Detection And Control Of Lectin Allergen From Kidney Beans (Phaseolus Vulgaris L.)

Kidney bean(Phaseolus vulgaris L.)lectin is the major allergen protein in the beans,which can stimulate allergy reaction within an extremely low dose and cause a high rate of death,affecting the consumption safety.Therefore,in this study,effects of superfine grinding on the pretreatment of lectin extraction were firstly studied using black kidney bean lectin(BKBL)as tested materials.A novel oriented antibody immobilization based the voltammetric immunosensor for the allergenicity detection of lectin was developed.In addition,based on food processing methods,the effects of combined pH and thermal/HHP treatments on the potential allergenicity and conformational structure of lectin were explored.The main results were summarized as follows,1. Effects of superfine grinding on the physicochemical and lectin extraction properties of black kidney bean powder were investigated.With decreased particle size and disrupted microstructure,increased hydratability as well as thermal stability of black kidney bean powder were found after superfine grinding processing.Moreover,the protein content of the extraction was improved.The superfine powder with a size of75?38?m was potential for extracting lectin because the extraction showed the highest hemagglutination activity.2. A new-designed label-free voltammetric immunosensor combined with one-pot synthesized Au NPs-PEI-MWCNTs nanocomposite and orientedly Ab immobilization was successfully developed for BKBL detection.Under the optimized condition,the immunosensor displayed a good linear response(R~2=0.978)to BKBL with a range from0.05 to 100?g/m L and a detection limit of 0.023?g/m L.Simultaneously,the immunosensor exhibited well selectivity,interference-resistant ability,stability(at 4°C),and reproducibility.Compared with the conventional enzyme-linked immunosorbent assay(ELISA)for lectin detection,the immunosensor was successfully applied to quantify the allergenic activity of lectin in raw and cooked kidney bean milk samples.3. Combined effects of pH adjusting and thermal treatments(60-100?,3-17 min)on BKBL were investigated by response surface methodology(RSM).Low pH(1.0,2.0,and3.0)treatment decreased hemagglutination activity(HA)and antigenicity,but the activities would be restored when the lectin was treated by pH shifting to 7.2(1.0?7.2,2.0?7.2,and 3.0?7.2).Conformational structure analyses indicated that low pH-induced protein unfolding,but pH-shifting treatment resulted in a limited structural rearrangement.Mild heating,such 60?for 3 min,slightly increased the HA and antigenicity of pH shifted BKBL,but it has no obvious effect in the pH 1.0 incubated BKBL.High-temperature and long-time heating caused the BKBL protein aggregation and further decreased the HA and antigenicity.BKBL with almost eliminated antigenicity could be obtained by the combined treatments of pH 1.0 and heating at 80?for 15 min as well as the combination of pH 1.0?7.2 and heating at 100?for 10 min based on the optimized processing condition.4. The changes in conformational structures related to potential allergenicity of BKBL after combining of pH adjusting and HHP treatments were also investigated by RSM.The secondary and tertiary structures of low pH induced and pH shifted BKBL did not change much after a relatively low-pressure treatment,such as 100 MPa,thus the HA and antigenicity tended to no change.However,the BKBL unfolded and then tended to aggregate after high-pressure and long-time treatments,which resulted in the antigenicity decreased.Lower-pH(such as pH 1.0)treated BKBL seemed to be resistant to HHP-induced unfolding.Overall,RSM analyses indicated that under the condition of combining pH 1.0 with HHP at 500 MPa for 17 min,the decrease in the antigenicity of BKBL was up to?90%.