Immunochemical-based method for detection of hazelnut proteins in processed foods

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.     

Detection of soy proteins in processed foods: literature overview and new experimental work

Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 microg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 microg/mL, corresponding to 0.4 microg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.     

Disposable electrochemical DNA-array for PCR amplified detection of hazelnut allergens in foodstuffs

An electrochemical low-density DNA-array has been designed and implemented to be used in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens (Cor a 1.04, Cor a 1.03) in foodstuff. Unmodified PCR products were captured at the sensor interface via sandwich hybridization with surface-tethered probes and biotinylated signalling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to a α-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the α-naphthol signal. The detection limits for Cor a 1.03 and Cor a 1.04 were 0.3 and 0.1 nmol L⁻¹, respectively (R.S.D. 10%). The optimized conditions were used to test several commercially available foodstuffs, claiming to contain or not the targeted nuts. The results were compared with those obtained with classical ELISA tests.     

Determination of deoxynivalenol in processed foods

Deoxynivalenol (DON), commonly referred to as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium fungi. The presence of DON in foods is a human health concern. The frequency of occurrence of DON in wheat is high, although cleaning prior to milling can reduce DON concentration in final products, and food processing can partially degrade the toxin. This paper describes a method for the determination of DON in some major wheat food products, including bread, breakfast cereals, pasta, pretzels, and crackers. Test samples containing 5% polyethylene glycol were extracted with water. After blending and centrifuging, the supernatant was diluted with water and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column and the toxins eluted with methanol. The toxins were then subjected to RPLC separation and UV detection. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON spiked at levels from 0.5 to 1.5 microg/g in the five processed foods were >70%. SD and RSD values ranged from 2.0 to 23.5% and from 2.0 to 23.2%, respectively. HorRat values were <2 for all of the matrixes examined. The method was found to be acceptable for the matrixes examined. LC/MS/MS with multiple-reaction monitoring was used to confirm the identity of DON in naturally contaminated test samples.     

A preamplification approach to GMO detection in processed foods

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.     

Immunoassay kit used to detect the presence of bovine material in processed foods

The Tepnel Bio Kit for the detection of beef in cooked foods was assessed to determine its validity in demonstrating if food being imported into New Zealand contains beef material. The test suffered no interference from the presence of other common nonbovine species meats accepted as food within New Zealand and it detected beef in cooked samples of mixed meats when the proportion of beef in the mixture was >2 or >1%, depending on other meat species present. The documentation supplied with the kit indicates that the specific proteins it measures in cooked beef are stable to 130 degrees C. This was confirmed in the literature when the kit was used to test meat and bone meal cooked to at least 133 degrees C. However, our results showed these proteins to be much less stable when heated to elevated temperatures in moist food under pressure, and samples containing beef ceased to be positive by the immunoassay test after being autoclaved to 121 degrees C. This suggests that the test may not be able to detect even relatively high levels of beef in low-acid canned foods, which are normally retorted under pressure to approximately 121 degrees C.     

Absolute quantitative analysis for sorbic acid in processed foods using proton nuclear magnetic resonance spectroscopy

An analytical method using solvent extraction and quantitative proton nuclear magnetic resonance (qHNMR) spectroscopy was applied and validated for the absolute quantification of sorbic acid (SA) in processed foods. The proposed method showed good linearity. The recoveries for samples spiked at the maximum usage level specified for food in Japan and at 0.13 g kg⁻¹ (beverage: 0.013 g kg⁻¹) were larger than 80%, whereas those for samples spiked at 0.063 g kg⁻¹ (beverage: 0.0063 g kg⁻¹) were between 56.9 and 83.5%. The limit of quantification was 0.063 g kg⁻¹ for foods (and 0.0063 g kg⁻¹ for beverages containing Lactobacillus species). Analysis of the SA content of commercial processed foods revealed quantities equal to or greater than those measured using conventional steam-distillation extraction and high-performance liquid chromatography quantification. The proposed method was rapid, simple, accurate, and precise, and provided International System of Units traceability without the need for authentic analyte standards. It could therefore be used as an alternative to the quantification of SA in processed foods using conventional method.     

Determination of ochratoxin A in foods: state-of-the-art and analytical challenges

The work presented describes the application of different analytical approaches for study of aluminium mobility in rock, soil, and sediment samples affected by mining activity (secondary quartzites with sulfidic deposits). For this purpose we used a combination of the single extractions, the optimized BCR three-step sequential extraction procedure (SEP), and reactive aluminium determination after chelating ion-exchange on Ostsorb (Iontosorb) Salicyl by a batch technique with flame atomic absorption spectrometry quantification. The single extraction agents H₂O, KCl, NH₄Cl, and BaCl₂ were found to be the best for the quantitative estimation of the aluminium mobility in rocks, soils, and sediments caused by acidification of the environment. This fact was confirmed by reactive aluminium determination in the same samples. The vast majority of the aluminium content of samples after application of the optimized BCR three-step SEP is in the residues. The available fraction of aluminium extracted by dilute CH₃COOH in the first step of this procedure correlates with the reactive aluminium content. The amounts of aluminium released in the second and the third steps and the sums from steps 1–3 of this procedure are closely associated with the aluminium content values obtained by the single dilute HCl leach. The accuracy of results obtained was verified with only informative values for individual fractions of the BCR three-step SEP because of the absence of suitable certified or standard reference materials. The amounts of the reactive aluminium determined in samples was in the range 12–82% of total soluble Al in the filtered H₂O extracts. It was confirmed that the acidified polluted samples contain the most of reactive Al content, which is responsible for its toxicity.     

A generic method for the determination of acrylamide in thermally processed foods

A generic sample preparation method for the determination of acrylamide in foods was developed. The method entails extraction with methanol, purification with Carrez I and II solutions, evaporation and solvent change to water, and cleanup with Oasis HLB solid-phase extraction (SPE) cartridge. The final extract was analyzed by liquid chromatography-mass spectrometry (LC-MS) for quantitation. The chromatographic separation was performed on ODS-3 column using the isocratic mixture of 0.01 mM acetic acid in 0.2% aqueous solution of formic acid at a flow rate of 0.6 ml/min at 25 degrees C. The recoveries of acrylamide from potato chips, biscuits and coffee ranged between 92.8 and 101.5% with relative standard deviations of 4.1% or less. The limit of detection (LOD) and the limit of quantitation (LOQ) were 2 ng/g and 6 ng/g in the basis of signal to noise ratios of 3:1 and 9:1, respectively.     

Detection of hazelnuts and almonds using commercial ELISA test kits

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.     

Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study

Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.     

The absorption and metabolism of modified amino acids in processed foods

The chemical reactions involved in the modifications of amino acids in processed food proteins are described. They concern the Maillard reaction, reaction with polyphenols and tannins, formation of lysinoalanine during alkaline and heat treatments, formation of isopeptides, oxidation reaction of the sulfur amino acids, and isomerization of the L-amino acids into their D-form. Information on the digestion, absorption, and urinary excretion of the reaction products obtained by using conventional nutritional tests is given. The studies that have been made on the metabolism of these molecules by using a radioisotopic approach to follow their kinetics in the organism after ingestion are also reviewed. This approach provides unique data on the quantitation of the metabolic pathways and on the kinetics of the metabolic processes involved.     

Determination of sulfur amino acids in foods as related to bioavailability

During the processing of feedstuffs and foods, methionine can be oxidized to methionine sulfoxide and methionine sulfone, and cysteine can be oxidized to cysteic acid. Methionine sulfone and cysteic acid are nutritionally unavailable, but methionine sulfoxide can be utilized, at least to some degree. The degree of utilization depends on the levels of methionine, cysteine, and methionine sulfoxide in the diet, but there is no consensus in the literature on the quantitative impact of these dietary constituents on methionine sulfoxide utilization. Methionine and cysteine are most often determined after quantitative oxidation to methionine sulfone and cysteic acid, respectively, using performic acid oxidation prior to hydrolysis. However, this method may overestimate the methionine content of processed foods, as it will include any methionine sulfoxide and methionine sulfone present. A selection of analytical methods has been developed to allow the separate determination of the 3 oxidized forms of methionine, the merits of which are discussed in this review. An additional consideration for determining methionine and cysteine bioavailability is that not all dietary methionine and cysteine is digested and absorbed from the small intestine. Selected methods designed to determine the extent of digestion and absorption are discussed. Finally, a concept for a new assay for determining methionine bioavailability, which includes determining the digestibility of methionine and methionine sulfoxide as well as the utilization of methionine sulfoxide, is presented.     

Analysis of erythritol in foods by polyclonal antibody-based indirect competitive ELISA

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol–BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1–100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22–24 mg/kg in watermelon and 130–300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86–105% (watermelon) and 85–93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol. Figure Indirect competitive ELISA for quantitation of erythritol     

Detection of processed genetically modified food using CIM monolithic columns for DNA isolation

The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pretreated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix-food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods.     

Detection of hidden hazelnut protein in food by IgY-based indirect competitive enzyme-immunoassay

The development of an indirect competitive enzyme-immunoassay for the detection of hidden hazelnut protein in complex food matrices is described. A sensitive and selective polyclonal antibody was raised by immunisation of laying hens with protein extracts from roasted hazelnuts. In contrast to traditional antibody generation in mammals, the antibody was not isolated from the blood of immunised mammals but from the egg yolk of immunised chickens. A standard calibration curve was optimised using immunoaffinity purified antibody extract and a coating antigen concentration of 10 μg ml⁻¹. One percent skim milk powder was chosen for blocking. The assay has a minimum detection limit of 10 μg l⁻¹, with an IC₅₀ of 618 μg l⁻¹ when a 50 mM phosphate buffer at pH 7.5 and 10 mM sodium chloride is used as assay buffer. The cross reactivity testing shows a high specificity for hazelnut proteins and various foods and food additives were found to be non reactive except beans, sunflower seed or poppy seed.     

Detection of adulterations in processed coffee with cereals and coffee husks using capillary zone electrophoresis

The proposed method for the identification of adulteration was based on the controlled acid hydrolysis of xylan and starch present in some vegetable adulterants, followed by the analysis of the resulting xylose and glucose, which are the monosaccharides that compose, respectively, the two polysaccharides. The acid hydrolysis with HCl increases the ionic strength of the sample, which impairs the electrophoretic separation. Thus, a neutralization step based on anion exchange resin was necessary. The best separations were obtained in NaOH 80 mmol/L, CTAB 0.5 mmol/L, and methanol 30% v/v. Because of the high value of pH, monosaccharides are separated as anionic species in such running electrolyte. The LOQ for both monosaccharides was 0.2 g for 100 g of dry matter, which conforms to the tolerable limits.     

Determination of Cry9C protein in processed foods made with StarLink corn

StarLink (Aventis CropScience US) hybrid corn has been genetically modified to contain a pesticidal protein, Cry9C, which makes it more resistant than traditional varieties to certain types of corn insect pests. Unlike other varieties of genetically engineered corn, the U.S. Environmental Protection Agency authorized the use of StarLink corn for animal feed and industrial use only, not for human consumption. However, some Cry9C-containing corn was mistakenly or inadvertently comingled with yellow corn intended for human food use. Because corn containing the Cry9C construct was not approved for human use, the U.S. Food and Drug Administration considers food containing it to be adulterated. Consequently, this regulatory violation resulted in hundreds of recalls of corn-based products, such as taco shells, containing cry9C DNA. Detecting the novel protein in StarLink corn is an emerging issue; therefore, there is no standardized or established analytical method for detecting Cry9C protein in processed foods. We developed a procedure for quantitation of Cry9C protein, with validation data, in processed food matrixes with a limit of quantitation at 1.7 ng/g (ppb), using a commercial polyclonal antibody-based Cry9C kit that was intended for corn grain samples. Intra- and interassay coefficients of variation were 2.8 and 11.8%, respectively. Mean recoveries were 73 and 85% at 2 and 5 ng/g Cry9C fortifications, respectively, for 19 control non-StarLink corn-based matrixes. Our data demonstrate only 0-0.5% of Cry9C protein survived the processing of tortilla chips and soft tortillas made from 100% StarLink corn, resulting in levels from below the detection limit to 45 ppb.     

Electrochemical ELISA for the screening of DDT related compounds: analysis in waste waters

An electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p′-DTT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE), 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane (p,p′-DDD) and o,p-DDT was developed. Optimization of the ELISA competition conditions, led to similar response for the p,p′-isomers. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3′,5,5′-tetramethylbenzidine as substrate. The use of purified antiserum for p,p′-DDT resulted in a sensitive assay with a detection limit of 40 pg ml⁻¹ and R.S.D. ranging from 1 to 3% intra-day and 3 to 6% inter-day. No matrix effect for waste water samples of different origin has been evidenced. The ELISA was used to detect DDTs in 20 samples after extraction in diethylether. This method appears suitable for routine screening of DDTs without sample pre-treatment other than dilution in PBS or after organic solvent extraction if high sensitivity is required.