Downstream processing of lactic acid-whey permeate fermentation broths by hollow fiber ultrafiltration

Membrane filtration is a suitable method for cell harvesting and clarification of fermentation broths. Hollow fiber ultrafilters gave essentially 100% rejection ofL. bulgaricus cells from a whey permeate fermentation broth. A combination of low pressures and high velocity generally gave the best permeate flux. Fermentation media components (in this case, from the whey permeate) contributed significantly to fouling. Considering the pressure limitations of the current generation of asymmetric hollow fiber modules and the changes in physical properties of the fermentation broths, a cell concentration of 100–150 g/L could be obtained with the flux still relatively high (above 20LMH), although the chemical compatibility of the membrane module itself under long-term exposure to high acid conditions should be considered.     

Resistance in series model for micellar enhanced ultrafiltration of eosin dye

A study has been performed to quantify the extent of flux decline during micellar enhanced ultrafiltration (MEUF) of an acid dye (eosin red) using hexadecyl (cetyl) pyridinium chloride as the cationic surfactant. Effects of the operating conditions, e.g., transmembrane pressure drop and feed-surfactant-to-dye ratio, on the permeate flux profile and observed retention have been investigated in an unstirred batch ultrafiltration (UF) cell. A simple resistance-in-series model has been used to quantify the flux decline. From the flux decline history, it has been found that the membrane permeability decreases rapidly due to reversible pore blocking and further flux decline is caused by the growth of a gel-type layer over the membrane surface. The different resistances and growth kinetics of the gel layer have been investigated as functions of the operating conditions.     

Rheological behavior of fermentation broths in antibiotic industry

An experimental study of rheological behavior of three morphological types of fermentation broths in relation to process conditions was carried out in industrial and pilot-plant bioreactors. Three industrially manufactured antibiotic broths were studied: Cephalosporin C (fungal), tylosin (actinomycetic), and apramycin (proactinomycetic), in which the rheology may cause real problems. A viscometer with coaxial cylinders and six-blade impeller was used for rheological measurements. The shear rates range in which both the instruments give similar results was determined. During the experiments rheological and morphological behavior, pH, activity, biomass concentration, fats, glucose, and nitrogen concentrations were studied. All the broths studied exhibited nonNewtonian behavior, which could be described with Ostvald-de-Waele power-law model. It was found that the rheological behavior of the broths during the process is closely related to biomass concentration and morphological changes of mycelia, so that viscosity could be used as a parameter for process monitoring and regulation. A set of experiments on oxygentransfer improvement in the case of the most viscous tylosin broths by water addition was also done.     

Separation of cells and proteins from fermentation broth in a shear-enhanced cross-flow ultrafiltration module as the first step in the refinement of lactic acid

A shear-enhanced, cross-flow ultrafiltration module was used to separate cells and proteins from the fermentation broth. Three (fermented) media were studied: rich medium, rich medium with hydrolytic enzymes added after fermentation, and wheat flour hydrolysate. To find a membrane with as high a flux as possible, but still capable of separating cells and proteins from the lactic acid containing broth, the performance of three hydrophilic membranes of varying cutoffs (10,000, 20,000, and 30,000) and one hydrophobic membrane (cutoff 25,000) was investigated. The proteins produced by the lactic acid bacteria during fermentation and the hydrolytic proteins were retained by the hydrophilic membrane with a cutoff of 20,000, whereas wheat flour proteins were detected in the permeate. In the permeates from the hydrophobic membrane (cutoff 25,000), almost no proteins were detected. The flux of the whole-wheat flour hydrolysate was significantly lower than that of rich medium, for both the hydrophilic and the hydrophobic membranes. The flux was, in all cases, higher for the hydrophilic membrane (12–85 L/[m²·h], depending, on which medium was treated) than for the hydrophobic one (8–45 L/[m²·h]), even though the nominal cutoffs of the hydrophobic and hydrophilic membranes were almost the same. However, the difference in flux was smaller when the whole-wheat flour hydrolysate was processed (12 vs 8 L/[m²·h]) than when the rich medium was processed (85 vs 45 L/[m²·h]). Protein retention was higher for the hydrophobic membrane than for the hydrophilic membrane (cutoff 20,000) owing to blocking of the pores by proteins adsorbed on to the hydrophobic membrane surface.     

Bacillus subtilis epoxide hydrolase-catalyzed preparation of enantiopure 2-methylpropane-1,2,3-triol monobenzyl ether and its application to expeditious synthesis of (R)-bicalutamide

Expeditious synthesis of (R)-bicalutamide (1), a synthetic antiandrogen, from enantiopure 2-methylpropane-1,2,3-triol monobenzyl ether (4) was achieved. An engineered Bacillus subtilis epoxide hydrolase worked enantioselectively on the racemic epoxide (7) to provide the above starting material in highly enantiomerically enriched state.     

Extraction of surfactin from fermentation broth with n-hexane in microporous PVDF hollow fibers: Significance of membrane adsorption

In order to avoid foaming behavior and the formation of stable emulsions in traditional extraction, non-dispersive extraction of surfactin from the fermentation broth of Bacillus subtilis ATCC 21332 culture with n-hexane was studied in microporous polyvinylidene fluoride (PVDF, pore size 0.2 μm) hollow fiber module. In this work, the broth was pretreated by acid precipitation and the precipitate was then dissolved in NaOH solution, and the treated broth was passed through the lumen side of the module and n-hexane was flowed across the shell side. Experiments were performed at a fixed pH of 8.0 and a flow rate of both phases of 2.5 mL min⁻¹ but at different surfactin concentrations (300–3000 mg L⁻¹). Under the conditions studied, it was shown that surfactin was adsorbed onto the surface of the fibers, instead of being extracted by n-hexane and transported through the pores of the fibers into bulk n-hexane phase. The adsorption capacity was determined and the adsorption dynamics was analyzed. The purity of surfactin desorbed from the fibers with ethanol was found to be higher than that obtained after solvent extraction with n-hexane.     

Study on the interaction of plasma protein binding rate between edaravone and taurine in human plasma based on HPLC analysis coupled with ultrafiltration technique

In this work, two high‐performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and L ‐glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o‐phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C₁₈ column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate–acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)–methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.     

The nebramycin aminoglycoside profiles of Streptomyces tenebrarius and their characterization using an integrated liquid chromatography-electrospray ionization-tandem mass spectrometric analysis

The development of an efficient analytical protocol for the reliable identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is both challenging and essential for further studies into gene-to-metabolite networks. A simple and highly selective method for detecting the biosynthetic intermediates involved in the aminoglycosidic nebramycin pathway of Streptomyces tenebrarius was developed and validated. Cleanup utilizing a solid-phase extraction (OASIS MCX SPE) technique provides a simple and reproducible method for extracting the nebramycin factors from a fermentation broth. The separation of each factor through a reversed-phase C₁₈ column using an ion-pairing reagent allowed the simultaneous profiling of the aminoglycosides. By employing the authentic tobramycin spiked into a blank fermentation medium, the combined use of acid extraction, OASIS MCX SPE cleanup, and HFBA (heptafluorobutyric acid)-mediated chromatographic separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection was proven to be sufficiently accurate and reliable to analyze the nebramycin factors produced in a culture broth. The detection limit of tobramycin spiked in the culture broth was approximately 1.8 ng mL⁻¹. The mean recovery ranged from 89 to 92%, the intra- and inter-day precision (RSD) was <6% and their accuracy ranged from 88 to 93%. A total of nine nebramycin factors including apramycin, 6″-O-carbamoylkanamycin B, 6″-O-carbamoyltobramycin, 3′-hydoxyapramycin, tobramycin, kanamycin B, NK-1012-1, nebramine, and neamine were identified. This is the first report on the integrated LC-ESI-MS/MS characterization of a wide range of nebramycin factors from a bacterial fermentation broth.     

Electrochemical biosensors for monitoring malolactic fermentation in red wine using two strains of Oenococcus oeni

Amperometric biosensors for the determination of l-malic and l-lactic acids were optimised and used to monitor micro-malolactic fermentations (micro-MLFs) in red wine. Platinum-based probes, coupled with appropriate enzymes, were assembled in electrochemical flow-injection analysis systems. A classical lactate oxidase based sensor was used for l-lactic acid, while l-malic acid was detected via a new biosensor based on the malic enzyme immobilised in a reactor using phenazine methosulphate as mediator. After a preliminary optimisation phase, a recovery study to evaluate the effect of the matrix (red wine) on biosensor performance was carried out by the addition of different standard solutions of the two analytes to the samples. Recoveries from 93 to 100% and from 94 to 102% were observed for l-malic acid and l-lactic acid, respectively. These optimised biosensors were finally employed to monitor micro-MLFs induced by inoculation of two different strains of Oenococcus oeni into red wine. During the micro-MLFs, samples of wine were collected and assayed for l-malic, l-lactic, and citric acids by use of both biosensors and spectrophotometric techniques. In parallel the viable bacterial cell count was also evaluated. The kinetics of bacterial growth, degradation of l-malic and citric acids, and production of l-lactic acid was found to be a function of the strains inoculated.     

Applications of in vivo and in vitro solid-phase microextraction techniques in plant analysis: A review

As a very popular sample preparation technique, solid-phase microextraction (SPME) coupled with various analytical instrumentation, has been widely used for the determination of trace levels of different plant compounds, such as volatile organic compounds (VOCs) emitted from the different plant organs, and environmental contaminants in plants. In this review, recent applications of in vitro and in vivo SPME in plant analysis are discussed and summarized according to the different organs of plants, including fruits, flowers, leaves, stems, roots and seeds, and the whole plant as well. Future developments and applications of SPME in plant analysis, especially in vivo sampling approaches, are also prospected.     

Speciation analysis of arsenic in Mya arenaria Linnaeus and Shrimp with capillary electrophoresis-inductively coupled plasma mass spectrometry

An improved sheath-flow interface used to couple capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS) and a microwave-assisted extraction used to extract each arsenic species in seafood were developed in this work. The improved sheath-flow interface completely avoids laminar flow in CE capillary caused by the suction from ICP-MS, makes electric supply more stable in CE, and transports analyte solution to ICP-MS easily and more efficiently. CE-ICP-MS coupled with our interface have two quantitative analysis modes: continuous sample-introduction mode and collective sample-introduction mode. The collective sample-introduction technique greatly reduced the dead volume of interface to approximately zero, obviously avoided the excessive dilution of analyte, and eventually led to a much lower detection limit and a much better electrophoretic resolution. This was demonstrated by the better symmetry and narrow peak widths (10-12 s) and much lower detection limits (0.030-0.042 μg/L) of four species of arsenic determined with collective sample-introduction mode.With the help of this improved sheath-flow interface and the microwave-assisted extraction, we have successfully separated and determined four arsenic species, As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in dried Mya arenaria Linnaeus and Shrimp samples using CE-ICP-MS within 10 min with a relative standard deviation of 2-4% (peak areas, n = 6) and a recovery of 96-105%.     

Simultaneous determination of amino acids and carbohydrates in culture media of Clostridium thermocellum by valve-switching ion chromatography

An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5–4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum.     

Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen)

This review summarizes the recent advances in the chemical analysis of Danshen and its finished products, including the introduction of the identified bioactive components, analytical methods for quantitative determination of target analytes and fingerprinting authentication, quality criteria of Danshen crude herb and its preparations, as well as the pharmacokinetic and pharmacodynamic studies on the active components of Danshen and its finished products. Danshen contains mainly two types of constituents, the hydrophilic depsides and lipophilic diterpenoidal quinones and both of them are responsible for the pharmacological activities of Danshen. In order to monitor simultaneously both types of components which have different physicochemical properties, numerous analytical methods have been reported using various chromatographic and spectrophotometric technologies. In this review, 110 papers on analysis of Danshen are discussed, various analytical methods and their chromatographic conditions are briefly described and their advantages/disadvantages are compared. For obtaining a quick, accurate and applicable analytical approach for quality evaluation and establishing a harmonized criteria of Danshen and its finished products, the authors’ suggestion and opinions are given, including the reasonable selection of marker compounds with high concentration and commercial availability, a simple sample preparation procedure with high recoveries of both the hydrophilic phenols and lipophilic tanshinones, and an optimized chromatographic condition with ideal resolutions of all the target components. The chemical degradation and transformation of the predominant constituent salvianolic acid B in Danshen during processing and manufacturing are also emphasized in order to assure the quality consistency of Danshen containing products.     

Crystallographic and infrared spectroscopic study of bond distances in Ln[Fe(CN)6]·4H2O (Ln=lanthanide)

Along with crystallographic data of Ln[Fe(CN)₆]·4H₂O (Ln=lanthanide), the infrared spectra are reassigned to examine bond length trends across the series of Ln. The changes in mean Ln-O, Ln-N, CN and Fe-C distances are discussed and the bond natures of Ln-N and Ln-O are studied by bond length linear or quadratic fitting and comparisons with relevant ionic radii. The two different CN bond distances have been simulated by the covalo-electrostatic model.     

Chemical and thermal analysis of the biopolymers in thyme (Thymus vulgaris)

Thyme (Thymus vulgaris) has been known, long time ago, for its aromatic properties. It contains essential oils and polymers such as cellulose (mixture of hemicellulose and cellulose) and lignin. The thyme, studied in this work, was gathered from the same place, in the period from November 1999 to October 2000. The chemical analysis (water, total ash, essential oils, extractive substances, cellulose, holocellulose and lignin) can be used roughly in the characterisation of the four periods that correspond to the four seasons of the year. The cellulose level was found to be more than lignin level in the wet periods (growth of the plant). The opposite was found in the dry periods. The total ash and essential oil levels were found to be high during the period of high pluviometry. The thermal decomposition of cellulose and holocellulose was found to fit well with the first-order kinetics. The activation energy, under air flow, was 185 and 196 kJ mol⁻¹ for cellulose and holocellulose, respectively. The maximum decomposition rate and thermal analysis heating rate of lignin were found to have a direct linear relationship.     

Application of J(C,H) coupling constants in conformational analysis

Conformational equilibria for a number of methyl substituted 1,3-dioxanes 1, 1,3-oxathianes 2 and 1,3-dithianes 3 were calculated at the HF and DFT levels of theory. In addition to the chair conformers also the energetically adjacent twist conformers were considered and the positions of the corresponding conformational equilibria estimated. On the basis of the global energy minima of conformers, participating in the conformational equilibria, the ¹J_C,Hax,equ coupling constants were calculated using the GIAO method and compared with the experimental values obtained from ¹³C,¹H coupled ¹³C NMR spectra. The Perlin effect, the influence of the solvent and the suitability of this NMR parameter for assigning the conformational equilibria present are critically discussed.     

Solvation of monovalent anions in acetonitrile and N,N-dimethylformamide: Parameterization of the IEF-PCM model

The present work reports the parameterization of the polarizable continuum model for predicting the free energies of solvation for monovalent anions in acetonitrile and N,N-dimethylformamide. The parameterization of the model for acetonitrile employed the experimental free energies of solvation for a set of 12 charged solutes, containing H, C, N, O, S, F, Cl, Br, and I atoms. For the N,N-dimethylformamide solutions, experimental solvation free energies for 11 monovalent anions were used. A mean absolute error of 0.7 kcal/mol in the solvation free energies has been achieved for the 12 anions in acetonitrile, whereas the mean absolute error for the 11 anions corresponds to 0.5 kcal/mol in N,N-dimethylformamide. These results indicate that the polarizable continuum model is a suitable methodology for the study of thermodynamic effects in solutions of monovalent anions in both solvents.     

Optimization of matrix solid-phase dispersion extraction method for the analysis of isoflavones in Trifolium pratense

A method based on matrix solid-phase dispersion (MSPD) has been developed for the determination of 12 isoflavones in Trifolium pratense L. Dried leaf samples were blended with C₁₈, placed in small columns and isoflavones extracted with dichloromethane–methanol. Analyses were performed by high performance liquid chromatography with diode array detection (HPLC-DAD) with 2-methoxyflavone as internal standard. Several dispersants, eluents and clean-up steps were tested during the optimization of the process in order to obtain the best selectivity and yields. Mean recoveries ranged from 70% to 119%, with relative standard deviations <18%. The limits of detection were between 0.006 mg/l for biochanin A and 0.108 mg/l for daidzin. The performance of the optimized method in real samples was compared with a conventional method based in solid–liquid extraction (SLE).     

Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry

This study presents a novel and rapid method to identify chemical markers for the quality control of Radix Aconiti Preparata, a world widely used traditional herbal medicine. In the method, the samples with a fast extraction procedure were analyzed using direct analysis in real time mass spectrometry (DART MS) combined with multivariate data analysis. At present, the quality assessment approach of Radix Aconiti Preparata was based on the two processing methods recorded in Chinese Pharmacopoeia for the purpose of reducing the toxicity of Radix Aconiti and ensuring its clinical therapeutic efficacy. In order to ensure the safety and effectivity in clinical use, the processing degree of Radix Aconiti should be well controlled and assessed. In the paper, hierarchical cluster analysis and principal component analysis were performed to evaluate the DART MS data of Radix Aconiti Preparata samples in different processing times. The results showed that the well processed Radix Aconiti Preparata, unqualified processed and the raw Radix Aconiti could be clustered reasonably corresponding to their constituents. The loading plot shows that the main chemical markers having the most influence on the discrimination amongst the qualified and unqualified samples were mainly some monoester diterpenoid aconitines and diester diterpenoid aconitines, i.e. benzoylmesaconine, hypaconitine, mesaconitine, neoline, benzoylhypaconine, benzoylaconine, fuziline, aconitine and 10-OH-mesaconitine. The established DART MS approach in combination with multivariate data analysis provides a very flexible and reliable method for quality assessment of toxic herbal medicine.