Optimization of the isoelectric precipitation method to obtain protein isolates from amaranth (Amaranthus cruentus) seeds

This research was conducted to evaluate the effect of extraction pH (7.8-9.2) and precipitation pH (4.3-5.7) on four selected quality attributes of protein isolates from amaranth seeds (Amaranthus cruentus) such as protein content (PC), whiteness index (WI), enthalpy of transition (EN), and denaturation temperature (DT). Ten different treatments involving extraction and precipitation pH combinations were analyzed by a central composite design; the experimental data were fitted by a second-order model using a least-squares method for each one of the four dependent variables. Response surface methodology was used for the optimization process; in addition, a common optimum value for the four dependent variables was obtained utilizing the desirability method. A confirmatory test showed that the generated regression equations could adequately predict performance of this isoelectric precipitation method. The results indicate that extraction pH and precipitation pH showed an important effect on PC, WI, and EN. However, the different combinations did not significantly affect the DT. Values of 9.2 and 8.0 for extraction pH and 5.7 for precipitation pH produced the best overall result for all responses. Finally, the results have shown that it is possible to obtain protein isolates from A. cruentus seeds at optimized values of extraction pH and precipitation pH, which presented a high protein content and good physicochemical properties.     

Chemical characterization of Klason lignin preparations from plant-based foods

To analyze the accuracy of the Klason lignin method as applied for the determination of lignin contents in plant based-food products, Klason lignin preparations from curly kale, pears, whole wheat grains, and corn bran were chemically characterized. Characterization included routine ash and protein determinations and the extraction of fat/waxes as well as cutin/suberin depolymerization and extraction of the liberated monomers. Fat/wax and cutin/suberin amounts in the Klason lignin preparations were determined gravimetrically, and their compositions were analyzed by using GC-MS. Typical fat, wax, and cutin (and suberin) constituents such as saturated and unsaturated fatty acids, hydroxy and/or epoxy fatty acids, and phenolic acids were identified in all samples, whereas the detection of long-chain hydrocarbons, alcohols, and ketones, sterols, stanols, and dioic acids was dependent on the sample analyzed. Estimation of the contribution of non-lignin compounds to the Klason lignin contents reduced the noncorrected Klason lignin contents of the insoluble fibers from 28.7% (kale), 22.8% (pear), 14.8% (wheat), and 9.9% (corn) to maximum lignin contents of 6.5% (kale), 16.4% (pear), 4.9% (wheat), and 2.3% (corn). These data demonstrate that certain commonly used statements such as "cereal brans are highly lignified" need to be revised.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Interaction Between Sorghum Protein Extraction and Precipitation Conditions on Yield,Purity, and Composition of Purified Protein Fractions

Sorghum proteins have the potential to be used as a bio‐industrial renewable resource for applications such as biodegradable films and packaging. This project was designed to evaluate the effect of interactions between sorghum protein extraction and precipitation conditions on the yield, purity, and composition of sorghum protein fractions. Proteins were extracted with 70% ethanol under nonreducing conditions, with ultrasound, or under reducing conditions using either sodium metabisulfite or glutathione as the reducing agent. Several conditions were used to isolate the extracted proteins through precipitation, including lowering ethanol concentrations alone or in combination with lowering to pH 2.5, or by adding 1M NaCl to the extract. Combinations of these conditions were also tested. All precipitation conditions effectively precipitated proteins and lowering the pH and adding 1M NaCl to the extracts enhanced precipitation in some cases. However, the conditions that precipitated the maxium amount of protein or highest purity of protein varied according to how the proteins were initially extracted. Precipitated proteins were characterized by RP‐HPLC, SEC, HPCE, and SDS‐PAGE to compare the protein fractions composition. Nonreduced and sonicated samples had a much wider M_w distribution than reduced extracts. Thus, extraction and precipitation conditions influenced the isolated proteins yield, purity, and composition. Because the extraction and purification processes influenced the composition, purity, and biochemical properties, it may be possible to prepare protein fractions with unique functionalities for specific end‐uses.     

Comparison of two alkaline treatments in the extraction of organic compounds associated with water repellency in soil under Pinus taeda

Isopropanol:NH₃ and Methanol:KOH (saponification) alkaline treatments are usually applied in the study of soil organic matter. The first is used in studies of soil water repellency, and the latter in the extraction of ester-bound lipids from soil. In this study, isopropanol:NH₃ and methanol:KOH treatments were applied separately in a solvent extracted repellent soil, in order to compare their efficiency in the extraction of water repellent compounds. The soil sample was taken from a site under a 16 year old Pinus taeda stand. The amount and class of organic compounds released by each treatment were compared using gas chromatography-mass spectrometry (GC-MS). Both treatments resulted in wettable soil after alkaline extraction. In general, alkaline treatments yielded extracts with the same class of organic compounds. Alkanoic acids, α,ω-alkanedioic acids, hydroxyalkanoic acids, aromatic compounds, and alkanols were identified, indicating the preservation of suberin and cutin biopolyester in the soil. Large differences were observed in the amounts of ω-hydroxyalkanoic acids, as well as in the quantity and distribution of dihydroxyalkanoic and trihydroxyalkanoic acids. In contrast to methanol:KOH, isopropanol:NH₃ was not efficient in the extraction of whole aliphatic biopolyesters, mainly pine cutin-related products. Methanol:KOH was more effective in hydrolysis. The presence of biopolyesters in water repellent soil under the P. taeda stand seems to play an important role in water repellency.     

Gas chromatographic determination of N-nitrosamines, aromatic amines, and melamine in milk and dairy products using an automatic solid-phase extraction system

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.     

Extractability of 2,4-D,Dicamba and MCPP from Soil

The adsorption of herbicides on soil colloids is a major factor determining their mobility, persistence, and activity in soils. Solvent extraction could be a viable option for removing sorbed contaminants in soils. This study evaluated the extractability of three herbicides: 2,4 dichlorophenoxy-acetic acid (2,4-D), 4-chloro-2-methylphenoxypropanoic acid (mecoprop acid or MCPP), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Three solvents (water, methanol, and iso-propanol) and three methods of extraction (column, batch, and soxhlet) were compared for their efficiencies in removing the herbicides from three soils (loamy sand, silt loam, and silty clay). Both linear and non-linear Freundlich isotherms were used to predict sorption intensity of herbicides on soils subjected to various extraction methods and conditions. High K_dand K_fr, and low N values were obtained for all herbicides in silty clay soil by batch extraction. Methanol was the best solvent removing approximately 97% of all added herbicides from the loamy sand either by column or soxhlet extraction method. Isopropanol ranked second by removing over 90% of all herbicides by soxhelet extraction from all three soils. However, water was ineffective in removing herbicides from any of the soils using any of the three extracting procedures used in this study. In general, the extent of herbicide removal depended on soil type, herbicide concentration, extraction procedure, solvent type and amount, and extraction time.     

Thin layer chromatographic identification and quantitation of niacin and niacinamide in pharmaceutical preparations

A safe and simple thin layer chromatographic method has been developed for the estimation of niacin and niacinamide in pharmaceutical preparations containing other vitamins, enzymes, herbs, antiamoebic drugs, etc. The method involves removal of excipients from the preparation by ethanol precipitation and isolation of niacin or niacinamide from other ingredients by TLC on silica gel with water as developing solvent, followed by extraction in 0.1N HCl, and spectrophotometric estimation of the vitamin at 262 nm. The percent recoveries for niacin and niacinamide were 100.1 +/- 1.9 and 100.2 +/- 1.5, respectively.     

Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts

Twenty-four plant lignans were analyzed by high-performance liquid chromatography-tandem mass spectrometry in bran extracts of 16 cereal species, in four nut species, and in two oilseed species (sesame seeds and linseeds). Eighteen of these were lignans previously unidentified in these species, and of these, 16 were identified in the analyzed samples. Four different extraction methods were applied as follows: alkaline extraction, mild acid extraction, a combination of alkaline and mild acid extraction, or accelerated solvent extraction. The extraction method was of great importance for the lignan yield. 7-Hydroxymatairesinol, which has not previously been detected in cereals because of destructive extraction methods, was the dominant lignan in wheat, triticale, oat, barley, millet, corn bran, and amaranth whole grain. Syringaresinol was the other dominant cereal lignan. Wheat and rye bran had the highest lignan content of all cereals; however, linseeds and sesame seeds were by far the most lignan-rich of the studied species.     

Processing of rice husk autohydrolysis liquors for obtaining food ingredients

Rice husks are of a lignocellulosic nature, with a hemicellulose fraction made up of substituted arabinoxylan. Rice husks were treated with hot, compressed water (autohydrolysis reaction) under optimized conditions to cause the hydrolytic degradation of arabinoxylan. The reaction products contained volatile components and nonvolatile components (NVC), which were made up of hemicellulose-derived products (substituted oligosaccharides and monosaccharides) and other nonvolatile solutes (ONVS). To decrease the content of ONVS, concentrated autohydrolysis liquors were first subjected to ethyl acetate extraction and then subjected to various alternative treatments (solvent precipitation, freeze-drying solvent extraction, or ion exchange). The resulting liquors were assayed for composition and yield determination. Material balances are presented for the several processes considered. The best results in terms of purification were obtained with sequential stages of ethyl acetate extraction and ion exchange, which led to concentrates with hemicellulose-derived compounds (sugars and substituted sugar oligomers) accounting for 92 wt % of the NVCs.     

Determination and distribution of 6-methoxymellein in fresh and processed carrot puree by a rapid spectrophotometric assay.

Isocoumarin or 6-methoxymellein (6-MM) was extracted from carrot tissue using alkali saponification to solubilize the lactone portion of its structure into an aqueous phase. Acidification and subsequent organic solvent extraction allowed isolates to be quantified and verified as 6-MM by spectrophotometric determination. 6-Methoxymellein was analyzed in carrot cross sections, as a function of depth and before and after thermal processing. A natural propensity for 6-MM accumulation was observed in root tip sections exposed to ethylene, and levels increased as a result of wounding. Consecutive layer peeling demonstrated that small-diameter roots accumulated greater amounts of 6-MM in periderm tissue compared to large roots. Processing carrots into a puree resulted in 10-25% greater extraction of 6-MM than grinding fresh carrot samples, whereas steam-cooked and thermally processed purees had 15% greater extraction than unheated purees. This analytical technique will allow carrot processors to accurately estimate raw and processed products for the bitter compound 6-MM.     

Biochemical changes across a carbon saturation gradient: Lignin,cutin, and suberin decomposition and stabilization in fractionated carbon pools

Soils that exhibit soil organic carbon (SOC) saturation provide an opportunity to examine mechanisms of C storage in soils with increasingly limited C-stabilization potential. A manure rate experiment in Lethbridge, Alberta, in which SOC responded asymptotically to long-term manure C additions, allowed us to assess changes in SOC biochemical composition in response to soil C saturation. By quantifying the cupric oxide oxidation products of lignin, cutin, and suberin in fractionated SOC pools that are characterized by chemical (i.e., mineral-associated), physical (i.e., microaggregate-associated), or no protection (i.e., free particulate organic matter), we evaluated the interaction between C saturation and the biochemical characteristics of SOC.We tested the specific responses of soil fraction lignin, cutin, and suberin to C saturation level by using the bulk soil to approximate C-input composition across manure input treatments. Carbon-normalized lignin (lignin-VSC/OC) in the chemically protected fractions did not differ, while in the non-protected and physically protected soil fractions, it decreased with C saturation level. Neither the stabilization of cutin and suberin, nor the lignin:cutin + suberin ratio, differed in any of the measured soil fractions in response to C saturation level.These results indicate that with C saturation and decreased C stabilization potential, lignin, cutin, or suberin were not preferentially stabilized or depleted in mineral protected soil C pools. The lack of evidence for biochemical preference in mineral associations with C saturation supports the existence of an outer kinetic zone of organomineral associations, in which partitioning of organic compounds, rather than sorption, controls mineral SOC accumulation at high SOC loadings. Furthermore, despite theories of inherent lignin recalcitrance, depleted lignin concentrations with C saturation in the non-protected and aggregate protected fractions indicate that lignin was, in this study, preferentially decomposed when not protected by association with mineral phases in the soil. In conclusion, C-input quantity, and not quality, combined with physical and chemical protection mechanisms that govern long-term C storage, appeared to control C saturation and stabilization at this site.     

Cutin composition of five finnish berries

The raw cutin (i.e., extractive-free isolated cuticular membrane) fraction from Finnish berries, sea buckthorn (Hippopha? rhamnoides), black currant (Ribes nigrum), cranberry (Vaccinium oxycoccos), lingonberry (Vaccinium vitis-idaea), and bilberry (Vaccinium myrtillus), was depolymerized by NaOMe-catalyzed methanolysis. The composition of cutin monomers was determined by GC-(EI)MS analysis either as methyl esters or as TMSi esters, with OH groups derivatized to TMSi ethers. There was a notable difference in the degree of depolymerization, ranging from 6 to 47%. The extractive-free berry cuticle, that is, raw cutin, thus contains <50% polyester polymer cutin. The predominant cutin monomers were C(16) and C(18) omega-hydroxy acids with midchain functionalities, mainly epoxy and hydroxyl groups. Typically, the major compounds were 9,10-epoxy-18-hydroxyoctadecanoic acid, 10,16-dihydroxyhexadecanoic acid, 9,10,18-trihydroxyoctadecanoic acid, 9,10-epoxy-18-hydroxyoctadec-12-enoic acid, and 18-hydroxyoctadec-9-enoic acid. The amount of epoxyacids was rather high in sea buckthorn ( approximately 70%) and cranberry ( approximately 60%), compared with the other berries. The black currant cutin differed from that of the other berries with a significant portion of hydroxyoxohexadecanoic acid ( approximately 12% of total monomers). This investigation of the cuticular hydroxy acids of five Finnish berries is part of the exploitation of the northern natural resources related to the chemical composition, nutritional value, and sensory properties.     

Extraction of Antioxidants from Wheat Bran Using Conventional Solvent and Microwave-Assisted Methods

Total phenolic and tocopherol contents and free radical scavenging capability of wheat bran extracted using conventional and microwave-assisted solvent extraction methods were studied. Three different solvents (methanol, acetone, and hexane) were used in the conventional solvent extraction. Methanol was the most effective solvent, producing higher extraction yield (4.86%), total phenolic compound content (241.3 μg of catechin equivalent/g of wheat bran), and free radical scavenging capability (0.042 μmol of trolox equivalent/g of wheat bran) than either acetone or hexane. However, there was no significant difference in the total tocopherol contents (13.6–14.8 μg/g of wheat bran) among the three different solvent extraction methods. Microwave-assisted solvent extraction using methanol significantly increased the total phenolic compound content to 467.5 and 489.5 μg of catechin equivalent; total tocopherol content to 18.7 and 19.5 μg; and free radical scavenging capability to 0.064 and 0.072 μmol of trolox equivalent/g of wheat bran at extraction temperatures of 100 and 120°C, respectively. However, extraction yields of conventional methanol solvent and microwave-assisted methanol extractions at different temperatures were not significantly different.     

Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples.

Accelerated solvent extraction (ASE) is a new sample extraction method offering a number of advantages such as low pe -extraction cost, reduced solvent and time consumption, and simplified extraction protocols. In this study, the ASE method was applied to the extraction of polycyclic aromatic hydrocarbons (PAHs) from biological samples. For recovery studies, fish tissues and ground pork were used as sample matrices. Sample aliquots fortified with 16 PAHs were extracted by ASE, and the extracts were treated with sulfuric acid and Florisil, followed by gas chromatography-mass spectrometry analysis. The PAH recoveries by the ASE method were found to be comparable with or better than those by Soxhlet extraction. The extraction and quantitation method was then applied to the determination of PAHs in several smoked meat samples obtained from a local market. Up to 12 PAHs were found to be present at concentrations ranging from 3 to 52 ng/g wet sample.     

Sequential fractionation of grape seeds into oils, polyphenols, and procyanidins via a single system employing CO2-based fluids

Pure supercritical CO(2) was used to remove >95% of the oil from the grape seeds. Subcritical CO(2) modified with methanol was used for the extraction of monomeric polyphenols, whereas pure methanol was used for the extraction of polyphenolic dimers/trimers and procyanidins from grape seed. At optimum conditions, 40% methanol-modified CO(2) removed >79% of catechin and epicatechin from the grape seed. This extract was light yellow in color, and no higher molecular weight procyanidins were detected. Extraction of the same sample after removal of the oils and polyphenols, but now under enhanced solvent extraction conditions using methanol as a solvent, provided a dark red solution shown via electrospray ionization HPLC-MS to contain a relatively high concentration of procyanidins. The uniqueness of the study is attested to by the use of CO(2)-based fluids and the employment of a single instrumental extraction system.     

Molecular and sensory studies on the umami taste of Japanese green tea

Aimed at defining the key drivers for the quality-determining umami taste of a high-grade powdered green tea, called mat-cha, a bioactivity-guided fractionation using solvent extraction, solvent precipitation, preparative chromatographic separations, and human psychophysical experiments was applied on freshly prepared mat-cha. Liquid chromatography-tandem mass spectrometry and one-/two-dimensional nuclear magnetic resonance studies on isolated fractions led to the identification of l-theanine, succinic acid, 3,4,5-trihydroxybenzoic acid (gallic acid), and (1R,2R,3R,5S)-5-carboxy-2,3,5-trihydroxycyclohexyl-3,4,5-trihydroxybenzoate (theogallin) as umami-enhancing compounds in the green tea beverage, and it can be shown by sensory studies that these compounds are able to raise the umami intensity of sodium l-glutamate proportionally.     

Determination of polychlorinated biphenyls in sediments, using sonication extraction and capillary column gas chromatography-electron capture detection with internal standard calibration

A sonication technique is presented for the extraction of polychlorinated biphenyls (PCBs) from sediments. In addition, a quantitation scheme is described which allows peak-specific and, in many cases, congener-specific determination of PCBs. PCBs are quantitated by capillary column gas chromatography-electron capture detection, with internal standard calibration. Results utilizing sonication extraction were compared with those obtained by Soxhlet and steam distillation extractions of 3 U.S. Environmental Protection Agency (EPA) quality control sediment samples and 3 lake sediments known to be contaminated with PCBs. Environmental lake sediments were extracted wet, with no drying prior to extraction. Recoveries by each technique varied depending on the sediment sample being extracted and degree of chlorination of PCB congeners. With proper selection of extraction solvent, the sonication technique can recover amounts of PCBs equivalent to and sometimes greater than recoveries by the Soxhlet or steam distillation techniques. A 24-h quiescent period in the extraction solvent between 2 sonications improved extraction efficiency for 2 freeze-dried sediments but did not affect results obtained for 3 environmentally contaminated sediments that were extracted without drying. Replacement of Soxhlet extraction with the sonication technique results in reduced sample preparation time, decreased volumes of solvents and sample, and substitution of common laboratory glassware in place of fragile, expensive Soxhlet glassware. Sonication extraction can also improve precision compared with Soxhlet extraction.     

STUDIES OF SOIL ORGANIC MATTER USING FORMIC ACID SOLVENTS: FACTORS AFFECTING COMPOSITION AND DEGREE OF FORMYLATION OF ORGANIC ISOLATES

Two soils were extracted under reflux with formic acid/H₂O for 0.25 h, followed by formic acid/HF for 0.25 h. In one set of experiments ¹⁴C-labelled formic acid was used in the first extraction and unlabelled formic acid in the second: in a second set this labelling sequence was reversed. After separation from the soil residues, the soil extracts were further refluxed for up to 3.75 h. Organic matter was then precipitated from the various extracts by addition of di-isopropyl ether. Prolonged boiling of the extracts reduced the recovery of organic matter and nitrogen, and increased the inorganic contamination of the organic isolates. The degree of formylation of the isolates from the first extraction with formic acid/H₂O increased with reflux time to 0.50 h and thereafter decreased, but overall it was appreciably greater than that of the isolates from the second extraction with formic acid/HF. Formylation of the latter increased slowly with reflux time up to 4.00 h. In both solvents hydrolysis and formylation of organic solutes occur concurrently during refluxing. Treatment of formic acid/H₂O extracts with a TiCl₄/HCl/propan-2-ol reagent to precipitate organic matter and washing the precipitate in a hydrous HF medium resulted in lower yields than precipitation with di-isopropyl ether, but the final washed precipitate contained much less formyl groups and inorganic material.     

Determination of altosid insect growth regulator in waters, soils, plants, and animals by gas-liquid chromatography.

Residues of isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate (Altosid) insect growth regulator are determined in waters, soils, plants, milk, eggs, fish, shellfish, poultry and cattle tissues, blood, urine, and feces. Acetonitrile is the primary extraction solvent for all samples. Residues are extracted by high-speed blending followed by vacuum filtration. Fatty extracts are subjected to cold-temperature precipitation and filtration. Samples are cleaned up by petroleum ether partitioning and Florisil and neutral alumina chromatography. The concentrated eluants are analyzed by gas-liquid chromatography (GLC) on columns of differing polarity, using hydrogen flame ionization detectors. The identity of suspected residues is confirmed by additional GLC and by mass fragmentography. The lower limits of detection were: water samples, 0.0004-0.001 ppm; soils, blood, and urine, 0.001 ppm; forage grasses, forage legumes, and rice foliage, 0.005 ppm; and milk, eggs, fish, shellfish, poultry and cattle tissues, and feces, 0.010 ppm.