Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48?±?0.02 g g^?1), theoretical yield (93?±?3 %), and extracellular invertase productivity (1,430?±?50 IU l^?1 h^?1), respectively, when fermenting 180 g sugars l^?1 in molasses medium at 43 °C in 300 m³ working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M^?1) and entropy (ΔS*) (?202.88 J M^?1 K^?1) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l^?1 h^?1) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.     

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l^?1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol^?1 added cysteine and a productivity of 6.1 ± 0.0 g l^?1 h^?1. This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.     

Phospholipid/Polydiacetylene Vesicle-Based Colorimetric Assay for High-Throughput Screening of Bacteriocins and Halocins

Multi-phase anaerobic reactor for H₂ and CH₄ production from paperboard mill wastewater was studied. The reactor was operated at hydraulic retention times (HRTs) of 12, 18, 24, and 36 h, and organic loading rates (OLRs) of 2.2, 1.5, 1.1, and 0.75 kg chemical oxygen demand (COD)/m³ day, respectively. HRT of 12 h and OLR of 2.2 kg COD/m³ day provided maximum hydrogen yield of 42.76?±?14.5 ml/g COD_removed and volumetric substrate uptake rate (?rS) of 16.51?±?4.43 mg COD/L h. This corresponded to the highest soluble COD/total COD (SCOD/TCOD) ratio of 56.25?±?3.3 % and the maximum volatile fatty acid (VFA) yield (Y_VFA) of 0.21?±?0.03 g VFA/g COD, confirming that H₂ was mainly produced through SCOD conversion. The highest methane yield (18.78?±?3.8 ml/g COD_removed) and ?rS of 21.74?±?1.34 mgCOD/L h were achieved at an HRT of 36 h and OLR of 0.75 kg COD/m³ day. The maximum hydrogen production rate (HPR) and methane production rate (MPR) were achieved at carbon to nitrogen (C/N) ratio of 47.9 and 14.3, respectively. This implies the important effect of C/N ratio on the distinction between the dominant microorganism bioactivities responsible for H₂ and CH₄ production.     

Determination of Nateglinide in Human Plasma by LC-ESI-MS and Its Application to Bioequivalence Study

To evaluate the bioequivalence of nateglinide, a rapid and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated to determine nateglinide for human plasma samples. The analyte was detected using electrospray positive ionization mass spectrometry in the selected ion monitoring mode. Tinidazole was used as the internal standard. A good linear relationship obtained in the concentration ranged from 0.05 to 16 μg mL^?1 (r ² = 0.9993). Lower limit of quantification was 0.05 μg mL^?1 using 100 μL of plasma sample. Intra- and inter-day relative standard deviations were 2.1–7.5 and 4.7–8.9%, respectively. Among the pharmacokinetic data obtained, T _max was 2.09 ± 1.06 h for reference formulation and 2.40 ± 0.97 h for test formulation. C _max was 4.17 ± 1.31 μg mL^?1 for reference formulation and 4.37 ± 1.53 μg mL^?1 for test formulation. The half-life (t _½) was 1.93 ± 0.44 h for reference formulation and 1.92 ± 0.29 h for test formulation. AUC_0–10h was 13.67 ± 4.36 μg h mL^?1 for reference formulation and 13.21 ± 4.09 μg h mL^?1 for test formulation. This method was successfully applied to the pharmacokinetic study in human plasma samples.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Evaluation of 222Rn and 226Ra concentrations in cement and limestone of Sheikh Buddin Hill,Pezu, Pakistan using different techniques

Lucky Cement Factory, Pezu is using limestone of Sheikh Buddin Hills as a raw material in cement. Workers of the factory have direct and general public have indirect exposure to radiological hazard due to natural radionuclides present in limestone. To address the radiological hazards, limestone, mixed (limestone+clay) and cement samples were evaluate for concentrations of ²²²Rn and ²²⁶Ra using CR-39, RAD7 and HPGe detectors. Maximum mean values of ²²²Rn using CR-39 and RAD7 detectors were found 1447 ± 198 and 1416 ± 74 Bq.m^?3 in cement samples and minimum were found in 536 ± 122 and 525 ± 45 Bq.m^?3 limestone samples, respectively. Maximum mean value of radon exhalation rate of 12.28 ± 1.68 Bq.m^?2 h^?1 in cement samples was found below the world average value of 57.6 Bq.m^?2 h^?1. Maximum mean values of ²²⁶Ra measured by CR-39 and HPGe detectors were found 24.25 ± 3.35 and 23.6 ± 0.70 Bq.kg^?1 in cement samples and minimum were found in 8.98 ± 2.02 and 9.19 ± 0.40 Bq.kg^?1 limestone samples, respectively. A positive correlations (R² = 0.9714) using CR-39 and RAD7 detectors and (R² = 0.9573) using CR-39 and HPGe detectors were obtained for the concentrations of ²²²Rn and ²²⁶Ra, respectively. Maximum mean value of annual effective dose of 347.78 ± 47.58 µSv.y^?1 in cement samples was found below the world average value of 1100 µSv.y^?1.     

Single-cell Protein and Xylitol Production by a Novel Yeast Strain <Emphasis Type="Italic">Candida intermedia</Emphasis> FL023 from Lignocellulosic Hydrolysates and Xylose

Yeasts are good candidates to utilize the hydrolysates of lignocellulose, the most abundant bioresource, for bioproducts. This study aimed to evaluate the efficiencies of single-cell protein (SCP) and xylitol production by a novel yeast strain, Candida intermedia FL023, from lignocellulosic hydrolysates and xylose. This strain efficiently assimilated hexose, pentose, and cellubiose for cell mass production with the crude protein content of 484.2 g kg^?1 dry cell mass. SCP was produced by strain FL023 using corncob hydrolysate and urea as the carbon and nitrogen sources with the dry cell mass productivity 0.86 g L^?1 h^?1 and the yield of 0.40 g g^?1 sugar. SCP was also produced using NaOH-pretreated Miscanthus sinensis straw and corn steep liquor as the carbon and nitrogen sources through simultaneous saccharification and fermentation with the dry cell productivity of 0.23 g L^?1 h^?1 and yield of 0.17 g g^?1 straw. C. intermedia FL023 was tolerant to 0.5 g L^?1 furfural, acetic acid, and syringaldehyde in xylitol fermentation and produced 45.7 g L^?1 xylitol from xylose with the productivity of 0.38 g L^?1 h^?1 and the yield of 0.57 g g^?1 xylose. This study provides feasible methods for feed and food additive production from the abundant lignocellulosic bioresources.     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min^?1. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL^?1 for isomer I and 0.40–2.40 µg mL^?1 for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL^?1 for isomer I and 0.0356 and 0.1078 µg mL^?1 for isomer II, respectively.     

Development and Validation of an HPLC–UV Method for the Determination of Insulin in Rat Plasma: Application to Pharmacokinetic Study

A simple, sensitive high performance liquid chromatographic method with UV detection was developed and validated for determination of insulin in rat plasma, using methyl paraben as an internal standard. Insulin was extracted from plasma by a liquid–liquid extraction with a mixture of dichloromethane and n-hexane (1:1, v/v) followed by an acidic back extraction. Chromatographic separation was achieved isocratically with a Phenomenex® C₁₈ analytical column (150 × 4.6 mm ID, 5 μm) at ambient room temperature. The calibration curves were linear within a concentration range of 0.7–8.4 μg mL^?1 (r ² = 0.9994). The inter-day and intra-day accuracy and precision were ≤3.33 and ≤5.55%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.35 and 0.7 μg mL^?1. The average recovery was 87.86% for insulin and 83.52% for methyl paraben. Insulin containing plasma samples were stable at ?20 °C for 7 days. Validated HPLC method was successfully applied to a pharmacokinetic study of insulin in streptozotocin induced diabetic rats.     

Human Saliva-Based Quantitative Monitoring of Clarithromycin by Flow Injection Chemiluminescence Analysis: A Pharmacokinetic Study

Human saliva quantitative monitoring of clarithromycin (CLA) by chemiluminescence (CL) with flow injection analysis was proposed for the first time, which was based on the quenching effect of CLA on luminol–bovine serum albumin (BSA) CL system with a linear range from 7.5?×?10^?4 to 2.0 ng/ml. This proposed approach, offering a maximum sample throughput of 100 h^?1, was successfully applied to the quantitative monitoring of CLA levels in human saliva during 24 h after a single oral dose of 250 mg intake, with recoveries of 95.2～109.0 % and relative standard deviations lower than 6.5 % (N?=?7). Results showed that CLA reached maximum concentration of 2.28?±?0.02 μg/ml at approximately 3 h, and the total elimination ratio was 99.6 % in 24 h. The pharmacokinetic parameters including absorption rate constant (0.058?±?0.006 h^?1), elimination rate constant (0.149?±?0.009 h^?1) and elimination half-life time (4.66?±?0.08 h) were obtained. A comparison of human saliva and urine monitoring was also given. The mechanism study of BSA–CLA interaction revealed the binding of CLA to BSA is an entropy driven and spontaneous process through hydrophobic interaction, with binding constant K _BSA–CLA of 4.78?×?10⁶ l/mol and the number of binding sites n of 0.82 by flow injection–chemiluminescence model. Molecular docking analysis further showed CLA might be in subdomain IIA of BSA, with K _BSA–CLA of 6.82?×?10⁵ l/mol and ΔG of ?33.28 kJ/mol.     

Isolation of Novel Microalgae from Acid Mine Drainage and Its Potential Application for Biodiesel Production

Microalgae were selected and isolated from acid mine drainage in order to find microalgae species which could be cultivated in low pH condition. In the present investigation, 30 microalgae were isolated from ten locations of acid mine drainage in South Korea. Four microalgae were selected based on their growth rate, morphology, and identified as strains of KGE1, KGE3, KGE4, and KGE7. The dry biomass of microalgae species ranged between 1 and 2 g L^?1 after 21 days of cultivation. The growth kinetics of microalgae was well described by logistic growth model. Among these, KGE7 has the highest biomass production (2.05?±?0.35 g L^?1), lipid productivity (0.82?±?0.14 g L^?1), and C16–C18 fatty acid contents (97.6 %). These results suggest that Scenedesmus sp. KGE 7 can be utilized for biodiesel production based on its high biomass and lipid productivity.     

Expression,Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)⁺ to NAD(P)H. The NADP⁺-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP⁺ as the cofactor. The K _m values for l-malate and NADP⁺ were 2.19?±?0.01 and 0.38?±?0.02 mM, respectively, while the V _max values were 147?±?2 and 302?±?14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺, and low concentrations of Zn²⁺ rather than Ca²⁺, Cu²⁺, or high concentrations of Zn²⁺. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K _i values were 0.08 and 0.6 mM, respectively.     

Study on thermal decomposition and oxidation kinetics of cation exchange resins using non-isothermal TG analysis

Kinetics of two successive thermal decomposition reaction steps of cationic ion exchange resins and oxidation of the first thermal decomposition residue were investigated using a non-isothermal thermogravimetric analysis. Reaction mechanisms and kinetic parameters for three different reaction steps, which were identified from a FTIR gas analysis, were established from an analysis of TG analysis data using an isoconversional method and a master-plot method. Primary thermal dissociation of SO₃H⁺ from divinylbenzene copolymer was well described by an Avrami–Erofeev type reaction (n = 2, g(α) = [?ln(1 ? α)]^1/2]), and its activation energy was determined to be 46.8 ± 2.8 kJ mol^?1. Thermal decomposition of remaining polymeric materials at temperatures above 400 °C was described by one-dimensional diffusion (g(α) = α ²), and its activation energy was determined to be 49.1 ± 3.1 kJ mol^?1. The oxidation of remaining polymeric materials after thermal dissociation of SO₃H⁺ was described by a phase boundary reaction (contracting volume, g(α) = 1?(1 ? α)^1/3). The activation energy and the order of oxygen power dependency were determined to be 101.3 ± 13.4 and 1.05 ± 0.17 kJ mol^?1, respectively.     

An electrochemical magneto immunosensor (EMIS) for the determination of paraquat residues in potato samples

An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite–epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18?±?0.09 μg L^?1. Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90 % of the maximum signal) of 2.18?±?2.08 μg kg^?1, far below the maximum residue level (20 μg kg^?1) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.     

Investigation of volatile metabolites during growth of Escherichia coli and Pseudomonas aeruginosa by needle trap-GC-MS

A new method for the growth-dependent headspace analysis of bacterial cultures by needle trap (NT)-gas chromatography-mass spectrometry (GC-MS) was established. NTs were used for the first time as enrichment technique for volatile organic compounds (VOCs) in the headspace of laboratory cultures. Reference strains of Escherichia coli and Pseudomonas aeruginosa were grown in different liquid culture media for 48 h at 36 °C. In the course of growth, bacterial culture headspace was analysed by NT-GC-MS. In parallel, the abiotic release of volatile organic compounds (VOC) from nutrient media was investigated by the same method. By examination of microbial headspace samples in comparison with those of uninoculated media, it could be clearly differentiated between products and compounds which serve as substrates. Specific microbial metabolites were detected and quantified during the stationary growth phase. P. aeruginosa produced dimethyl sulfide (max. 125 μg L^?1??1) and 2-nonanone (max. 200 μg L^?1), whereas E. coli produced carbon disulfide, butanal and indole (max. 149 mg L^?1). Both organisms produced isoprene. Graphical Abstract

MVOCs produced by P. aeruginosa and E. coli at T = 36 °C in autoclaved LB + TRP medium      

HPLC Analysis and Pharmacokinetic Study of Paeoniflorin after Intravenous Administration of a New Frozen Dry Powder Formulation in Rats

A simple and specific high performance liquid chromatographic (HPLC) method with UV detection using picroside II as the internal standard was developed and validated to determine the concentration of paeoniflorin in rat plasma and study its pharmacokinetics after an single intravenous administration of 40 mg kg^?1 paeoniflorin to Wistar rats. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 0.05 mol L^?1 NaH₂PO₄ solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB C₁₈ column (250 × 4.6 mm I.D., 5 μm) with a Shim-pack GVP-ODS C₁₈ guard column (10 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–water–acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 mL min^?1. The UV detection was performed at a wavelength of 230 nm. The linear calibration curves were obtained in the concentration range of 0.05–200.0 μg mL^?1 in rat plasma with the lower limit of quantification (LLOQ) of 0.05 μg mL^?1. The intra- and inter-day precisions in terms of % relative standard deviation (RSD) were lower than 5.7 and 8.2% in rat plasma, respectively. The accuracy in terms of % relative error (RE) ranged from ?1.9 to 2.6% in rat plasma. The extraction recoveries of paeoniflorin and picroside II were calculated to be 69.7 and 56.9%, respectively. This validated method was successfully applied to the pharmacokinetic study of a new paeoniflorin frozen dry power formulation. After single intravenous administration, the main pharmacokinetic parameters t _1/2, AUC_0-∞, CL_TOT, V _Z, MRT_0-∞ and V _ss were 0.739 ± 0.232 h, 43.75 ± 6.90 μg h mL^?1, 15.50 ± 2.46 L kg^?1 h^?1, 1.003 ± 0.401 L kg^?1, 0.480 ± 0.055 h and 0.444 ± 0.060 L kg^?1, respectively.     

Rapid screening and detection of XOD inhibitors from S. tamariscina by ultrafiltration LC-PDA–ESI-MS combined with HPCCC

Xanthine oxidase (XOD) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction of which could cause hyperuricemia, a risk factor for gout. Inhibition of XOD is a major treatment for gout, and biflavonoids have been found to act as XOD-inhibitory compounds. In this study, ultrafiltration liquid chromatography with photodiode-array detection coupled to electrospray-ionization tandem mass spectrometry (UF-LC-PDA–ESI-MS) was used to screen and identify XOD inhibitors from S. tamariscina. High-performance counter-current chromatography (HPCCC) was used to separate and isolate the active constituents of these XOD inhibitors. Furthermore, ultrahigh-performance liquid chromatography (UPLC) and triple-quadrupole mass spectrometry (TQ-MS) was used to determine the XOD-inhibitory activity of the obtained XOD inhibitors, and enzyme kinetics was performed with Lineweaver–Burk (LB) plots using xanthine as the substrate. As a result, two compounds in S. tamariscina were screened as XOD inhibitors: 65.31 mg amentoflavone and 0.76 mg robustaflavone were isolated from approximately 2.5 g?S. tamariscina by use of HPCCC. The purities of the two compounds obtained were over 98 % and 95 %, respectively, as determined by high-performance liquid chromatography (HPLC). Lineweaver–Burk plot analysis indicated that amentoflavone and robustaflavone were non-competitive inhibitors of XOD, and the IC ₅₀ values of amentoflavone and robustaflavone for XOD inhibition were 16.26 μg mL^?1 (30.22 μmol L^?1) and 11.98 μg mL^?1 (22.27 μmol L^?1), respectively. The IC ₅₀ value of allopurinol, used as the standard, was 7.49 μg mL^?1 (46.23 μmol L^?1). The results reveal that the method for systematic screening, identification, and isolation of bioactive components in S. tamariscina and for detecting their inhibitory activity using ultrafiltration LC–ESI-MS, HPCCC, and UPLC–TQ-MS is feasible and efficient, and could be expected to extend to screening and separation of other enzyme inhibitors. Graphical Abstract

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Flow Injection Spectrophotometric Determination of the Antibacterial Levofloxacin in Tablets and Human Urine

Abstract

A sensitive and fast flow‐injection (FI) spectrophotometric method for the determination of levofloxacin based on the formation of a colored product upon oxidation with N‐bromosuccinimide (NBS) in acidic medium is proposed. Optimization of chemical and FI variables has been made. Under the optimized conditions, the sampling rate was over 90 h^?1, the calibration curve obtained was linear over the range 10–300 µg·mL^?1, and the detection limit was 3 µg·mL^?1. The proposed method was successfully applied to the determination of levofloxacin in pharmaceuticals and human urine samples. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. Results are precise (RSD<2.7%; n =10) and in agreement with those found by the reference high pressure liquid chromatography (HPLC) procedure.     

Cell-Free Supernatants Obtained from Fermentation of Cheese Whey Hydrolyzates and Phenylpyruvic Acid by Lactobacillus plantarum as a Source of Antimicrobial Compounds, Bacteriocins, and Natural Aromas

Cheese whey hydrolyzates supplemented with phenylpyruvic acid (PPA) and commercial nutrients can be efficiently metabolized by Lactobacillus plantarum CECT-221 to biosynthesize some compounds with attractive applications in the food market. The main metabolites of cell-free extracts were antimicrobial compounds such as phenyllactic acid (PLA) and lactic acid (LA). The production of PLA by L. plantarum CECT-221 was evaluated in the Man–Rogosa–Sharpe broth supplemented with two biosynthetic precursors: phenylalanine or PPA. Using 30.5 mM PPA, the microorganism increased sevenfold the concentration of PLA producing 16.4 mM PLA in 46 h. A concentration of 40 mM PPA was a threshold to avoid substrate inhibition. The biosynthesis of whey hydrolyzates as a carbon source was enhanced by fed-batch fermentation of PPA; the average productivity of PLA increased up to 45.4?±?3.02 mM after 120 h with a product yield of 0.244 mM mM^?1; meanwhile, LA reached 26.1?±?1.3 g L^?1 with a product yield of 0.72 g g^?1. Cell-free fed-batch extracts charged in wells showed bacteriocin activity with halos of 7.49?±?1.44 mm in plates inoculated with Carnobacterium piscicola and antimicrobial activity against Staphylococcus aureus (11.54?±?1.14 mm), Pseudomonas aeruginosa (10.17?±?2.46 mm), Listeria monocytogenes (7.75?±?1.31 mm), and Salmonella enterica (3.60?±?1.52 mm). Additionally, the analysis of the volatile composition of the headspace of this cell-free extract revealed that L. plantarum is a potential producer for natural aromas, such as acetophenone, with high price in the market. This is the first report of PLA production from cheese whey and PPA. The extracts showed bacteriocin activity and potential to be applied as an antimicrobial in the elaboration of safer foods.     

Analysis of drug interactions with very low density lipoprotein by high-performance affinity chromatography

High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein’s non-polar core. This partitioning was described by overall affinity constants of 1.2 (±0.3)?×?10⁶ M^?1 for R-propranolol and 2.4 (±0.6)?×?10⁶ M^?1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (±2.3)?×?10⁴ M^?1 for R-propranolol and 9.6 (±2.2)?×?10⁴ M^?1 for S-propranolol. Comparable results were obtained at 20 and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes.