Indirect competitive ELISA for determination of traces of peanut (Arachis hypogaea L.) protein in complex food matrices

An indirect competitive ELISA was developed allowing the detection of hidden peanut protein residues down to 2 ppm (micorgrams per gram) in various foods. The high-titer, peanut-specific polyclonal antiserum used recognized potentially allergenic proteins in both native and roasted peanuts. In the absence of a food matrix, extractable protein from roasted peanuts was detected at 104 +/- 13%. From various food items, peanut protein at > or =13 ppm was recovered between 84 and 126%, and at 2 ppm of peanut protein recovery was 143 +/- 6%. Intra- and interassay precision was <15%. In 5 of 17 commercial food products without declaration of peanut components, between 2 and 18 ppm of peanut protein was detected. This is the first assay based on commercially available reactants that allows the reliable determination of trace amounts of hidden peanut allergens in a variety of complex food matrices.     

Detection of potentially allergenic hazelnut (Corylus avellana) residues in food: a comparative study with DNA PCR-ELISA and protein sandwich-ELISA

Allergen detection is of increasing interest for food labeling purposes. A comparative study with a commercial hazelnut-specific PCR-ELISA and a sandwich-type ELISA detecting hazelnut protein was performed to investigate to what extent immunochemical and DNA-based techniques would correlate in the detection of trace amounts of potentially allergenic hazelnut residues. Both methods were highly sensitive and allowed the detection of even <10 ppm of hazelnut in complex food matrixes. The protein-ELISA was highly specific for hazelnut. However, some foods could lead to false-positive results at the 10 ppm level. The PCR-ELISA did not show any cross-reactions with non-hazelnut foods, thus reducing the probability of having false positives at the trace level. Forty-one commercial food products with and without hazelnut components on their labels were analyzed for the presence of hazelnut. Of the 27 products in which hazelnut components were detected, two samples were not identified by the protein-ELISA, and only one sample, namely one white chocolate having <1 ppm of hazelnut protein, was not detected by PCR-ELISA. The good correlation of the results of PCR-ELISA and protein-ELISA suggested that both PCR-based and immunochemical techniques are suitable for reliable detection of potentially allergenic hazelnut residues in foods at the trace level.     

A novel approach for the detection of potentially hazardous pepsin stable hazelnut proteins as contaminants in chocolate-based food

Contamination of food products with pepsin resistant allergens is generally believed to be a serious threat to patients with severe food allergy. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to measure pepsin resistant hazelnut protein in food products. Capturing and detecting rabbit antibodies were raised against pepsin-digested hazelnut and untreated hazelnut protein, respectively. The assay showed a detection limit of 0.7 ng/mL hazelnut protein or <1 microg hazelnut in 1 g food matrix and a maximum of 0.034% cross-reactivity (peanut). Chocolate samples spiked with 0.5-100 microg hazelnut/g chocolate showed a mean recovery of 97.3%. In 9/12 food products labeled "may contain nuts", hazelnut was detected between 1.2 and 417 microg hazelnut/g food. It can be concluded that the application of antibodies directed to pepsin-digested food extracts in ELISA can facilitate specific detection of stable proteins that have the highest potential of inducing severe food anaphylaxis.     

Development of a stable isotope dilution assay for the quantification of 5-methyl-(E)-2-hepten-4-one: application to hazelnut oils and hazelnuts.

A stable isotope dilution assay was developed for the quantitation of the hazelnut odorant 5-methyl-(E)-2-hepten-4-one by mass chromatography using synthesized [(2)H](2)-5-methyl-(E)-2-hepten-4-one as the internal standard. Application of the method on two batches of commercial hazelnut oils, processed from either roasted or unroasted nuts, revealed 6.4 microg 5-methyl-(E)-2-hepten-4-one per kg of unroasted oil whereas 315.8 microg per kg was determined in the roasted nut oil. The about 50-fold higher amount of 5-methyl-(E)-2-hepten-4-one in roasted hazelnut oil suggested the necessity of a thermal treatment to generate the flavor compound. Pan frying of raw hazelnuts (9 to 15 min) or boiling of the crushed nut material for 1 h in water led to an increase of 5-methyl-(E)-2-hepten-4-one by factors of 600 and 800, respectively, thereby corroborating that the major part of the nut flavorant is formed during heat treatment from a yet unknown precursor in hazelnuts.     

Browning indicators in bread

Bread is the most important food in the Spanish household and represents the largest proportion of products produced by commercial bakeries. The browning indicators furosine, hydroxymethylfurfural (HMF), and color were determined to evaluate heat effects induced during manufacture of these foods. The breads analyzed were common, special, sliced toasted, and snack breads. Identical sample preparation and HPLC conditions were used to determine HMF in all breads. The precision tested at high and low HMF concentration in breads was 2.60% and 1.57%, respectively. Recovery of HMF was 96.2%. The HMF values ranged from 2.2 to 68.8 mg/kg. Color index (100 - L) ranged from 17.0 to 38.2. The linear correlations (r(2)) between 100 - L/HMF were above 0.70 for common, special, and snack breads. Similar correlation was obtained between 100 - L/HMF in a dough baking at different times. The furosine content in common bread ranged between 125 and 208 mg/100 g of protein. No linear correlation was found between furosine and HMF. Moreover, HMF and furosine were also determined in crumb and crust. Levels of HMF had a wide range (0.9-1.76 mg/kg) and furosine was between 43 and 221 mg/100 g of protein.     

Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR

Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.     

Effects of roasting on the antioxidant status and phenolic profiles of commercial Turkish hazelnut varieties (Corylus avellana L.)

The effect of roasting on the antioxidant status and phenolic profiles of seven commercial Turkish hazelnut varieties (namely, ?ak?ldak, Fo?a, Karaf?nd?k, Mincane, Palaz, Sivri, and Tombul) was assessed. Samples were examined for their total phenolics, oxygen radical absorbance capacity (ORAC) values, condensed tannins, and phenolic acids (free and bound forms). Significant losses (p < 0.05) in total phenolics (~66.3%), ORAC values (~41.6%), condensed tannins (~75.2), and phenolic acids (~42.7) were noted when the hazelnuts were roasted. Some variations both between and within natural and roasted hazelnuts were observed (p < 0.05). Phenolic acids were mainly found in the bound form. Gallic, protocatechuic, p-coumaric, and ferulic + sinapic acids were present in all hazelnut varieties, albeit to different extents, and the first two were dominant. Mincane, in roasted form, had the highest total phenolics, ORAC values, condensed tannins, and phenolic acids. This was due to the presence of some skin in roasted Mincane. No skin was left in all other varieties upon roasting. The present work suggests that roasting results in a significant loss in the antioxidant status and phenolic profiles because of the removal of the skin, which is a rich source of phenolics. It is highly recommended to consume natural hazelnut instead of the roasted counterpart to take advantage of all of the functional benefits of this nut.     

Sandwich immunoassays for the determination of peanut and hazelnut traces in foods

People suffering from food allergies are dependent on accurate food labeling, as an avoidance diet is the only effective countermeasure. Even a small amount of allergenic protein can trigger severe reactions in highly sensitized patients. Therefore, sensitive and reliable tests are needed to detect potential cross-contamination. In this paper two fast sandwich immunoassays are described for the determination of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) traces in complex food matrices. Mouse monoclonal antibodies were used as capture antibodies, and labeled rabbit polyclonal antibodies were used as detection antibodies in both assays. The assay time was 30 min in total, and cross-reactivities against a variety of fruits and seeds were found to be in the low 10(-4)% (ppm) level or in some cases not detectable. The recoveries in all tested food matrices ranged from 86 to 127%, and the limits of detection were in the range of 0.2-1.2 mg/kg (ppm) in food for both peanut and hazelnut, respectively.     

Nutritional composition of hazelnut (Corylus avellana L.) as influenced by basic fertilization

Nitrogen (N), phosphorus (P), and potassium (K) deficiencies are a widespread phenomenon throughout the world, and are one of the most common nutritional disorders in Turkish hazelnut (Corylus avellana L.) cultivation. In this research, the effects of macronutrient fertilizers on mineral contents and some biochemical contents of ‘Tombul’ hazelnut (Corylus avellana L.) variety cultivated in the Black Sea Region of Turkey were investigated, and the contribution of these nuts to human nutrition was determined. The trials were carried out at ‘Tombul’ hazelnut orchards, and the hazelnuts were fertilized with five different doses of nitrogen, phosphorus, and potassium in each year for three consecutive years. The basic fertilizers (N, P₂O₅, and K₂O) significantly affected some biochemical contents and mineral compositions of the hazelnuts. In order to improve the biochemical and mineral compositions of hazelnut, 200 kg ha^?1 and 400 kg ha^?1 N fertilizations, 120 kg ha^?1 and 160 kg ha^?1 P₂O₅ fertilizations, and 400 kg ha^?1 and 600 kg ha^?1 K₂O fertilizations could be recommended for practice. According to the daily mineral element requirements, 100 g of hazelnuts provided about 43.5% P, 13.2% K, 19.4% Ca, 37.0% Mg, 0.2% Na, 53.8% Fe, 24.5% Zn, 14.5% B, and 66.7% Mo of the recommended dietary allowances. Cu and Mn contents of 100 g hazelnut were higher than the respective daily requirements. These results indicated that the mineral composition of hazelnut depended not only on the variety but also on the cultivation techniques such as soil condition and especially basic fertilization practices.     

Variations in isoflavone levels in soy foods and soy protein isolates and issues related to isoflavone databases and food labeling

The reliability of databases on the isoflavone composition of foods designed to estimate dietary intakes is contingent on the assumption that soy foods are consistent in their isoflavone content. To validate this, total and individual isoflavone compositions were determined by HPLC for two different soy protein isolates used in the commercial manufacture of soy foods over a 3-year period (n = 30/isolate) and 85 samples of 40 different brands of soy milks. Total isoflavone concentrations differed markedly between the soy protein isolates, varying by 200-300% over 3 years, whereas the protein content varied by only 3%. Total isoflavone content varied by up to 5-fold among different commercial soy milks and was not consistent between repeat purchases. Whole soybean milks had significantly higher isoflavone levels than those made from soy protein isolates (mean +/- SD, 63.6 +/- 21.9 mg/L, n = 43, vs 30.2 +/- 5.8 mg/L, n = 38, respectively, p < 0.0001), although some isolated soy protein-based milks were similar in content to "whole bean" varieties. The ratio of genistein to daidzein isoflavone forms was higher in isolated soy protein-based versus "whole bean" soy milks (2.72 +/- 0.24 vs 1.62 +/- 0.47, respectively, p < 0.0001), and the greatest variability in isoflavone content was observed among brands of whole bean soy milks. These studies illustrate large variability in the isoflavone content of isolated soy proteins used in food manufacture and in commercial soy milks and reinforce the need to accurately determine the isoflavone content of foods used in dietary intervention studies while exposing the limitations of food databases for estimating daily isoflavone intakes.     

Analysis for total sulfite in foods by using rapid distillation followed by redox titration

A rapid and accurate analysis for total sulfite as sulfur dioxide has been developed for foods and food products. The method, which combines a selective distillation cleanup procedure with the selective redox titration of sulfite ion by iodine, has been applied to a variety of foods and food products over a period of time with no significant interference encountered in any matrixes other than garlic and leeks. For the foods analyzed, the method typically shows a detection limit of 10 ppm, a relative standard deviation of 7.5% (compared with 10.4% for similar matrixes by the Monier-Williams method), and recoveries of 97.9 +/- 6.4%. Comparison of results for this method with those obtained using the Monier-Williams method showed a mean value for the distillation/titration method of 241 ppm compared with 242 ppm for the Monier-Williams method. A correlation of 0.991 and odds of a difference between methods of 10.7% (Student's paired t-test (1-alpha) X 100) were obtained for those matrixes where no interferences were encountered with either method.     

Liquid chromatographic determination of polydextrose in food matrixes

A liquid chromatographic (LC) method has been developed to determine the content of polydextrose, a water-soluble 1 calorie/g bulking agent, in food matrixes such as cookies, cakes, fruit spreads, and chocolate toppings. This analysis, which requires use of a blank matrix, provides a feasible means to control the manufacture of foods containing this additive and provides a component for the accurate determination of the caloric value of a particular food product. The method involves aqueous extraction of the polydextrose from the food matrix followed by separation on a carbohydrate analysis column. The LC system uses a mobile phase of 0.005M CaSO4.2H2O and a refractive index detector for quantitation. Polydextrose recoveries from the food matrixes varied from 91.5 to 100.9% with assay precision, expressed as coefficient of variation, ranging from 0.7 to 4.3%. Each error estimate was derived from 5 parallel determinations. The present methodology is precise and selective in contrast to the modified classical phenol-sulfuric acid colorimetric method for assaying carbohydrates, which had been used for polydextrose determination in food matrixes in the past. Because the coefficient of variation frequently exceeded 10%, replicate analyses were necessary to achieve quantitation.     

Solid-phase microextraction for studies on the enantiomeric composition of filbertone in hazelnut oils

The enantiomeric distribution of filbertone was determined in unroasted and roasted hazelnut oils of different geographical origins by using solid-phase microextraction (SPME) and capillary gas chromatography. An optimization procedure including SPME fiber, extraction time, exposure temperature, and sample volume enabled the best conditions to be selected. Under the optimized conditions, detection limits were in the micrograms per liter level for both enantiomers of filbertone with relative standard deviation values of 7.1 and 4.9% for R-filbertone and S-filbertone, respectively. The proposed approach allowed the rapid determination of the enantiomeric composition of filbertone and demonstrated that its variability is an inherent property of the natural compound. Analysis of two batches of hazelnut oils obtained from either unroasted or roasted hazelnuts showed, in general, significantly higher amounts of filbertone in roasted hazelnut oils.     

Single-tube nested real-time PCR as a new highly sensitive approach to trace hazelnut

Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a wide variety of processed foods. However, it is a source of allergens capable of inducing mild to severe allergic reactions in sensitized individuals. Hence, the development of highly sensitive methodologies for hazelnut traceability is essential. In this work, we developed a novel technique for hazelnut detection based on a single-tube nested real-time PCR system. The system presents high specificity and sensitivity, enabling a relative limit of detection of 50 mg/kg of hazelnut in wheat material and an absolute limit of detection of 0.5 pg of hazelnut DNA (1 DNA copy). Its application to processed food samples was successfully achieved, detecting trace amounts of hazelnut in chocolate down to 60 mg/kg. These results highlight the adequacy of the technique for the specific detection and semiquantitation of hazelnut as potential hidden allergens in foods.     

Triacylglycerol and phospholipid composition of hazelnut (Corylus avellana L.) lipid fraction during fruit development

We analyzed the triacylglycerol and phospholipid contents of hazelnuts from early development to maturity. Both were analyzed by high-performance liquid chromatography coupled to a light scattering detector. Trioleylglycerol, linoleyl-dioleylglycerol, and palmitoyl-dioleylglycerol were the most predominant triacylglycerols throughout development. Triacylglycerols showed small variation during hazelnut development. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were the most abundant phospholipids. Traces of phosphatidic acid were also detected. The statistical analysis showed that the positive correlation among the individual phospholipid contents was significant. Phospholipid contents showed a steep decrease during hazelnut development. Triacylgycerols and phosphatidylcholine were isolated by preparative thin-layer chromatography, and their fatty acid profile was determined by gas-liquid chromatography. Triacylglycerols showed a high percentage of monounsaturated fatty acid moieties, whereas phosphatidylcholine had the highest percentage of saturated and monounsaturated fatty acid moieties. The polyunsaturated fatty acid moiety showed low percentages in the triacylglycerol and phospholipid backbone.     

Detection of allergenic ingredients using real-time PCR: a case study on hazelnut (Corylus avellena) and soy (Glycine max)

Compliance with the European allergen labeling legislation (Directive 2007/68/EC) is only possible when coupled with appropriate methods to detect allergens in food. The aim of the current study was to develop new real-time PCR assays for the detection of hazelnut and soy and evaluate these assays via comparison with commercially available kits. Although the new assays were not as sensitive as the commercial qualitative assays, they proved to be more specific. Moreover, the cross-reactivity study indicated contamination of some of the food products used with either hazelnut or soy, which presents a risk for the allergic consumer. The assays were able to quantify as few as 5-15 genome copies. This unit, used to express analytical results for allergen detection by means of PCR, needs to be converted to a unit expressing the amount of allergenic ingredient in order to be informative. This study emphasizes that the use of real-time PCR for allergen quantification is complicated by the lack of appropriate reference materials for allergens.     

Inulin determination for food labeling

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.     

Quantification of the group B soyasaponins by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins alphag, betag, and betaa, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 micromol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 micromol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 micromol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.     

Thermal stability of organophosphorus pesticide triazophos and its relevance in the assessment of risk to the consumer of triazophos residues in food

The degradation of triazophos in aqueous solutions was monitored at 205 and 254 nm after separation using high-performance liquid chromatography. An ODS column was used with a mobile phase of 60% acetonitrile and 0.04% phosphoric acid at a flow rate of 1.4 cm(3) min(-)(1). When dissolved in distilled water, approximately 30% of the original triazophos was detected. The effect of heating time and temperature on a 0.5 mg dm(-3) standard was investigated. Over a 150 min period at 100 degrees C the peak area detected for the standard decreased by 58.67 +/- 6.19 and 65.03 +/- 4.61% when measured at 254 and 205 nm, respectively. The precision of the absorbance detected at 205 and 254 nm was 3.54 +/- 2.8 and 3.86 +/- 3.9%, respectively. There was a significant difference (P = 0.10) between the precision of the results obtained at each wavelength. The t(calcd) value was -2.236 and the t(crit) value was 1.94. The most sensitive wavelength was 205 nm. A 54% difference in the gradients of the calibration graphs obtained at each wavelength was observed. The results suggest that approximately 72% of triazophos is degraded during a 20 min cooking period at 100 degrees C, due to ambient and elevated temperature hydrolysis. Therefore, the dose to the consumer of triazophos residues in cooked food is likely to be approximately 72% lower than in the raw food, with a concomitant reduction in toxicological risk.