as biopharmaceutical classification (BCS) class II because of its poor solubility and high permeability, which retards dissolution rate and drug absorption, and decreases bioavailability. METHODS: Three-factors and two-level (23) multifactorial design have been employed to study the effect of independent variables which are drug concentration; (X1), stabilizer type (X2), and stabilizer concentration (X3) on responses; saturated solubility of EP in distilled water (Y1), saturated solubility in acidic media pH 1.2 (Y2), particle size (Y3), and polydispersity index, PDI (Y4). Also, they were characterized by Fourier transformed infrared spectroscopy (FTIR), Powder X-ray diffraction (PXRD), Differential scanning calorimetry (DSC), and yield percentage. The optimum formula was further subjected to an in-vitro release study. RESULTS: The optimized formulation showed a saturated solubility of EP as 1.29, and 1.86 (mg/ml) in distilled water and acidic media (pH 1.2) respectively. Also, the particle size of 133 nm, and PDI of 0.824 with a small percentage of the difference between the observed and predicted values. Ninety-one percent of EP was released within 10 min., and it was completely released within 45 min. with a significantly higher release rate compared to raw drug. CONCLUSION: This work resulted in a satisfactory enhancement of solubility and dissolution rate which, is suitable for further in-vivo analysis.

Optimization of Meloxicam Solid Dispersion Formulations for Dissolution Enhancement and Storage Stability Using 33 Full Factorial Design Based on Response Surface Methodology.

This study aimed to formulate and optimize solid-dispersion of meloxicam (MX) employing response-surface-methodology (RSM). RSM allowed identification of the main effects and interactions between studied factors on MX dissolution and acceleration of the optimization process. 33 full factorial design with 27 different formulations was proposed. Effects of drug loading percentage (A), carriers' ratio (B), method of preparation (C), and their interactions on percent MX dissolved after 10 and 30 min (Q10min & Q30min) from fresh and stored samples were studied in distilled water. The considered levels were 2.5%, 5.0%, and 7.5% (factor A), three ratios of Soluplus®/Poloxamer-407 (factor B). Physical mixture (PM), fusion method (FM), and hot-melt-extrusion (HME) were considered factor (C). Stability studies were carried out for 3 months under stress conditions. The proposed optimization design was validated by 3-extra checkpoints formulations. The optimized formulation was selected via numerical optimization and investigated by DSC, XRD, PLM, and in vitro dissolution study. Results showed that HME technique gave the highest MX dissolution rate compared to other techniques (FM & PM). At constant level of factor (C), the amount of MX dissolved increased by decreasing MX loading and increasing Soluplus in carriers' ratio. Actual responses of the optimized formulation were in close consistency with predicted data. Amorphous form of MX in the optimized formulation was proved by DSC, XRD, and PLM. Selected factors and their levels of the optimization design were significantly valuable for demonstrating and adapting the expected formulation characteristics for rapid dissolution of MX (Q10min= 89.09%) from fresh and stored samples.

A novel quality by design approach for development and validation of a green reversed-phase HPLC method with fluorescence detection for the simultaneous determination of lesinurad, febuxostat, and diflunisal: Application to human plasma.

A novel and eco-friendly reversed-phase HPLC method with fluorescence detection was developed for simultaneous estimation of two co-administered antigout drugs (lesinurad and febuxostat) with diflunisal as a nonsteroidal anti-inflammatory drug. Unlike routine methodology, the developed method was optimized using analytical quality by design approach. A full factorial design was applied to optimize the effect of variable factors on chromatographic responses. The chromatographic separation was performed using isocratic elution on the Hypersil BDS C18 column at 40°C. The mobile phase consisted of acetonitrile:potassium phosphate buffer (30.0 mM; pH 5.5, 32.2:67.8% v/v) pumped at a flow rate of 1.0 mL/min and injection volume of 20.0 µL was employed. The proposed method was able to separate the ternary mixture in <10 min. The calibration curves of diflunisal, lesinurad, and febuxostat were linear over concentration ranges of 50.0-500.0, 50.0-700.0, and 20.0-700.0 ng/mL, respectively. Recovery percentages ranging from 98.1 to 101.3% with % relative standard deviation of <2% were obtained upon spiking to human plasma samples, indicating high bioanalytical applicability. Furthermore, the method was found to be excellent green when it was assessed according to Green Analytical Procedure Index and analytical Eco-Scale guidelines.

Toxicity inhibition strategy of microplastics to aquatic organisms through molecular docking, molecular dynamics simulation and molecular modification.

In the present study, the combined toxic effect of microplastics and their additives (five) on aquatic organisms (zebrafish) was studied using full factorial design method, molecular docking, and molecular dynamics (MD) simulation technology. The aquatic toxicity control programmer was designed to improve the optimal combination of plasticizer and microplastics based on the design of environment-friendly phthalic acid ester (PAE) derivatives. First, a total of 64 groups of microplastic-additives were designed using the full factorial design method. Next, the microplastic-additives and aquatic receptor protein were docked together, and the binding energy of these complexes was calculated using the MD simulation method. The results revealed that the aquatic toxicity effects of different microplastic-additive combinations were variable; therefore, the optimal combination of microplastics exhibiting the lowest aquatic toxicity effect could be screened out. Base on the analyzing the bonding effect and surrounded amino acid residues between the microplastic additives and receptor protein, the main driving forces for the binding of the microplastic-additive and the protein were hydrophobic force, hydrogen bonding force and electrostatic force. The main effects and the second-order interaction of the microplastic-additives combination were analyzed using the fixed-effect model. The main additives that affect the aquatic toxicity of the microplastics can be known. In addition, based on the MD simulation of the molecular replacement of PAE derivatives, the optimal level of component combination of low aquatic toxicity effect of microplastics was constructed.

A low-cost brewery waste as a carbon source in bio-surfactant production.

This work aims to produce bio-surfactant using a brewery waste (trub) as a strategy to reduce production costs related to the substrate, as well as to provide an eco-friendly destination for this residue. Trub is obtained during the boiling of the wort, being mainly composed of proteins and reducing sugars. To evaluate important process parameters on bio-surfactant production, a full factorial design (24) was elaborated, having agitation rate and concentrations of trub, yeast extract, and peptone as independent variables. The highest bio-surfactant concentration achieved was 100.76 mg L-1, where FTIR and Maldi-ToF-MS confirmed functional groups characteristic of peptides and isomers of surfactin in the bio-surfactant extract. Trub, agitation and yeast extract showed statistically significant effects on the response variable (surface tension), where an increase in the agitation rate and in the concentration of yeast extract demonstrated a positive impact on the production of bio-surfactant.

Green Tea Waste as an Efficient Adsorbent for Methylene Blue: Structuring of a Novel Adsorbent Using Full Factorial Design.

Adsorptive removal of methylene blue (MB) from contaminated water samples was achieved using green tea waste (GTW). Adsorption of MB onto raw (RGTW) and thermally treated waste (TTGTW250-TTGTW500) was explored. The performance of the tested adsorbents was assessed in terms of percentage removal of MB (%R) and adsorption capacity (qe, mg/g). A full factorial design (FFD) was employed to optimize the adsorption of MB onto both RGTW and TTGTW500. Four factors were studied: pH, adsorbent dose (AD), dye concentration (DC), and contact time (CT). Value for %R of 96.58% and 98.07% were obtained using RGTW and TTGTW500, respectively. FT-IR and Raman analyses were used to study the surfaces of the prepared adsorbents, and the IR spectrum showed the existence of a variety of functionalities on the surfaces of both the RGTW and thermally treated samples. BET analysis showed the presence of mesopores and macropores in the case of RGTW and micropores in the case of thermally processed adsorbents. Equilibrium studies indicated that the Freundlich isotherm best described the adsorption of MB onto both adsorbents. The maximum adsorption capacity (qmax) was found to be 68.28 and 69.01 mg/g for RGTW and TTGTW500, respectively, implying the superior capacity of TTGTW500 in removing MB. Adsorption of MB was found to proceed via chemisorption (RGTW) and physisorption (TTGTW500), as indicated by the Dubinin-Radushkevich (D-R) isotherm. A pseudo-second order (PSO) model best demonstrated the kinetics of the MB adsorption onto both adsorbents.

Application of experimental designs and response surface methods in screening and optimization of reverse micellar extraction.

Reverse micellar extraction (RME) has emerged as a versatile and efficient tool for downstream processing (DSP) of various biomolecules, including structural proteins and enzymes, due to the substantial advantages over conventional DSP methods. However, the RME system is a complex dependency of several parameters that influences the overall selectivity and performance of the RME system, hence this justifies the need for optimization to obtain higher possible extraction results. For the last two decades, many experimental design strategies for screening and optimization of RME have been described in literature. The objective of this article is to review the use of different experimental designs and response surface methodologies that are currently used to screen and optimize the RME system for various types of biomolecules. Overall, this review provides the rationale for the selection of appropriate screening or optimization techniques for the parameters associated with both forward and backward extraction during the RME of biomolecules.

Combined Effects of Temperature, Irradiance, and pH on Teleaulax amphioxeia (Cryptophyceae) Physiology and Feeding Ratio For Its Predator Mesodinium rubrum (Ciliophora)1.

The cryptophyte Teleaulax amphioxeia is a source of plastids for the ciliate Mesodinium rubrum and both organisms are members of the trophic chain of several species of Dinophysis. It is important to better understand the ecology of organisms at the first trophic levels before assessing the impact of principal factors of global change on Dinophysis spp. Therefore, combined effects of temperature, irradiance, and pH on growth rate, photosynthetic activity, and pigment content of a temperate strain of T. amphioxeia were studied using a full factorial design (central composite design 23 *) in 17 individually controlled bioreactors. The derived model predicted an optimal growth rate of T. amphioxeia at a light intensity of 400 µmol photons · m-2 · s-1 , more acidic pH (7.6) than the current average and a temperature of 17.6°C. An interaction between temperature and irradiance on growth was also found, while pH did not have any significant effect. Subsequently, to investigate potential impacts of prey quality and quantity on the physiology of the predator, M. rubrum was fed two separate prey: predator ratios with cultures of T. amphioxeia previously acclimated at two different light intensities (100 and 400 µmol photons · m-2 s-1 ). M. rubrum growth appeared to be significantly dependent on prey quantity while effect of prey quality was not observed. This multi-parametric study indicated a high potential for a significant increase of T. amphioxeia in future climate conditions but to what extent this would lead to increased occurrences of Mesodinium spp. and Dinophysis spp. should be further investigated.

Optimizing ectoine biosynthesis using response surface methodology and osmoprotectant applications.

PURPOSE: Numerous applications of compatible salts (osmolytes) as ectoine in food and pharmaceutical industries have been intensively increased nowadays. Decreasing the cost of industrial production of ectoine using low-cost cultivation media and improving the yield through modeling procedures are the main scopes of the present study. METHODS: Three statistical design experiments have been successfully applied for screening the parameters affecting the production process, studying the relations among parameters and optimizing the production using response surface methodology. RESULTS: A novel semi-synthetic medium based on hydrolyzed corn gluten meal has been developed to cultivate moderate halophilic bacterial strains; Vibrio sp. CS1 and Salinivibrio costicola SH3, and support ectoine synthesis under salinity stress. Two regression equations describe the production process in the new medium have been formulated for each bacterial strain. Response surface optimizer of the central composite model predicts the maximum ectoine production is achieved at incubation time; 63.7 h, pH; 7.47 and salinity; 7.27% for Vibrio sp. CS1 whereas these variables should be adjusted at 56.95 h, 7.089 and 10.34%; on the same order regarding Salinivibrio costicola SH3. In application studies, 50 µg ectoine decreases RBCs hemolysis due to streptolysin O toxin by 21.7% within ten minutes. In addition, 2% ectoine succeeds to increase the viability of lactic acid bacteria in Yogurt as a classic example of functional food during the storage period (7 days). CONCLUSION: The present study emphasizes on modeling the process of ectoine production by halophilic bacteria as well as its activity as a cryoprotectant agent.