Effects of Maillard Reaction on the Immunoreactivity of Almond Major Protein in the Food Matrices

In the US, among edible tree nut seeds consumed, almonds are ranked number one. Almonds are considered heart healthy and approved by FDA for qualified health claim. Almonds are consumed as natural raw unprocessed or as variously processed seeds such as blanched, roasted, and fried. Additionally, almond seeds, in various forms (such as chopped, diced, slivered, powdered and others) are used as an ingredient in many foods including baked goods and confectionery items, granola bars, breakfast cereals, and several snack mixes. Inclusion of almonds in these products is valued because almonds provide desirable crunchy texture, sweet mellow flavor, and several micro- and macronutrients. Although safely enjoyed by most, almonds may induce adverse reactions, such as allergic reaction, in sensitive individuals. Almond allergy has been identified as the third most frequent tree nut allergy in the US and anaphylaxis to almond has been reported. Amandin, a globular storage protein that accounts for ~65% of the extractable proteins is the major allergen in almond seeds. Amandin is a hexameric protein and consists of two trimers. Each trimer is composed of three polypeptides each of which is composed of 40-42 kDa acidic chain (α) linked by a disulfide bond to 20 kDa basic chain (β). Foods, containing almonds, subjected to thermal processing typically experience Maillard reaction. Maillard reaction, chemical reaction between reducing sugars and the amino groups on amino acids/polypeptides/proteins cause non-enzymatic browning of foods. This browning reaction is desirable in many food products due to color and flavor formation. Maillard reaction also may glycate food proteins. As a result of Maillard reaction, destruction of amino groups, glycation, and/or denaturation of proteins may therefore alter amandin immunoreactivity. The current research therefore focused on amandin immunoreactivity in variously processed almonds and almond containing foods. In the selected commercial and laboratory prepared food matrices, the occurrence of non-enzymatic Maillard browning was objectively assessed by determining Hunter L* (lightness), a* (green-red), b* (blue-yellow) values. L* values used for different degrees of browning (roasting) were: Light = 53±1, Medium = 48.5±1, Dark = 43±1. The L* values for the tested samples were in the range 31.75-85.28 consistent with Maillard browning or the natural product color (e.g. white chocolate). Immunoassays (ELISA, dot-blot, and Western blot) were used to determine the immunoreactivity. Three murine monoclonal antibodies (mAbs), 4C10 4F10 and 2A3 were used to probe the desired samples. The mAb 4C10 targeted conformational and 4F10 and 2A3 recognized two independent linear amandin epitopes. The tested food matrices that did not contain almond exhibited no cross-reactivity in the immunoassays indicating their amandin specificity. For sandwich ELISAs, R = protein concentration of sample for 50% of the maximum signal for the corresponding standard curve/ the protein concentration required to register 50% of the maximum signal for the standard curve. The Fisher's Least Significant Difference (LSD) at p ≤ 0.05 was calculated for appropriate data. None of the tested food matrices exhibited increased immunoreactivity. The range of R values for tested food matrices containing almonds were 0.67-15.19 (4C10, LSD = 1.90), 1.00-11.83 (4F10, LSD = 0.86) and 0.77-23.30 (2A3, LSD = 1.34). Results of dot blots and Westerns blots were consistent with the ELISA results. Certain bakery and confectionary samples exhibited significantly decreased immunoreactivity. The observed decrease in immunoreactivity may be due to Maillard reaction, epitope degradation due to loss of amandin disulfide bond(s), loss of protein solubility, amandin thermal aggregation; or a combination thereof. Results of the study indicate that the immunoassays using the murine mAbs are specific, sensitive and robust for amandin detection in the tested food matrices and amandin incurred samples.