Determination of fat in raw milk: comparison of mojonnier (ether extraction) and Gerber method

Results are compared for the approved Gerber method that specifies a TC 11.07 mL pipet and 2 modifications that specify a TC 10.77 mL pipet or a weighed 11.125 g sample of milk rather than a pipetted sample. All results were also compared with results obtained with the Mojonnier method for determining the fat content of raw milk. The standard deviation is somewhat lower for the Mojonnier method than for any of the Gerber methods; among the Gerber methods, the standard deviations were lower for the TC 10.77 mL and 11.125 g modifications. It is recommended that one of these modifications replace the current specified TC 11.07 mL pipet method.     

Separation and identification of phenolic compounds in olive oil by coupling high-performance liquid chromatography with postcolumn solid-phase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR)

This study reports the first application of the hyphenated LC-SPE-NMR technique using postcolumn solid-phase extraction to the direct analysis of phenolic compounds in the polar part of olive oil. Apart from the identification and structure elucidation of simple phenols (hydroxytyrosol, tyrosol, vanillic acid, vanillin, p-coumaric acid, hydroxytyrosol, and tyrosol acetates), lignans (pinoresinol and 1-acetoxypinoresinol), flavonoids (apigenin and luteolin), and a large number of secoiridoid derivatives, this technique enables the identification of several new phenolic components, which had not been reported previously as constituents in the polar part of olive oil.     

Development of a CE method to analyze organic acids in dairy products: application to study the metabolism of heat-shocked spores

Organic acids are relevant in dairy products for nutritional reasons and because they contribute to the flavor and aroma. They are the major products of carbohydrate catabolism of lactic acid bacteria and nonstarter bacteria associated with milk. In several research and quality programs, it is very important to develop a rapid and sensitive method for their quantitative determination in dairy products to monitor bacterial growth and activity. A capillary electrophoresis method for the simultaneous determination of oxalic, citric, formic, succinic, orotic, uric, pyruvic, acetic, propionic, lactic, and butyric acid in less than 18 min has been developed. Various parameters affecting analysis, including capillary length, type, composition, and pH of the electrolyte have been optimized. Some alternatives are given to improve the separation of particular organic acids of special interest. Its application to analyze the quality of some dairy products has been investigated. In addition, the suitability of the technique to determine profiles of organic acids generated during the metabolism of heat-shocked spores has been demonstrated.     

A capillary electrophoresis laser-induced fluorescence method for analysis of potato glycoalkaloids based on a solution-phase immunoassay. 1. Separation and quantification of immunoassay products

Solution-phase immunoassays are typically faster and more precise than ELISAs. This research developed a solution-phase for the immunoassay of potato glycoalkaloids (GAs) based on quantification by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Solanidine coupled to 4'-(aminomethyl)fluorescein and a polyclonal antibody solution were used as the immunoreagents. Unbound fluorescent solanidine was detected by CE-LIF (excitation 488 nm, emission 520 nm). Optimum resolution of immunoassay products was achieved with a buffer consisting of 50 mM phosphate, 10% (v/v) methanol, and 1.5 mM SDS, pH 7.5. A plot of signal vs log [GA] produced a sigmoidal curve typical of immunoassays. Analysis of extracts of sprouted Yukon Gold potato tubers and nonsprouted Yukon Gold tubers resulted in total [GA] of 98 microg/g (RSD 9%) and 55 microg/g (RSD 9%), respectively. The findings indicated that CE-LIF coupled with a solution-phase immunoassay can be used to quantify total GA in potatoes.     

Acetonitrile as a buffer additive for free zone capillary electrophoresis separation and characterization of maize (Zeamays L. ) and sorghum (Sorghum bicolor L. Moench) storage proteins

An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (<0.3% relative standard deviation, measured over 15 consecutive injections) for migration time. Subclasses of maize and sorghum storage proteins were identified, and genotypes of each cereal were successfully differentiated using ACN-containing buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.     

Ascorbic acid as a reductant for extraction of iron-bound phosphorus in soil samples: a method comparison study

Sequential extraction is commonly used to identify and quantify different forms of phosphorus (P) associated with particulate samples. Iron (Fe)-bound P is an important fraction of total particulate phosphorus because iron (Fe) is ubiquitous in natural environments. Three reductant solutions, i.e., sodium hydroxide (NaOH), dithionite, and ascorbic acid, have been used to extract solid phase reactive iron and associated phosphorus from sediments and soils. This study compares the efficiencies of three different methods in extracting Fe and Fe-bound P and evaluates the potential and limitation of each method. Based on the results of this comparative study it is recommended that the ascorbic acid reduction method is used for extraction of Fe-bound P in particulate samples, such as soil and sediment.     

Extraction of rutin from buckwheat (Fagopyrum esculentumMoench) seeds and determination by capillary electrophoresis.

The content of the flavonoid rutin was determined in different milling fractions of buckwheat seeds and in buckwheat stems, leaves, and flowers. The extraction was performed by using a solvent containing 60% of ethanol and 5% of ammonia in water. The extracts were analyzed by capillary electrophoresis (running buffer of 50 mM borate (pH 9.3), 100 mM sodium dodecyl sulfate; determination at 380 nm). In bran fractions the concentration of rutin was 131-476 ppm, and in flour fractions 19-168 ppm. On average, about 300, 1000, and 46000 ppm of rutin were found in leaves, stems, and flowers, respectively. The results indicate that buckwheat could be an important nutritional source of flavonoids, especially in countries with a low mean daily flavonoid intake.     

A simple wet chemical extraction procedure to characterize soil organic matter (Som): 2. Reproducibility and verification

Abstract

In a previous communication in this journal, a fractionation scheme of soil organic matter (SOM) was presented (1). The goal of this paper is to discuss the reproducibility and verification of this procedure with an expanded data set of 150 samples. Litter compound analysis (LCA) is appropriate to detect small differences in the decomposition degree at a quantitative level which are not detectable with SOM morphology. In contrast, humic compound analysis (HCA) is not appropriate to characterize SOM with regard to quantitative data, because the detected carbon (C) (C recovery rate <800 mg/g TOC) reflects only parts of the total SOM. In addition selected, SOM fractions are determined with both extraction procedures. When counting C as polysaccharides in the LCA and as fulvic acid in the HCA, this gives recovery rates of much more than 100% (>1,200 mg/g TOC). These errors induce both an under‐ or an over‐estimation of C within the combination of the litter and humic compound analyses (LCA+HCA) and the conversion to 100% should not be used. Because of the method problems and limited chemical information provided with HCA, we propose using LCA and additional analytical instuments (e.g. NMR, pyrolysis) to further characterize structures in the non‐litter substances of the SOM pool.     

Static-dynamic superheated liquid extraction of hydroxytyrosol and other biophenols from alperujo (a semisolid residue of the olive oil industry)

Hydroxytyrosol and other olive biophenols (OBPs) such as tyrosol, verbascoside, apigenin-7-glucoside, and alpha-taxifolin have been extracted from alperujo by using static-dynamic superheated liquids. Multivariate methodology has been used to carry out a detailed optimization of the extraction. Under the optimal working conditions no further extraction of the target analytes was achieved after 27 min (up to 2800 and 1500 mg/kg of hydroxytyrosol and tyrosol, respectively), so complete removal of them within this interval was assumed. The extract was injected into a chromatograph-photodiode array detector assembly for individual separation-quantification. The efficacy of ethanol/water mixtures to extract OBPs from alperujo has been demonstrated and compared with that of a conventional stirring-based method. These less toxic extractant mixtures are of interest with a view to future human uses of OBPs.     

A simple wet chemical extraction procedure to characterize soil organic matter (SOM). I. application and recovery rate

Abstract

The knowledge of soil organic matter (SOM) composition is important for research in soil science. This is why two classical wet chemical extraction procedures were tested and combined to characterize SOM. Twenty‐five samples from typical forest and arable soils in Schleswig‐Holstein, Northwest Germany, were investigated in the laboratory. Lipids were extracted using a pre‐step method. Several polysaccharide fractions were extracted sucessively with inorganic acids in a litter compound analysis (LCA). Proteins and lignins were determined in the bulk soil sample. In a humic compound analysis (HCA), fulvic and humic acids were extracted in the classical way with NaOH, and the non‐humic substances were removed with the aid of the “Sulfacetolysis” from the residues (= usually “humins")‐ The combination of these two wet chemical extraction proce dures (LCA and HCA) permitted quantitative estimations of the SOM composition in several soil horizons. The LCA method produced a better recovery rate (104%±4%) than the HCA methode (95%±15%). The litter compound/humic compound ratio of both analyses, and the combination of both correlated with visible humification grades in a significant way (r = ‐0.733 to ‐0.742***). LCA may be sufficient for solving special pedogenetic problems, because of its high recovery rate and the strong correlation between LCA and HCA.     

Development of a solid-phase extraction method for phenoxy acids and bentazone in water and comparison to a liquid-liquid extraction method

A rapid solid-phase extraction (SPE) method was developed for the determination of bentazone and the phenoxy acids 2,4-D, dichlorprop, MCPA, and mecoprop in Norwegian environmental water samples. Cartridges with a high-capacity cross-linked polystyrene-based polymer were used for off-line preconcentration. The effects of elution solvent, elution volume, sample volume, sorbent mass, pH, and flow rate on the recoveries of the pesticides were investigated using HPLC. Average recovery of >90% was achieved with 500 mg sorbents using 2 mL of methanol with 5% NH3 as elution solvent. The recoveries were independent of sample pH in the tested range of pH 1-7. Using a sample volume of 200 mL, the limits of determination for the phenoxy acids and bentazone are 0.02 microg/L. Sample volumes up to 2000 mL at a flow rate of 60 mL/min could be handled without any loss of analytes, which makes it possible to lower the limits of determination. The SPE method was compared to a routinely used liquid-liquid extraction method. Three different water matrices spiked at 1.0 and 0.05 microg/L were extracted, and the quantification was performed by GC-MS. Both methods permitted the determination of phenoxy acids and bentazone in distilled water, creek water, and well water down to a level of 0.05 microg/L with recoveries >80% for 200 mL samples. Important advantages of the SPE method compared to the liquid-liquid extraction method were the short extraction times, lack of emulsions, use of disposable equipment, and reduced consumption of organic solvents.     

SFE-plus-C(18) lipid cleanup and selective extraction method for GC/MS quantitation of polycyclic aromatic hydrocarbons in smoked meat

In biological matrixes lipid material often poses an interference problem for determinations of nonpolar compounds, e.g., polycyclic aromatic hydrocarbons (PAHs). A newly developed supercritical fluid extraction plus adsorbent method, "SFE-plus-C(18)", offers selective extraction of PAHs in lipid-rich biological matrixes without the need for supplementary cleanup. This method eliminates the use of large volumes of toxic solvent and lengthy lipid removal procedures. This study reports the first application of the SFE-plus-C(18) method to the analysis of a genuine food product, i.e., smoked meat (beef). The procedure employs the addition of C(18) adsorbent beads to the initial sample slurry of pureed smoked meat prior to supercritical CO(2) extraction and GC/MS quantitation. During SF extraction, indigenous lipids are preferentially retained on the beads, and PAHs are selectively extracted with supercritical CO(2). In a comparison of determinations of PAHs by SFE-plus-C(18) vs the conventional SFE method, only 11-17% of the indigenous lipids observed by the conventional SFE method were co-extracted using the SFE-plus-C(18) method. The PAHs in smoked meat could thus be determined efficiently in the presence of a reduced background of co-extracted lipids. Out of 10 targeted PAHs, seven were detected with a range of 10.0-26.0 ng/g in the smoked meat sample. The other three PAHs were not present above the detection limit of the instrument (2.5-4.1 pg). The recoveries of PAHs obtained using the conventional SFE method were 63-94% lower than those achieved by SFE-plus-C(18).     

Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study

Identification of 10 white fish species associated with U.K. food products was achieved using PCR-RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end-point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials.     

Foliar application of urea to almond and olive: Leaf retention and kinetics of uptake

Regression equations relating volume of urea solutions retained on leaves initially and leaf area were developed to assess urea deposition nondestructively and facilitate measurement of urea uptake by leaves of olive (Olea europaea L.) and almond [Prunus dulcis (Mill.) D. A. Webb], Foliar uptake of urea was slower in olive than it was in almond, but uptake in both species was proportional to the concentration of the urea applied. Foliar uptake of urea was not influenced by previous applications. No phytotoxicity was apparent in almond and olive following single applications of 0.5% w/v urea and 4% (w/v), respectively. Approximately 15 times more urea could be applied per cm of an olive leaf than per cm of an almond leaf at the threshold of phytotoxicity. Leaf N content in olive was increased 47% with minimal phytotoxicity following 5 successive foliar applications of 2% urea within ten days.     

Separation of food grade antioxidants (synthetic and natural) using mixed micellar electrokinetic capillary chromatography.

A capillary electrophoretic method, for the determination of antioxidants present in food, has been developed using mixed micellar electrokinetic capillary chromatography. The buffer consists of sodium cholate (40 mM), sodium dodecyl sulfate (15 mM), 10% methanol, and 10 mM borate at pH 9.3. A separation was obtained for nine antioxidants (synthetic and natural) commonly found in food. High-performance liquid chromatography and capillary electrophoresis were applied to the analysis of sesame oil and wine. Ascorbic acid was identified in wine.     

Genetic fingerprinting of grape plant (Vitis vinifera) using random amplified polymorphic DNA (RAPD) analysis and dynamic size-sieving capillary electrophoresis

Dynamic size-sieving capillary electrophoresis with laser-induced fluorescence detection (DSCE-LIF) was combined with random amplified polymorphic DNA (RAPD) analysis to demonstrate the feasibility of the genetic analysis of grape plant varieties and clones within a variety. Parameters of the genomic DNA extraction process, as well as those of the RAPD analysis, were optimized specifically for this application. Polymorphic DNA fragments were generated for four different grape plant varieties including Cabernet Franc, Cabernet Sauvignon, Merlot, and Chardonnay. Relative to slab gel electrophoresis (SGE) with ethidium bromide staining, DSCE-LIF provided superior separation efficiency and detection limits in the analysis of DNA polymorphic bands. Optimal DSCE-LIF analyses were achieved using a 10-fold RAPD sample dilution, hydrodynamic sample injection, and 100 ng/mL of YO-PRO-1 DNA intercalator in the dynamic size-sieving buffer solution. In addition, the reproducibility of both the DSCE-LIF and RAPD analyses were demonstrated.     

The use of Lactobacillus pentosus 1MO to shorten the debittering process time of black table olives (Cv. Itrana and Leccino): a pilot-scale application

Fifty lactobacilli isolated from black table olive brines were evaluated for their salt tolerance, resistance to oleuropein and verbascoside, and ability to grow in modified filter-sterilized brines. A strain of Lactobacillus pentosus was selected and used as a starter to ferment, in pilot plant, black olives (Itrana and Leccino cv.) in brines modified for pH, carbohydrate, and growth factor concentrations, at 28 degrees C. The temperature-controlled fermentation of Leccino cv. olives resulted in obtaining ready-to-eat, high-quality table olives in a reduced-time process. HPLC analysis of phenolic compounds from fermented olives showed a decrease of oleuropein, a glucoside secoiridoid responsible for the bitter taste of olive drupes, and an increase of the hydroxytyrosol concentration. The selected strain of L. pentosus (1MO) allowed the reduction of the debittering phase period to 8 days.     

Proteomics-based approach to detect and identify major allergens in processed peanuts by capillary LC-Q-TOF (MS/MS)

An MS-based method, combining reversed-phase capillary liquid chromatography (capillary LC) with quadrupole time-of-flight tandem mass spectrometry (nano-ESI Q-TOF MS/MS), was developed with the aim of identifying a set of peptides that can function as markers for peanut allergens. Emphasis was given to the identification of the three major peanut allergens Ara h 1, Ara h 2, and Ara h 3, because these proteins are considered to represent >30% of the total protein content of peanut and are directly relevant for the allergenic potential of this food. The analytical data obtained were used to perform databank searching in combination with de novo sequencing and led to the identification of a multitude of sequence tags for all three peanut allergens. Food processing such as roasting of peanuts is known to affect the stability of proteins and was shown to influence the detection of allergen sequence tags. The analysis of raw and roasted peanuts allowed the identification of five peanut-specific sequence tags that can function as markers of the specific allergenic proteins. For Ara h 1, two peptide markers were proposed, namely, VLEENAGGEQEER (m/z 786.88, charge 2+) and DLAFPGSGEQVEK (m/z 688.85, charge 2+), whereas for Ara h 2 only one peptide, RQQWELQGDR (m/z 439.23, charge 3+), was found to satisfy the required conditions. For Ara h 3, the two specific peptides, SPDIYNPQAGSLK (m/z 695.35, charge 2+) and SQSENFEYVAFK (m/z 724.84, charge 2+), were selected. Other peptides have been proposed as indicative for food processing.     

Volatile compounds and phenolic composition of virgin olive oil: optimization of temperature and time of exposure of olive pastes to air contact during the mechanical extraction process

The operative conditions of malaxation such as temperature and time of exposure of olive pastes to air contact (TEOPAC) affect volatile and phenolic composition of virgin olive oil (VOO) and, as a consequence, its sensory and healthy qualities. In this paper, optimal temperature and TEOPAC during malaxation were studied, in lab scale, in two Italian cultivars using phenolic compounds, volatile composition, and sensory analysis of VOO as markers. The optimal temperature and TEOPAC, selected by response surface modeling,were cultivar-dependent being 30 min of TEOPAC at the lowest temperature investigated (22 degrees C) and 0 min of TEOPAC at 26 degrees C for Frantoio and Moraiolo cultivars, respectively.     

Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: application to Echium vulgare honeys

A method for analyzing honey samples was developed that enabled the simultaneous detection and identification of pyrrolizidine alkaloids and their N-oxides. Honey samples were treated with methanol or dilute sulfuric acid and then centrifuged to remove insoluble material. Subsequent strong cation exchange, solid-phase extraction of the supernatant provided a fraction that was analyzed for the presence of pyrrolizidine alkaloids and their N-oxides using high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry. The procedure was validated using extracts of Echium plantagineum and authenticated standards of pyrrolizidine alkaloids and their N-oxides from other plant sources. Of several variations of the solid-phase extraction method assessed in this study, the best combination for generic use involved the dilution of honey with 0.05 M sulfuric acid and the subsequent application of the centrifuged solution to solid-phase extraction columns at the rate of a maximum of 10 g of honey per solid-phase extraction column. The method was applied to the analysis of nine floral honeys, five of which were attributed by the apiarist to Echium vulgare. Seven of the honey samples were positive for pyrrolizidine alkaloids and N-oxides characteristic of E. vulgare.