Detección de trazas de soja y de leche en productos libres de gluten: desarrollo de dos enzimoinmunoensayos competitivos / Detection of traces of soy and milk in gluten-free products: development of two competitive enzyme immunoassays / Detecção de vestígios de soja e leite em produtos livres de glúten: desenvolvimento de dois enzimoimunoensaios competitivos

Resumen El objetivo del presente trabajo fue el desarrollo de dos enzimoinmunoensayos competitivos (EIC) para la detección de trazas de soja y de leche en productos libres de gluten. Como anticuerpos primarios se utilizaron antisueros policlonales de conejo específicos contra proteínas de soja o de leche. Se determinaron las concentraciones óptimas de antígenos a inmovilizar en la placa y las concentraciones de anticuerpos primarios a utilizar en la competencia. Las curvas de calibración se ajustaron utilizando concentraciones crecientes de un extracto de producto de soja y de un extracto de leche descremada en polvo. El producto de soja y la leche descremada se extrajeron con buffer Tris-HCl 0,0625 M con dodecilsulfato de sodio al 3% y sulfito de sodio 0,1 M al 2%. Se evaluaron los parámetros de validación: linealidad, límites de detección y de cuantificación, recuperación y precisión en el día y entre días, los cuales resultaron adecuados. Se analizaron 9 productos libres de gluten con los EIC desarrollados y con kits de ELISA comerciales. Ambos EIC se comportaron de manera similar con respecto a los kits comerciales. Los EIC permitieron confirmar la presencia de leche en las muestras que la declaraban. En algunas muestras que no declaraban ni leche ni soja, ambos EIC detectaron su presencia (resultados confirmados con los kits comerciales). Los EIC desarrollados poseen menor costo que los kits y, por lo tanto, éstos podrían utilizarse como métodos de screening. Cuando esta metodología resulte negativa, debe confirmarse con un método más sensible (comercial) para garantizar la ausencia de proteínas de soja o de leche.

Abstract The aim of this study was to develop two competitive enzyme immunoassays (CEI) to detect the presence of traces of soy and milk in gluten-free products. Specific rabbit polyclonal antiserums against soy protein and other against elemilk protein were used as primary antibodies. Optimal antigen concentrations to be immobilized on the plate and primary antibody concentrations to be used in competition were determined. The calibration curves were fitted using increasing concentrations of an extract of soy product and of defatted milk powder. The soy product and the defatted milk were extracted with Tris-HCl buffer 0,0625 M with 3% sodium dodecyl sulfate and 2% sodium sulfite 0.1 M. The validation parameters were evaluated: linearity, limit of detection and quantification, recovery and precision on the day and in between days. They were appropriate. Nine commercial samples of gluten-free products were analyzed with these developed CEI and commercial ELISA kits. It was observed that both CEI behaved similarly with respect to the commercial kits. The enzyme immunoassays confirmed the presence of milk in samples that declared it. In some samples that did not declare the presence of milk or soy, both enzyme immunoassays detected their presence -these results were confirmed using commercial kits. The developed CEI have a lower cost than the commercial kits, so these could be used as screening methods. When this methodology is negative, it should be confirmed with a more sensitive (commercial) method to ensure the absence of soy or milk protein.

Resumo O objetivo do presente trabalho foi o desenvolvimento de dois enzimoimunoensaios competitivos (EIC), para a detecção de vestígios de soja e leite em produtos livres de glúten. Antissoros policlonais de coelho específicos contra proteínas de soja ou de leite foram utilizados como anticorpos primários. Foram determinadas as concentrações ótimas de antígenos a serem imobilizados na placa e as concentrações de anticorpos primários a serem utilizadas na competição. As curvas de calibração foram ajustadas usando concentrações crescentes de um extrato de produto de soja e de um extrato de leite em pó desnatado. O produto de soja e o leite desnatado foram extraídos com tampão Tris-HCl 0,0625 M com dodecil sulfato de sódio a 3% e sulfito de sódio 0,1 M a 2%. Os parâmetros de validação foram avaliados: linearidade, limite de detecção e quantificação, recuperação e precisão no dia e entre os dias, os quais resultaram adequados. Nove produtos livres de glúten foram analisados com os EIC desenvolvidos e com kits de ELISA comerciais. Os dois EICs se comportaram de maneira semelhante em relação aos kits comerciais. Os EIC permitiram confirmar a presença de leite nas amostras que o declararam. Em algumas amostras que declaravam nem leite nem soja, ambos os EIC detectaram sua presença (resultados confirmados usando kits comerciais). Os EIC desenvolvidos têm um custo menor que os kits, portanto, eles poderiam ser utilizados como métodos de triagem. Quando esta metodologia é negativa, deve ser confirmada com um método mais sensível (comercial) para garantir a ausência de proteínasda soja ou do leite.

Assessment of Phenotypic Variations and Correlation among Seed Composition Traits in Mutagenized Soybean Populations.

Soybean [Glycine max (L.) Merr.] seed is a valuable source of protein and oil worldwide. Traditionally, the natural variations were heavily used in conventional soybean breeding programs to select desired traits. However, traditional plant breeding is encumbered with low frequencies of spontaneous mutations. In mutation breeding, genetic variations from induced mutations provide abundant sources of alterations in important soybean traits; this facilitated the development of soybean germplasm with modified seed composition traits to meet the different needs of end users. In this study, a total of 2366 'Forrest'-derived M2 families were developed for both forward and reverse genetic studies. A subset of 881 M3 families was forward genetically screened to measure the contents of protein, oil, carbohydrates, and fatty acids. A total of 14 mutants were identified to have stable seed composition phenotypes observed in both M3 and M4 generations. Correlation analyses have been conducted among ten seed composition traits and compared to a collection of 103 soybean germplasms. Mainly, ethyl methanesulfonate (EMS) mutagenesis had a strong impact on the seed-composition correlation that was observed among the 103 soybean germplasms, which offers multiple benefits for the soybean farmers and industry to breed for desired multiple seed phenotypes.

Ultrafast Multiplexed-Allergen Detection through Advanced Fluidic Design and Monolithic Interferometric Silicon Chips.

A silicon-based miniaturized sensor chip combined with an advanced microfluidic module for the simultaneous, label-free immunochemical determination of four allergens, bovine milk protein, peanut protein, soy protein, and gliadin, is presented. The sensor chip consists of an array of 10 broad-band Mach-Zehnder interferometers (BB-MZIs) monolithically integrated on silicon, along with their respective broad-band light sources. The BB-MZIs were biofunctionalized with the targeted allergens and their responses during immunoreaction were monitored by multiplexing their transmission spectra through an external miniaturized spectrometer. The assay is performed by running mixtures of calibrators or samples with the antibodies against the four allergens followed by an antispecies specific antibodies solution. Employing a fluidic module of nearly one-dimensional geometry, that provided for uniform delivery of the reagents, CV values <6% were achieved for the responses of the 10 BB-MZIs, allowing for reliable multianalyte determinations. The analysis is completed in 6.5 min, and the detection limits were 0.04 µg/mL for bovine k-casein, 1.0 µg/mL for peanut protein, 0.80 µg/mL for soy protein, and 0.10 µg/mL for gliadin. The assays were accurate (recoveries 88-118%) and repeatable (intra- and interassay CVs <7% for all four allergens). Finally, the sensor was evaluated by analyzing samples from a cleaning in place system (CIP) of a dairy industry and the results obtained were in good agreement with those received by the respective ELISAs. The analytical characteristics of the sensor combined with the short analysis time and the small chip size make the proposed system an ideal tool for on-site multianalyte determinations.

Variance of Commercial Powdered Milks Analyzed by Proton Nuclear Magnetic Resonance and Impact on Detection of Adulterants.

Proton nuclear magnetic resonance spectra for 66 commercial powdered milk samples were analyzed by principal component analysis, soft independent modeling of class analogy, and pooled, crossed analysis of variance. It was found that the sample type (skim milk powder or non-fat dry milk), the supplier, the production site, the processing temperature (high, medium, or low temperature), and the day of analysis provided statistically significant sources of variation. Interestingly, inexact alignment (deviations of ±0.002 ppm) of the spectral reference peak was a significant source of variation, and fine alignment was necessary before the variation arising from the other experimental factors could be accurately evaluated. Using non-targeted analysis, the lowest detectable adulteration for dicyandiamide, melamine, and sucrose was 0.05%, the lowest detectable adulteration for maltodextrin and urea was 0.5%, the lowest detectable adulteration for ammonium sulfate and whey was 5%, and the lowest adulteration for soy protein isolate was undetectable using methods described herein. The measurement of variance and detection of adulteration were relatively unaffected by the resolution. Similar results were obtained with unbinned data (0.0003 ppm resolution) and binning of 333 data points (0.1 ppm resolution).

Assessment of adulteration of soybean proteins in dairy products by 2D microchip-CE device.

To determine the adulteration of soybean proteins in dairy product, a microchip-CE device was developed to isolate selected fraction of soybean and milk proteins in pI range from 5.5 ∼ 7.0 by 1D IEF, followed by ITP/CZE in the embedded capillary for preconcentration, separation and UV detection at 280 nm. Compared to IEF-CZE without ITP preconcentration, the enhancement factor (EF) in detection of soybean proteins was 20 times. Adulteration of 0.1% soybean protein in total dairy proteins can be detected in less than 10 min.

Compositional differences in soybeans on the market: glyphosate accumulates in Roundup Ready GM soybeans.

This article describes the nutrient and elemental composition, including residues of herbicides and pesticides, of 31 soybean batches from Iowa, USA. The soy samples were grouped into three different categories: (i) genetically modified, glyphosate-tolerant soy (GM-soy); (ii) unmodified soy cultivated using a conventional "chemical" cultivation regime; and (iii) unmodified soy cultivated using an organic cultivation regime. Organic soybeans showed the healthiest nutritional profile with more sugars, such as glucose, fructose, sucrose and maltose, significantly more total protein, zinc and less fibre than both conventional and GM-soy. Organic soybeans also contained less total saturated fat and total omega-6 fatty acids than both conventional and GM-soy. GM-soy contained high residues of glyphosate and AMPA (mean 3.3 and 5.7 mg/kg, respectively). Conventional and organic soybean batches contained none of these agrochemicals. Using 35 different nutritional and elemental variables to characterise each soy sample, we were able to discriminate GM, conventional and organic soybeans without exception, demonstrating "substantial non-equivalence" in compositional characteristics for 'ready-to-market' soybeans.

Soy in wheat--contamination levels and food allergy risk assessment.

In the United States, packaged food ingredients derived from allergenic sources must be clearly labeled. However, no requirement exists to declare the presence of residues of raw agricultural commodities due to agricultural commodity comingling. Clinical reports of allergic reactions to undeclared soy in wheat-based products do not exist suggesting that a rather low degree of risk is posed by wheat-based products that are comingled with soy. Detectable soybean residues (>2.5 ppm soy flour) were found in 62.8% of commercially available wheat flours at concentrations of 3-443 ppm soy flour (1.6-236 ppm soy protein). Conservative probabilistic risk assessments predict a risk of allergic reaction among the most sensitive soy-allergic individuals of 2.8±2.0 per 1000 soy-allergic user eating occasions of foods containing wheat flour. However, the predicted reactions occur at exposure levels below the lowest eliciting dose observed to provoke objective reactions in clinical oral soy challenges. Given this low level of predicted risk and the lack of evidence for allergic reactions among soy-allergic consumers to wheat-based products, the avoidance of wheat-based products by soy-allergic consumers does not appear to be necessary.

Hypoallergenicity of various miso pastes manufactured in Japan.

Miso paste (miso), a fermented soybean food, is popular in Japan and other Asian countries. However, the soybean is known to induce an allergenic response in some individuals. In the present study, we evaluated the allergenicity of various kinds of miso available in Japan. Total proteins were extracted from Amakuti-kome miso, Karakuti-kome miso, Mugi-miso and Mame-miso, and the protein profiles were analyzed. The major protein bands detected in the intact soybean extract were not present in any of the miso samples, which instead showed various low molecular weight protein bands of approximately 10-25 kDa. The existence levels of six major soybean allergens were determined by Western blotting using specific antibodies. We found that the allergen levels varied among miso and allergen types; however, allergen levels were consistently lower in miso than in the soybean extract. We obtained similar results for IgE-ELISA experiments using serum IgE from soybean allergy patients. Taken together, these results indicate that compared to soybean extract, various types of miso contain small quantities of intact soybean allergens. Additionally, several lines of evidence indicated that the allergen levels were exceptionally low in the dark-colored Karakuti-kome miso and Mame-miso, which are produced with relatively long fermentation periods, suggesting that the duration of fermentation might be a key factor in the hypoallergenicity of miso.

Assessment of soy genotype and processing method on quality of soybean tofu.

Protein quality in six soybean varieties, based on subunit composition of their protein, was correlated with quality of the produced tofu. Also, protein changes due to a pilot plant processing method involving high temperature/pressure and commercial rennet as coagulant were assessed. In each soybean variety, glycinin (11S) and ß-conglycinin (7S) as well as 11S/7S ratio significantly changed from beans to tofu. Between varieties, the 11S/7S protein ratio in seed indicated genotypic influence on tofu yield and gel hardness (r = 0.91 and r = 0.99, respectively; p < 0.05). Also, the 11S/7S ratio correlated with soymilk pH (r = 0.89, p < 0.05), leading to a relationship between soymilk pH with protein recovery and yield of tofu (r = 0.94 and r = 0.91, respectively; p < 0.05). The soybean ß'-subunit of 7S protein negatively influenced tofu hardness (r = -0.91, p < 0.05). Seed protein composition and proportion of 7S protein subunits under the applied production method had an important role in defining tofu quality.

Sensory optimization of a mayonnaise-type spread made with rice bran oil and soy protein.

The optimum formulation for mayonnaise-type spreads containing rice bran oil (RBO) and soy protein concentrate (SPC) was determined based on sensory acceptability. RBO and SPC were used due to their health benefit claims such as lowering risk of heart disease. The effects of the proportions of high-impact ingredients (RBO, SPC, and water) on sensory acceptability of the spreads were determined. The 10 spread formulations, prepared following a 3-component constrained simplex lattice mixture design, were subjected to a consumer acceptance test to identify sensory attributes driving consumer acceptance and purchase intent. Predictive regression models were used to plot mixture response surfaces of the critical sensory attributes (taste, mouthfeel, and overall liking) that influenced purchase intent. Areas within the contour plots of these critical sensory attributes, having acceptability scores > or = 68 ("moderately like" on the 100-mm bidirectional labeled affective magnitude scale) were chosen and superimposed to obtain a predicted optimum formulation range (37% to 43% RBO, 4% to 7% SPC, and 52% to 57% water). The formulation containing 37% RBO, 6% SPC, and 57% water, which was located within the optimum region, was selected as a base for further developing flavored (sour cream & onion, cheddar & sour cream, or Monterrey Jack dried cheese) products. All flavored spreads were significantly more acceptable than the plain formulation. Purchase intent of all flavored products also significantly increased once consumers had been given the information about potential health benefits associated with RBO and SPC in the spreads.

Consumer acceptance of an extruded soy-based high-protein breakfast cereal.

Studies have shown the beneficial effects of soy and high-protein diets on weight loss. The objective of this study was to determine consumer acceptance of a soy-based high-protein breakfast cereal developed to be utilized for weight loss and control. Four formulations with soy flour content of 41%, 47%, 54%, and 60% (w/w) were processed by extrusion. The formulations met the Food and Drug Administration (FDA) guidelines to claim the role of soy protein in reducing the risk of cardiovascular diseases and guidelines for high-protein and high-fiber foods. The effects of soy flour level, addition of cinnamon flavor, and evaluation with or without milk on acceptance were investigated. Overall acceptance of 3 of 8 cereal products was also compared to the acceptance of 5 commercial products in the "healthy" cereal category. Addition of up to 54% (w/w) soy flour resulted in comparable acceptance ratings to products with lower soy flour content. Addition of milk improved aroma and texture acceptance scores and addition of cinnamon flavor improved overall, aroma, and taste acceptance scores. Acceptance of the developed cereal products was not as high as the commercial products; however, it significantly increased when nutritional and cost information was presented. The results of this study demonstrated that with modification of the formulations, an acceptable high-protein soy-based cereal can be developed to increase protein consumption during breakfast meals, which can consequently aid in weight loss and control.

Assessment of soy aeroallergen levels in different work environments.

BACKGROUND: Airborne soybean hull proteins are known causes of asthma epidemics around harbours and soy processing plants. Soy flour dust proteins may cause occupational allergy in food and feed industries. OBJECTIVE: To compare enzyme immunoassays (EIAs) for soy hull and soy flour aeroallergens, exposure assessment in various work environments. METHODS: Airborne dust samples (n=324) from soy unloading and/or processing plants, the animal feed industry and pig stables were analysed by two soy flour assays: one assay for measuring complete soy hull proteins and two assays for measuring the purified low-molecular-weight (LMW) soy hull allergens. RESULTS: Immunoblotting confirmed strong differences between antibody specificities and soy preparations. The results of the two soy flour assays and the assay for measuring complete soy hull proteins were highly correlated (r>0.85). The two LMW soy hull assays also showed a strong mutual correlation (r=0.91), but much less correlation with assays for measuring soy flour and complete soy hull. The levels of LMW soy hull proteins were the highest at sites of soybean unloading or processing, while soy flour levels were particularly high in the soy and animal feed industry. CONCLUSIONS: The optimal EIA procedure for soy aeroallergen exposure assessment depends on the type of work environment and the local soy dust composition. Thus, the type of work environment should always be taken into account in future soy allergy studies in order to prevent a possible underestimation of the workers' actual risk of developing soy allergy.

Assessment of indigenous Nepalese soybean as a potential germplasm resource for improvement of protein in North American cultivars.

Soybeans contain approximately 40% protein and 20% oil and represents an important source of protein in animal rations and human diets. Attempts are being made to increase further the overall protein content of soybeans by utilization of exotic germplasms. In this study, soybean cultivars from Nepal have been characterized and their potential as a germplasm resource for improvement of the protein content and quality of North American cultivars assessed. Soybean cultivars 'Sathia', 'Seti', 'Kavre', and 'Soida Chiny', indigenous to various regions of Nepal, contained 42-45% protein, which is significantly higher in comparison to that of the North American cultivar 'Williams 82' (39%). Fractionation of seed protein by high-resolution two-dimensional gel electrophoresis revealed differences in the protein profiles of these cultivars. Various isoelectric forms of glycinin and beta-conglycinin were identified by comparing the matrix-assisted laser desorption ionization time-of-flight mass fingerprinting data against the National Center for Biotechnology Information nonredundant database. Nepalese cultivar Sathia was distinct, lacking some isoelectric forms of acidic and basic glycinin subunits while expressing other unique forms. The contribution of these unique protein spots present in either Sathia or Williams 82 to the total protein content was quantified using scanning laser densitometry. Distinct restriction fragment length polymorphisms (RFLP) for group 1 glycinin genes were observed among the tested Nepalese genotypes, indicating sequence variation among the cultivars. Conversely, evaluation of RFLP for the genes encoding group 2 glycinins, beta-conglycinin, and Bowman-Birk proteinase inhibitors indicated a high degree of conservation in these genes. Determination of amino acid composition, a reflection of protein quality, indicated that the arginine content of the Nepalese soybeans ranged from 7.7 to 8.1%, which was 5-10% higher than the 7.4% expressed in Williams 82. Additionally, Karve and Seti contained significantly more cysteine than Williams 82. Nepalese high-protein soybeans having a desirable amino acid composition hold potential to increase the protein quality and diversity of North American cultivars.

Quality, labeling accuracy, and cost comparison of purified soy isoflavonoid products.

OBJECTIVES: To evaluate the labeling accuracy and product uniformity of available soy isoflavone products in the eastern Washington State region for 13 products and to perform a cost comparison based on the isoflavone content in milligrams for 11 products. DESIGN: Thirteen (13) isoflavone products (7 tablet and 6 capsule formulations) were randomly obtained from health food, grocery, and pharmacy outlets in eastern Washington State. Four different samples of the same product were extracted using 75% ethanol. Each sample was analyzed for isoflavone content using a gradient high-performance liquid chromatography (HPLC) method. Pure isoflavonoid standards were used to quantify the content of genistein, daidzein, and glycitein and their respective glycosides. The amount of total isoflavonoids per purchased product was then divided into its purchase price in order to make cost comparisons between the products based on a 50 mg/d dose. RESULTS: The weight variation within each product was generally small (<4%). However, there was significant variability in the composition of the products. Only 4 of the 13 products contained at least 90% of the isoflavone content claimed on the label and 2 of products contained impurities in the HPLC chromatogram that exceeded 40%. There was no difference in the total isoflavonoid contents of the 2 identical products made by the same manufacturer, but the percentages of the main components had changed significantly over time. Based on a 50-mg isoflavone per day dose, the cost of a 30-day supply ranged from $3.20 to $65.88. CONCLUSION: There is a lack of uniformity among the products tested and the majority (67%) of the products contained less than 90% of labeled amounts. There is significant variability in the compositions between the products and in the composition of the same product over time. Consumers cannot trust isoflavonoid product labels to represent the product's content accurately or that product pricing is a reflection of isoflavone content.

Assessment of the protein quality of nine northern adapted yellow and brown seed coated soybean cultivars by amino acid analysis.

Accurate and detailed amino acid determinations were carried out on nine northern adapted soybean cultivars to ascertain whether their amino acid profiles could be used as potentially useful indices for assessing their protein quality. The cultivars were Maple Amber, Maple Donovan, Maple Glen, Maple Isle, Maple Presto, Maple Ridge, and three brown seed coat near-isogenic lines, Maple Presto Brown, Maple Ridge Brown, and Maple Arrow Brown. Their total protein and amino acid composition were compared with those of an established cultivar, Maple Arrow. Mean protein values for the new cultivars ranged from 30.1 to 33.1% compared to Maple Arrow, which was 33.2%. The total nitrogen content was also variable among these cultivars, ranging from 5.0 to 5.4%. All nine Maple series soybean cultivars were higher in their essential amino acid (EAA) content, that is, EAA(9) = 45.2-46.5%, than the FAO/WHO reference protein pattern value of EAA(9) = 33.9%, for a 2-5-year-old child. Each of the nine new soybean cultivars was limited only in methionine and to a lesser extent in valine and isoleucine and had a protein digestibility corrected amino acid score of 91% for all cultivars, compared to the value of egg protein (97%). These results suggest that the most accurate evaluation of protein quality in soybeans, and possibly other legumes and cereals, is by the protein digestibility-corrected amino acid score.

[Immunologic analysis of milk, hen egg, and soybean proteins in butter and margarine, and clinical assessment for availability of hypoallergenic margarine (HAM)].

To determine the allergenic proteins in commercially available butter and margarine, protein fractions were extracted and immunologically analyzed against milk, hen egg, and soybean antigens. Butter and 10 kinds of margarine were proved to contain these proteins in various concentrations by ELISA and immunoblotting methods by use of the rabbit antisera developed against each food proteins. However, hypoallergenic margarine was found to contain no such proteins at all. Using sera obtained from atopic dermatitis patients, previously detected high levels of IgE antibodies to milk, hen egg, or soybeans, the extracted protein fraction from butter and margarine was analyzed whether these proteins react with IgG antibodies in patients' sera. The sera with high levels of specific IgE recognized protein antigens in these extracts except those from hypoallergenic margarine, suggesting that proteins in butter and margarine may become allergenic for the patients sensitive to the proteins, and that hypoallergenic margarine will be a reasonable alternative for the allergic patients to milk, hen egg, or soybeans.