High-speed Blender Research Articles

Evidence that homogenization of BSA-stabilized hexadecane-in-water emulsions induces structure modification of the nonadsorbed protein.

The structural modification of globular proteins (bovine serum albumin, BSA) in the aqueous phase of emulsions produced by homogenization was studied using front-face fluorescence spectroscopy (FFFS). A series of hydrocarbon oil-in-water emulsions (30 wt % n-hexadecane, 0.35 wt % BSA, pH 7.0) were homogenized to differing degrees with a high-speed blender and a high-pressure valve homogenizer. The wavelength of the maximum in the tryptophan emission spectrum (lambda(max)) of serum phases collected from the emulsions by centrifugation was measured and compared to lambda(max) values of BSA solutions subjected to the same homogenization conditions. There was no significant (p < 0.05) change in lambda(max) with homogenization conditions for BSA solutions. In contrast, lambda(max) of serum phases from emulsions blended for 2 min in a high-speed blender was significantly smaller (p < 0.05) than nontreated BSA solutions (Deltalambda(max) = 2 nm). In addition, there was a further significant decrease in lambda(max) of the serum phases with an increasing number of passes of the emulsion through the high-pressure valve homogenizer (e.g., Deltalambda(max) = 4 nm for 12 passes). This study shows that globular proteins present in the aqueous phase of a hexadecane-in-water emulsion after homogenization could be altered, which is probably caused by surface modification of the protein structure during temporary adsorption to emulsion droplet surfaces during homogenization.

Rapid Determination of Fosetyl-Aluminum Residues in Lettuce by Liquid Chromatography/Electrospray Tandem Mass Spectrometry

This paper describes a new method for the sensitive and selective determination of fosetyl-aluminum (Al) residues in vegetable samples. The method involves extraction with water by using a high-speed blender and subsequent injection of the 5-fold diluted extract into the liquid chromatograph. Fosetyl-Al is determined by liquid chromatography with electrospray tandem mass spectrometry after the addition of tetrabutylammonium acetate as the ion-pairing reagent. The method has been used to assay lettuce samples spiked at 2 and 0.2 mg/kg. Recoveries were satisfactory, with mean values of 98 and 106%, respectively, and relative standard deviations were < 10%. The limit of quantitation was 0.2 mg/kg, and the limit of detection was as low as 0.05 mg/kg. Matrix-matched calibration was used for quantitation, and the addition of an internal standard improved repeatability. The developed method allows the accurate and rapid determination of low levels of fosetyl-Al residues in lettuce with very little sample handling and good sensitivity; it was shown to be robust by the analysis of almost 100 samples.

Direct determination of paclobutrazol residues in pear samples by liquid chromatography-electrospray tandem mass spectrometry.

A rapid and sensitive liquid chromatography/electrospray ionization/tandem mass spectrometry (LC-ESI-MS-MS) method has been developed for the determination of the plant growth regulator paclobutrazol in pear samples. Extraction was performed with methanol by using a high-speed blender Ultra-Turrax, and 10 microL of pear extract was directly injected in the LC-ESI-MS-MS system without any previous sample treatment. The highest sensitivity of the method was achieved under MS-MS conditions obtaining a limit of detection of 0.7 microg/kg and a quantification limit of 5 microg/kg, with a run time of only 5.5 min. Recoveries for paclobutrazol from spiked pear samples at 0.005, 0.05, and 0.5 mg/kg were around 82-102% with relative standard deviations between 2 and 7%. The method was applied to real treated and untreated samples of pears, using quality control samples as a evaluation of the method reliability. Two MS-MS transitions were selected, one for quantification (294 > 70) and the other for confirmation of the analyte (296 > 70). All the experiments were performed in compliance with good laboratory practices.

Formation of intermolecular β-sheet structures: a phenomenon relevant to protein film structure at oil–water interfaces of emulsions

Oil-in-water emulsions stabilized with β-lactoglobulin ( β-lg) were made using a homogenizer or a high-speed blender. The protein was studied by Fourier transform infrared (FTIR) spectroscopy in the raw emulsion, in the bulk phase, and at the interface, as a function of pH, oil content, and homogenizing pressure. Results show that the amount of adsorbed protein varies with the available interfacial area. The protein that remains in the aqueous phase exhibit no spectral change, which suggests that homogenization causes no conformational modification or reversible ones. Strong and irreversible changes were observed in the adsorbed protein. Our findings reveal the formation of intermolecular antiparallel β-sheets upon adsorption due to the protein self-aggregation. As deduced from transmission electronic microscopy, this surface aggregation leads to the formation of continuous and homogeneous membranes coating the globules. The structure of the adsorbed proteins is unaffected by the homogenizing pressures used in our study and slightly modified by the pH. FTIR spectroscopy allows to characterize the type of aggregates formed at the interface. An analysis of the spectra of β-lg heat-induced gels shows that the aggregates at the interface are very close at a molecular scale to those that constitute particulate gels near the protein's isoelectric point. Since the type of aggregates is similar when the emulsion water phase is pure D 2O and D 2O at pD 4.4, the interface not only seems to induce aggregation, but seems to determine the type of aggregation as well. The mechanism that drives the formation of particulate aggregates (rather than fine-stranded ones) may reside in strong protein–protein interactions that are promoted by adverse oil–protein interactions.

Determination of abamectin and azadirachtin residues in orange samples by liquid chromatography–electrospray tandem mass spectrometry

A rapid and sensitive LC–ESI-MS–MS method has been developed for the determination of azadirachtin and abamectin residues in orange samples. Samples were extracted with acetonitrile, in a high-speed blender. After the addition of sodium acetate, an aliquot of extract was directly injected into the LC–ESI-MS–MS system. The highest sensitivity of the method was achieved under MS–MS conditions using [M+Na] + adducts as precursor ions. Recoveries for both compounds from spiked orange samples at 0.01 and 0.1 mg/kg were above 80%, with good repeatability (<10%). Method detection limits achieved (<0.007 mg/kg) were adequate for the determination of these pesticides in this kind of sample from the regulatory point of view. The importance of the solvent used for extraction, as well as the addition of sodium acetate to the extracts and the selection of adequate chromatographic conditions are discussed.

Method for the determination of water content in sultana raisins using a water activity probe

A new method for the fast (less than 5 min) and accurate (better than 0.5% error in water content) measurement of water in sultana raisins without the need for sample weighing has been developed. The method is based on a commercially available water sensor and probe. Additionally, a raisin press has been developed to prepare the sample for analysis in one step. The results obtained with the proposed method correlate well ( R 2=0.999) with those obtained using a high speed blending Karl Fischer auto-titrator. The method shows good analytical characteristics, the analysis time is short, and no expensive or dangerous chemical consumables are used. The method can thus be used easily by non-experienced personnel, either on site, or in the laboratory.

Protein extraction from heat-stabilized defatted rice bran. 1. Physical processing and enzyme treatments.

Physical processing with or without enzyme treatments on protein extraction from heat-stabilized defatted rice bran (HDRB) was evaluated. Freeze-thaw, sonication, high-speed blending, and high-pressure methods extracted 12%, 15%, 16%, and 11% protein, respectively. Sonication (0-100%, 750 W), followed by amylase and combined amylase and protease treatments, extracted 25.6-33.9% and 54.0-57.8% protein, respectively. Blending followed by amylase and protease treatment extracted 5.0% more protein than the nonblended enzymatic treatments. High-pressure treatments, 0-800 MPa, with water or amylase-protease combinations, extracted 10.5-11.1% or 61.8-66.6% protein, respectively. These results suggest that physical processing in combination with enzyme treatments can be effective in extracting protein from HDRB.

Hot Hardness of WC-Co/TiC-Al2O3 Composite Materials.

WC particle was coated with TiC and Al2O3 particles using Co binding one by means of high-speed rotational blending to enhance the degree of mixing. Then the sintered composite material of WC-Co/TiC-Al2O3 was prepared, and the hot hardness was measured up to 800°C to be compared with conventional WC-Co alloy. It was found that the addition of TiC and Al2O3 components in the WC-Co alloy has a strong influence on the hot hardness. The hardness increased with higher amount of TiC from 5 up to 30 by weight percentage, whereas increasing Al2O3 additions reduced the hardness. Particularly, Al2O3 was effective due to its higher chemical stability than TiC at elevated temperature. Then, the highest hardness was obtained with addition of 30TiC-5Al2O3 by weight. This suggests that these materials can further be utilized for high temperature applications.

Developing a standard spiking procedure for the introduction of hydrophobic organic compounds into field-wet soil

The purpose of this study was to reevaluate a number of spiking procedures we had previously investigated for spiking dry soil, determine their suitability for spiking radiolabeled hydrophobic organic compounds in field-wet soil, and identify the optimal spiking procedure. In the first part of this study, two radiolabeled polycyclic aromatic hydrocarbons (PAHs), phenanthrene (Phe) and benzo[a]pyrene (BaP), were introduced into field-wet soil by four different spiking procedures and the recovery and homogeneity of compound distribution assessed by analysis of replicate subsamples. The identified optimal spiking procedure involved adding an acetone spike solution to 10 ml acetone in a blender base, adding 250 g field-wet soil, and mixing with a high-speed blender. The relative standard deviations obtained by this procedure were 2.7 and 5.3% for 14C-Phe and 14C-7-BaP, respectively, and were comparable to those we obtained in a previous study for spiking dry soil. The persistence of acetone used as a carrier solvent in the optimal spiking procedure was assessed at various times after spiking to identify a suitable equilibration time to allow for solvent volatilization. The equilibration time, together with the identified optimal spiking procedure, were combined to develop a standard spiking procedure. The standard spiking procedure was used to prepare multiple soil microcosms individually spiked with radiolabeled Phe, pyrene, and BaP for a long-term aging study on the formation of PAH-bound residues in arable soil. The mean spike activity and homogeneity of spike distribution in 54 sterilized microcosms (3 × 18) stored over 525 d was determined. Mean spike recovery and relative standard deviations (RSDs) for the three radiolabeled PAHs in the individually spiked and prepared microcosms were very good. However, this data illustrates the importance of determining the concentration and homogeneity of spikes within all individual test portions in experiments where organic compounds are spiked into soil or sediment.

DEVELOPING A STANDARD SPIKING PROCEDURE FOR THE INTRODUCTION OF HYDROPHOBIC ORGANIC COMPOUNDS INTO FIELD-WET SOIL

The purpose of this study was to reevaluate a number of spiking procedures we had previously investigated for spiking dry soil, determine their suitability for spiking radiolabeled hydrophobic organic compounds in field-wet soil, and identify the optimal spiking procedure. In the first part of this study, two radiolabeled polycyclic aromatic hydrocarbons (PAHs), phenanthrene (Phe) and benzo[a]pyrene (BaP), were introduced into field-wet soil by four different spiking procedures and the recovery and homogeneity of compound distribution assessed by analysis of replicate subsamples. The identified optimal spiking procedure involved adding an acetone spike solution to 10 ml acetone in a blender base, adding 250 g field-wet soil, and mixing with a highspeed blender. The relative standard deviations obtained by this procedure were 2.7 and 5.3% for 14C-Phe and 14C-7-BαP, respectively, and were comparable to those we obtained in a previous study for spiking dry soil. The persistence of acetone used as a carrier solvent in the optimal spiking procedure was assessed at various times after spiking to identify a suitable equilibration time to allow for solvent volatilization. The equilibration time, together with the identified optimal spiking procedure, were combined to develop a standard spiking procedure. The standard spiking procedure was used to prepare multiple soil microcosms individually spiked with radiolabeled Phe, pyrene, and BaP for a long-term aging study on the formation of PAH-bound residues in arable soil. The mean spike activity and homogeneity of spike distribution in 54 sterilized microcosms (3 × 18) stored over 525 d was determined. Mean spike recovery and relative standard deviations (RSDs) for the three radiolabeled PAHs in the individually spiked and prepared microcosms were very good. However, this data illustrates the importance of determining the concentration and homogeneity of spikes within all individual test portions in experiments where organic compounds are spiked into soil or sediment.

Mixing and Lightness Characteristics of Fine Particles Coated by High-Speed Rotational Impact Blending.

Coarse white polyethylene particles were coated by fine pigment particles of red iron oxide by high-speed rotational impact blending and by hand mixing in a mortar. As the dark pigments became progressively distributed over the coarse particle surfaces, the color characteristics of the resulting powder changed. First, the effects of the mixing ratio of fine particles, the treatment time, the rotational speed of a rotor and the mean diameter of coarse particles were investigated with respect to the degree of mixing of the total mixture. The value of the degree of mixing was determined directly and precisely by chemical analysis. It was found that the degree of mixing increased with the treatment time at a certain mixing ratio of fine particles, corresponding to a coating ratio over coarse particles of less than 100% of closest packing on a number basis, but decreased at the mixing ratio of more than 100% by rotational impact. Micromixing to coat individual coarse particles was more easily and rapidly achieved by rotational impact blending as compared with hand mixing. Coating coarser particles at higher rotor speeds was more suitable for achieving homogenous mixing. In addition, it was found that the lightness of mixed particles decreased to lower than that by hand mixing due to the increased degree of mixing, but remained almost constant during the process of rotational impact blending, with its slight increase being due to embedment of fine particles by frequent impact. This means that the lightness of particles reached a steady state in a very short mixing time, resulting in the reduction of the amount of fine particles for the same lightness effect when using the high-speed rotational impact blender.

Atmospheric Methane Consumption by Forest Soils and Extracted Bacteria at Different pH Values.

The effect of pH on atmospheric methane (CH4) consumption was studied with slurries of forest soils and with bacteria extracted from the same soils. Soil samples were collected from a mixed hardwood stand in New Hampshire, from jackpine and aspen stands at the BOREAS (Boreal Ecosystem Atmosphere Study) site near Thompson, northern Manitoba, from sites in southern Québec, including a beech stand and a meadow, and from a site in Ontario (cultivated humisol). Consumption of atmospheric CH4 (concentration, approximately 1.8 ppm) occurred at depths of >5 cm in both acidic (pH 4.5 to 5.2) and alkaline (pH 7.2 to 7.8) soils. In slurries of acidic soils, maximum activity occurred at different pH values (pH 4.0 to 6.5). Bacteria extracted from these soils by high-speed blending and density gradient centrifugation showed pH responses different from the pH responses of the slurries. In all cases, these bacteria had a methanotrophy pH optimum of 5.8 and exhibited no activity at pH 6.8 to 7.0, the pH optimum range for known methanotrophs. This difference in pH responses could be useful in modifying media currently used for isolation of these organisms. Methanotrophic activity was induced in previously non-CH4-consuming soils by preincubation with 5% (vol/vol) CH4 (50,000 µl of CH4 per liter) or by liquid enrichment with 20% CH4. The bacteria showed pH responses typical of known methanotrophs and not typical of preexisting consumers of ambient CH4. Furthermore, methanotrophs induced by high CH4 levels were more readily extracted from soil than preexisting ambient CH4 consumers were. In the alkaline soils, preexisting activity either was destroyed or resisted extraction by the procedure used. The results support the hypothesis that consumers of ambient CH4 in soils are physiologically distinct from the known methanotrophs.

Automated sample clean-up and fractionation of chlorpyrifos, chlorpyrifos-methyl and metabolites in mussels using normal-phase liquid chromatography

An automated method based on normal-phase LC has been developed for the sample clean-up of mussel extracts prior to gas chromatographic analysis of residues of chlorpyrifos, chlorpyrifos-methyl and their metabolites chlorpyrifos-methyl-oxon and 3,5,6-trichloro-2-pyridinol. Pesticides were extracted by means of a high speed blender using acetonitrile-acetone (90:10, v/v). The extract obtained was filtered and concentrated using rotavapor and the residue was dissolved in hexane. One ml of the hexanoic extract was injected on the silica-gel column, using hexane as mobile phase. Pesticides and metabolites were eluted in fat-free fractions with different mixtures of hexane-ethyl acetate. Diode array detection allowed monitoring on-line the elution of lipids. Purified extracts were analyzed by GC using nitrogen-phosphorus detection for quantitation and MS for confirmatory purposes. The method is fully automated from the injection of the extract to the collection of fractions, which are directly injected into the GC system. In this way, neither further clean-up nor solvent exchange were necessary prior to GC analysis. Recoveries obtained from fortified mussel samples at two concentration levels −100 and 20 ng g −1 for parent pesticides and 200 and 40 ng g −1 for metabolites — were higher than 90%. Limits of detection of the whole procedure of analysis were lower than 1 ng g −1 for parent pesticides and than 10 ng g −1 for metabolites. This method has been successfully applied to bioconcentration studies with mussels exposed to chlorpyrifos. Chlorpyrifos and its metabolic derivative 3,5,6-trichloro-2-pyridinol were detected and confirmed by MS in analyzed samples.

Comparison of Cleanup Techniques for Simple Method for Analysis of Selected Organophosphorus Pesticide Residues in Molluscs

Abstract A simple method for determination of 5 organo phosphorus pesticides (dimethoate, chlorfenvin phos, chlorpyrifos, methidathion, and phosmet) in molluscs (Mytilus galloprovincialis and Venus gallina) was developed, with special attention to cleanup. Organophosphorus pesticides were extracted with acetonitrile-acetone (90 + 10, v/v) in a high-speed blender. Two cleanup procedures were used to defat extracts prior to injection into a gas chromatograph: liquid-liquid partition with acetoni trile-hexane and adsorption column chromatography with silica gel. The latter was more efficient for elimination of fat and fractionation of pesticides with different polarities. Limits of detection of the overall procedure including extraction and cleanup ranged from 0.2 to 1 ng/g. Quantitative recoveries for pesticide concentrations ranging from 1 to 10 000 ng/g were obtained.

Slurry preparation by high-pressure homogenization for cadmium, copper and lead determination in cervine liver and kidney by electrothermal atomic absorption spectrometry.

Homogenization with a flat valve homogenizer in combination with high-speed blending was evaluated for the preparation of slurries suitable for the ETAAS determination of cadmium, copper and lead concentrations in six SRMs and in frozen cervine liver and kidney. Fresh tissue (approximately 2 g) or powdered SRM (approximately 0.1 g) was dispersed, at high speed, in 20 ml of ethanol-water (1 + 9 v/v) containing 0.25% m/m tetramethylammonium hydroxide. The resulting suspension was passed through a high-pressure flat valve homogenizer. Determinations performed on the resulting homogenate, provided estimates for Cd, Pb and Cu concentrations that were within 27, 23 and 18% of the certified values, respectively, for the six SRMs. In all instances, the experimental results did not differ significantly from the certified values. For frozen tissues there was good agreement between the concentrations as determined by slurry homogenization-ETAAS and conventional digestion-ICP-MS. In addition, no significant differences were detected between the slopes of the calibration curves for external standards and standard additions to homogenized sample (SRMs or fresh tissue). Moreover, replicate determinations of analyte concentrations in slurries at various times post-preparation did not detect any segregation of the homogenates during 6 d. For these matrices at least, short-term sample storage had no discernible effect on the analyte apparent concentrations. The applicability of the process was limited only by the levels of contaminating Pb and Cu introduced into the sample by the homogenizer.

Isolation, Purification, and Liquid Chromatographic Determination of Stilbene Phytoalexins in Peanuts

Abstract A liquid chromatographic (LC) method for determination of stilbene phytoalexins (SPs) in peanuts has been developed. SPs were extracted with acetonitrile–water (90 + 10, v/v) by high-speed blending. An aliquot of extract was applied to a minicolumn packed with AI2O3–ODS (C18) mixture and eluted with acetonitrile-water (90 + 10, v/v). Eluate was evaporated under nitrogen, and residue was dissolved in LC mobile phase. SPs in an aliquot of purified extract were quantitated by normal-phase partition LC on silica gel with n-heptane–2-propanol–water–acetonitrile–acetic acid (1050 + 270 + 17 + 5 + 1, v/v) as mobile phase. Recoveries of SPs [frans-4-(3-methyl-but-1-enyl)-3,5,4′-trihy-droxystilbene (trans-arachidin-3; t-Ar-3), trans-3-isopentadienyl-4,3′,5′-trihydroxystilbene (t-IPD), and trans-3,5,4′-trihydroxystilbene (trans-resveratrol; t-Res)] from peanuts spiked at 300 ppb were 96.05 ± 2.8%. Limits of quantitation for t-Ar-3 and t-IPD were about 100 ppb. t-Ar-3, t-IPD, and t-Res were found in all parts of the peanut plant as major SPs produced in response to fungal invasion.

The effect of high speed blending upon Penicillium chrysogenum

During high speed blending of two strains of Penicillium chrysogenum, fragmentation of the mycelia was accompanied by considerable (strain dependent) biomass loss. The mean clump size decreased significantly, and the freely-dispersed hyphae became shorter and less branched on average. The homogeneity of the samples increased with duration of blending. The more recent Panlabs P1 strain was significantly more resistant to blending with respect to biomass loss than NRRL 1951.

Enumeration of Toxic Compound Degrading Bacteria in a Multi-Species Activated Sludge Biomass

A most probable number (MPN) method has been described for the enumeration of bacteria responsible for 2,4-dinitrophenol (DNP) degradation in activated sludge cultures. Because of the encapsulation of cells within the activated sludge floc, the method of pretreatment may be critical for producing a homogeneous distribution of bacteria to insure accurate dilution and enumeration. In these experiments, pretreatments using mechanical shear and incubation of cultures in EDTA have been tested. The combination of mechanical shearing in a high speed blender and incubation in a 0.5% EDTA solution resulted in significantly higher MPN values for DNP-degrading organisms growing in activated sludge in sequencing batch reactors (SBRs) which had been supplemented with glucose. Shearing of flocs and the combination of shearing and EDTA treatment produced the identical MPN values in both pure and activated sludge cultures grown only in DNP.

Viability, germination and infection potential of oospores of Phytophthora infestans

Matings between five A1 and five A2 wild‐type isolates of Phytophthora infestans from potato and tomato crops in the United Kingdom produced oospores in vitro in all cases examined. Oospores from the majority of crosses germinated, albeit at a low level (max 13‐4%), after extraction from agar cultures by high‐speed blending and treatment with novoZym 234. Viability of oospores from 20 crosses was tested by three methods. Two methods involved stains, either tetrazolium bromide (MTT) or phloxine B, and the third measured plasmolysis in 2 M NaCl. Both staining methods indicated a high percentage viability but gave false‐positive results with heat‐killed oospores. The plasmolysis method gave a lower percentage viability but no false positives. Oospores produced in vitro and stored either in sterile H2O or in soil at temperatures between 0 and 20 C survived for between 5 and 7 months, the length of the experiments. Oospores buried in non‐sterile field soil survived for up to 8 months (January‐August). Inoculation of potato with zoospores of AI and A2 isolates produced oospores in stems but not in leaf tissue. In some, but not all cases, rapidly growing potato shoots (15‐mm long) were successfully infected with oospores produced in vitro.

Kinetic study of gelation of solventless alkoxide-water mixtures

A very high-speed rotatory blender was used to enhance the homogenization of TEOS + H 2O mixtures. The effects of several reaction parameters on the gelation rate of these solutions and those submitted to ultrasonic treatment were studied. Comparison of the results points to differing accelerating mechanisms in each case.