Harshal H. Kshirsagar

Solubilization and electrophoretic characterization of select edible nut seed proteins.

The solubility of almond, Brazil nut, cashew nut, hazelnut, macadamia, pecan, pine nut, pistachio, walnut, and peanut proteins in several aqueous solvents was qualitatively and quantitatively assessed. In addition, the effects of extraction time and ionic strength on protein solubility were also investigated. Electrophoresis and protein determination (Lowry, Bradford, and micro-Kjeldahl) methods were used for qualitative and quantitative assessment of proteins, respectively. Depending on the seed, buffer type and ionic strength significantly affected protein solubility. The results suggest that buffered sodium borate (BSB; 0.1 M H(3)BO(3), 0.025 M Na(2)B(4)O(7), 0.075 M NaCl, pH 8.45) optimally solubilizes nut seed proteins. Qualitative differences in seed protein electrophoretic profiles were revealed. For a specific seed type, these differences were dependent on the solvent(s) used to solubilize the seed proteins. SDS-PAGE results suggest the polypeptide molecular mass range for the tree nut seed proteins to be 3-100 kDa. The results of native IEF suggested that the proteins were mainly acidic, with a pI range from >4.5 to <7.0. Western immunoblotting experiments indicated that rabbit polyclonal antibodies recognized substantially the same polypeptides as those recognized by the corresponding pooled patient sera IgE.

Effects of processing on immunoreactivity of cashew nut (Anacardium occidentale L.) seed flour proteins.

Cashew nut seeds were subjected to processing including autoclaving (121 degrees C for 5, 10, 20, and 30 min), blanching (100 degrees C for 1, 4, 7, and 10 min), microwave heating (1 and 2 min each at 500 and 1000 W), dry roasting (140 degrees C for 20 and 30 min; 170 degrees C for 15 and 20 min; and 200 degrees C for 10 and 15 min), gamma-irradiation (1, 5, 10, and 25 kGy), and pH (1, 3, 5, 7, 9, 11, and 13). Proteins from unprocessed and processed cashew nut seeds were probed for stability using anti-Ana o 2 rabbit polyclonal antibodies and mouse monoclonal antibodies directed against Ana o 1, Ana o 2, and Ana o 3 as detection agents. Results indicate that Ana o 1, Ana o 2, and Ana o 3 are stable regardless of the processing method to which the nut seeds are subjected.

Biochemical and spectroscopic characterization of almond and cashew nut seed 11S legumins, amandin and anacardein.

Native, undenatured amandin and anacardein secondary structures were estimated to be, respectively, 56.4 and 49% β-sheet, 14 and 23.7% α-helix, and 29.6 and 27.4% random coil. Circular dichroic (CD) and fluorescence spectroscopy were used to assess structural changes in amandin and anacardein subjected to denaturing treatments that included heat (100 °C, 5 min), guanidium HCl (GuHCl), urea, sodium dodecyl sulfate (SDS), and reducing agent, 2% v/v β-mercaptoethanol (βME) + heat. Mouse monoclonal antibodies (mAbs) 4C10 and 4F10 directed against amandin and 1F5 and 4C3 directed against anacardein were used to assess the influence of denaturing treatments on the immunoreactivity of amandin and anacardein. Among the denaturing treatments investigated, SDS and β-ME caused a significant reduction in the immunoreactivity of amandin and anacardein when probed with mAb 4C10 and 4C3, respectively.

Immunoglobulin E-Reactive Proteins in Cashew (Anacardium occidentale) Apple Juice Concentrate

Cashew apple juice has the potential to be a natural source of vitamin C and sugar in processed foods. The juice of the cashew apple is obtained by pressing the fleshy peduncle or receptacle, which forms a rounded apple that sits above the true fruit, the cashew nut. Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. To determine if cashew apple juice contains cashew nut allergens, immunoblotting was performed using a cashew apple juice 6X concentrate that was extracted and further concentrated through dialysis, lyophilization, and resuspension. Serum IgE of individuals allergic to cashew nut bound proteins in the cashew apple juice concentrate extract. For some serum samples, IgE reactivity could be inhibited by preincubation of the serum with cashew nut extract, suggesting the presence of cashew nut-related allergens. Using monoclonal antibodies specific for cashew nut allergens, the concentrate was found to contain Ana o 1 (vicilin) and Ana o 2 (legumin). Neither IgE from cashew nut allergic sera nor the monoclonal antibodies bound any peptides in 5 kDa filtered cashew apple juice concentrate. The cashew apple juice concentrate used in these studies contains proteins with IgE-reactive epitopes, including cashew nut legumin and vicilin. No IgE-binding peptides remained after 5 kDa filtration of the concentrate.

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.