Process Development for the Detoxification of Fermentation Inhibitors from Acid Pretreated Microalgae Hydrolysate

The aim of this study was to remove 5-hydroxymethyl furfural (5-HMF) and furfural, known as fermentation inhibitors, in acid pretreated hydrolysates (APH) obtained from Scenedesmus obliquus using activated carbon. Microwave-assisted pretreatment was used to produce APH containing glucose, xylose, and fermentation inhibitors (5-HMF, furfural). The response surface methodology was applied to optimize key detoxification variables such as temperature (16.5–58.5 °C), time (0.5–5.5 h), and solid–liquid (S-L) ratio of activated carbon (0.6–7.4 w/v%). Three variables showed significant effects on the removal of fermentation inhibitors. The optimum detoxification conditions with the maximum removal of fermentation inhibitors and the minimum loss of sugars (glucose and xylose) were as follows: temperature of 36.6 °C, extraction time of 3.86 h, and S-L ratio of 3.3 w/v%. Under these conditions, removal of 5-HMF, furfural, and sugars were 71.6, 83.1, and 2.44%, respectively, which agreed closely with the predicted values. When the APH and detoxified APH were used for ethanol fermentation by S. cerevisiae, the ethanol produced was 38.5% and 84.5% of the theoretical yields, respectively, which confirmed that detoxification using activated carbon was effective in removing fermentation inhibitors and increasing fermentation yield without significant removal of fermentable sugars.     

The equilibrium characteristics of pyridoxine hydrochloride sorption on clinoptilolite

Pyridoxine hydrochloride (vitamin B₆) sorption by clinoptilolite from aqueous solutions has been studied at 283, 295, and 317 K. Enhancement of the sorption with decreasing temperature has been found. Isotherm of pyridoxine hydrochloride adsorption from dilute solutions may be described by the Langmuir theory. It has been shown that the monolayer coverage of clinoptilolite surface with pyridoxine occurs via the equivalent exchange with extra-framework cations of the sorbent. Thermodynamic parameters of vitamin monolayer adsorption have been determined. It has been concluded that the polymolecular adsorption is due to the formation of pyridoxine associates in the sorbent mesopores.     

Multisyringe Flow Injection Analysis of Tropomyosin Allergens in Shellfish Samples

This paper presents the development and the application of a multisyringe flow injection analysis system for the fluorimetric determination of the major heat-stable known allergen in shrimp, rPen a 1 (tropomyosin). This muscle protein, made up of 284 amino acids, is the main allergen in crustaceans and can be hydrolyzed by microwave in hydrochloric acid medium to produce glutamic acid, the major amino acid in the protein. Glutamic acid can then be quantified specifically by thermal conversion into pyroglutamic acid followed by chemical derivatization of the pyroglutamic acid formed by an analytical protocol based on an OPA-NAC reagent. Pyroglutamic acid can thus be quantified between 1 and 100 µM in less than 15 min with a detection limit of 1.3 µM. The method has been validated by measurements on real samples demonstrating that the response increases with the increase in the tropomyosin content or with the increase in the mass of the shrimp sample.     

Multiresidue determination of pesticides in fruit and vegetables by gas chromatography/tandem mass spectrometry

Pesticide residues in fruit and vegetables were determined by gas chromatography/tandem mass spectrometry (GC/MS/MS). Electron impact (EI)/MS/MS and chemical ionization (CI)/MS/MS were developed for 80 compounds, including organochlorine, organophosphorus, organonitrogen, and pyrethroids, providing unambiguous spectral confirmation for these complex matrixes. Residues were extracted from samples with acetone followed by a mixture of dichloromethane-petroleum ether. Two injections per sample were required for analysis of the entire pesticide list by EI/MS/MS and CI/MS/MS. Initial steps involving cleanup and concentration of extracts were eliminated. The excellent selectivity and good linearity allowed quantification and identification of low levels of pesticides in the most difficult matrixes. The method has been used for routine analysis of many vegetables.     

Putative Role of a Streptomyces coelicolor-Derived α-Mannosidase in Deglycosylation and Antibiotic Production

SCO0948 was found to be the single open reading frame annotated to encode an α-mannosidase (AM1) in Streptomyces coelicolor M145. To characterize the protein, we overexpressed SCO0948 in Escherichia coli BL21(DE3). Recombinant AM1, with a molecular weight of 110 kDa, exhibited α-mannosidase activity toward 4-nitrophenyl-α-d-mannopyranoside with a K _m of 4.61 mM, a V _max of 101.6 mM/min, and a specific activity of 47.96 U/mg. Treatment of ovalbumin, a glycoprotein, with AM1 resulted in partial deglycosylation, as assessed by glycostaining and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The S. coelicolor deletion mutant for SCO0948 failed to produce α-mannosidase activity, confirming AM1 as the only α-mannosidase in S. coelicolor M145. Interestingly, the deletion mutant and a complementation strain produced lower levels of the antibiotics actinorhodin and undecylprodigiosin in glucose minimal media. The results indicate that AM1 as an α-mannosidase influences deglycosylation and antibiotic production in S. coelicolor M145.     

Controlled adhesion of Salmonella Typhimurium to poly(oligoethylene glycol methacrylate) grafts

Poly(oligoethylene glycol methacrylate), POEGMA, brushes were prepared by surface‐initiated atom transfer radical polymerization (SI‐ATRP) on gold‐coated silicon wafers. Prior to ATRP, the substrates were grafted by brominated aryl initiators via the electrochemical reduction of a noncommercial parent diazonium salt of the formula BF₄^?, ⁺N₂‐C₆H₄‐CH(CH₃)Br. The diazonium‐modified gold plates (Au‐Br) served as macroinitiators for ATRP of OEGMA which resulted in hydrophilic surfaces (Au‐POEGMA) that could be used for two distinct objectives: (i) resistance to fouling by Salmonella Typhimurium; (ii) specific recognition of the same bacteria provided that the POEGMA grafts are activated by anti‐Salmonella. The Au‐POEGMA plates were characterized by XPS, polarization modulation‐infrared reflection‐absorption spectroscopy (PM‐IRRAS) and contact angle measurements. Both Beer‐Lambert equation and Tougaard's QUASES software indicated a POEGMA thickness that exceeds the critical ～10 nm value necessary for obtaining a hydrophilic polymer with effective resistance to cell adhesion. The Au‐POEGMA slides were further activated by trichlorotriazine (TCT) in order to covalently bind anti‐Salmonella antibodies (AS). The antibody‐modified Au‐POEGMA specimens were found to specifically attach Salmonella Typhimurium bacteria. This work is another example of the diazonium salt/ATRP process to provide biomedical polymer surfaces. Copyright © 2011 John Wiley & Sons, Ltd.     

Proteome Analysis of Abundant Proteins Extracted from the Leaf of Gynura procumbens (Lour.) Merr

Gynura procumbens (Lour.) Merr. is a traditionally used medicinal plant to decrease cholesterol level, reduce high blood pressure, control diabetics, and for treatment of cancer. In our present study, a proteomic approach was applied to study the proteome of the plant that had never analyzed before. We have identified 92 abundantly expressed proteins from the leaves of G. procumbens (Lour.) Merr. Amongst the identified proteins was miraculin, a taste-masking agent with high commercial value. Miraculin made up ～0.1% of the total protein extracted; the finding of miraculin gave a great commercial value to G. procumbens (Lour.) Merr. as miraculin's natural source is limited while the production of recombinant miraculin faced challenges of not being able to exhibit the taste-masking effect as in the natural miraculin. We believe the discovery of miraculin in G. procumbens (Lour.) Merr., provides commercial feasibility of miraculin in view of the availability of G. procumbens (Lour.) Merr. that grow wildly and easily in tropical climate.     

Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection. An analytical comparison

Carbaryl, carbofuran and methiocarb are three of the most important N-methylcarbamate pesticides. In the present work, the application of laboratory-developed monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) to the determination of these compounds in fruits and vegetables is described. Cucumbers and strawberries were spiked with the three carbamates at 10, 50 and 200 ppb. After extraction and clean-up, samples were analyzed by immunoassay and by HPLC with post-column derivatization and fluorescence detection (US Environmental Protection Agency Method 531.1). Results obtained by ELISA correlated well with those obtained by HPLC, both in terms of accuracy and precision. Recoveries were in the 60-90% range by ELISA and in the 50-90% range by HPLC, depending on the particular combination of commodity, pesticide, and fortification level under consideration. ELISAs were also applied to the analysis of non-purified sample extracts with excellent results: recoveries close to 100% were obtained, while maintaining similar precision values. This approach avoids the use of solid-phase extraction columns, saves time, and considerably increases the sample throughput. Results clearly indicate that the developed immunoassays may be suitable for the quantitative and reliable determination of carbaryl, carbofuran and methiocarb in fruits and vegetables even without including clean-up steps. These considerations make these ELISAs very useful analytical tools for monitoring and regulatory programs, without the need of complex and expensive instrumentation.