Effects of the Maillard Reaction on the Immunoreactivity of Amandin in Food Matrices.

Amandin is the major storage protein and allergen in almond seeds. Foods, containing almonds, subjected to thermal processing typically experience Maillard browning reaction. The resulting destruction of amino groups, protein glycation, and/or denaturation may alter amandin immunoreactivity. Amandin immunoreactivity of variously processed almond containing foods was therefore the focus of the current investigation. Commercial and laboratory prepared foods, including those likely to have been subjected to Maillard browning, were objectively assessed by determining Hunter L* , a* , b* values. The L* values for the tested samples were in the range of 31.75 to 85.28 consistent with Maillard browning. Three murine monoclonal antibodies, 4C10, 4F10, and 2A3, were used to determine the immunoreactivity of the targeted samples using immunoassays (ELISA, Western blot, dot blot). The tested foods did not exhibit cross-reactivity indicating that the immunoassays were amandin specific. For sandwich ELISAs, ratio (R) of sample immunoreactivity to reference immunoreactivity was calculated. The ranges of R values were 0.67 to 15.19 (4C10), 1.00 to 11.83 (4F10), and 0.77 to 23.30 (2A3). The results of dot blot and Western blot were consistent with those of ELISAs. Results of these investigations demonstrate that amandin is a stable marker protein for almond detection regardless of the degree of amandin denaturation and/or destruction as a consequence of Maillard reaction encountered under the tested processing conditions. PRACTICAL APPLICATION: Foods containing almond are often subjected to processing prior to consumption. Amandin, the major allergen in almond, may experience Maillard reaction. Understanding the change in amandin immunoreactivity as a result of Maillard reaction is important for amandin detection and production of hypoallergenic food products.

Effects of processing and storage on almond (Prunus dulcis L.) amandin immunoreactivity.

A murine monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) was used to assess amandin immunoreactivity in processed and long-term stored almonds. The results demonstrated that amandin immunoreactivity is stable in variously processed almond seeds. Using the ELISA, amandin immunoreactivity could be detected in commercial whole raw and processed (blanched, sliced, dry roasted, and indicated combinations thereof) almond seeds stored for eleven years and eight months, defatted almond seed flours from several almond varieties/hybrids and their borate saline buffer-solubilized protein extracts stored for ten years and seven months, and several almond varieties grown in different California counties (full fat flours and their defatted flour counterparts). Roasting Nonpareil whole full fat almond seeds, full fat flour, and defatted flour at 170°C for 20min each with 2, 5, 10, and 20% w/w corn syrup or sucrose did not prevent amandin detection by ELISA. Similarly, amandin detection in select food matrices spiked with Nonpareil almond protein extract was not inhibited. In conclusion, amandin is a stable target protein for almond detection under the tested processing and storage conditions.

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Conformational epitope mapping of Pru du 6, a major allergen from almond nut.

Tree nuts are a widely consumed food. Although enjoyed safely by most individuals, allergic reactions to tree nuts, including almond, are not uncommon. Almond prunin (Pru du 6), an 11S globulin (legumin), is an abundant nut seed protein and a major allergen. Conformational epitope mapping studies of prunin have been performed with a murine monoclonal antibody (mAb) 4C10. This mAb reacts with non-reduced but not reduced prunin in immunoblotting assays, indicating the recognition of a conformational epitope. 4C10 competes with patient IgE, as assessed by ELISA, indicating clinical significance of the epitope. To characterize the 4C10 epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (MS) was performed on the native prunin-4C10 complex and on uncomplexed native prunin. Several epitope candidate peptides that differ in deuterium uptake between the complexed and uncomplexed forms were identified. The epitope was further mapped by analyzing chimeric molecules incorporating segments of the homologous soybean allergen, Gly m 6, in immunoassays. These data indicate that the 4C10 epitope overlaps with a subset of patient IgE binding epitopes on almond prunin and further supports HDX-MS as a valid technique for mapping conformational epitopes.

Functional properties of select edible oilseed proteins.

Borate saline buffer (0.1 M, pH 8.45) solubilized proteins from almond, Brazil nut, cashew nut, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean seeds were prepared from the corresponding defatted flour. The yield was in the range from 10.6% (macadamia) to 27.4% (almond). The protein content, on a dry weight basis, of the lyophilized preparations ranged from 69.23% (pine nut) to 94.80% (soybean). Isolated proteins from Brazil nut had the lightest and hazelnut the darkest color. Isolated proteins exhibited good solubility in aqueous media. Foaming capacity (<40% overrun) and stability (<1 h) of the isolated proteins were poor to fair. Almond proteins had the highest viscosity among the tested proteins. Oil-holding capacity of the isolated proteins ranged from 2.8 (macadamia) to 7 (soybean) g of oil/g of protein. Least gelation concentrations (% w/v) for almond, Brazil nut, cashew, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean were, respectively, 6, 8, 8, 12, 20, 12, 10, 14, 14, and 16.

Effects of long-term frozen storage on electrophoretic patterns, immunoreactivity, and pepsin in vitro digestibility of soybean (Glycine max L.) proteins.

Soybean flours stored for 20 years at -20 degrees C retained protein polypeptide profile integrity. Proteins in stored soybean flours retained their immunoreactivity. Long-term frozen storage of seed flours at -20 degrees C did not adversely affect seed protein in vitro pepsin digestibility.