Reducing of salivary α-amylase inhibition by using bovine serum albumin and calcium chloride for forensic saliva screening

Inhibiting salivary α-amylase is a critical issue of forensic saliva identification using the catalytic method. This study aims to identify human α-amylase inhibitors in forensic saliva screening by using a blue starch amylase test and to measure the extent of enzyme inhibition. Thus, in order to demonstrate the presence of inhibitors, we prepared positively charged metal ion sources or chelators that were mixed into the saliva stains. The results of this study show that ferric chloride (FeCl3), magnesium chloride, ethylenediaminetetraacetic acid, and citric acid significantly decrease the α-amylase activity of saliva stains. We also verified this approach using blood, a magnesium-containing liquid supplement, and two citric acid-containing soft drinks that were contaminated with saliva stains as forensic mock samples; these samples also showed a significant reduction in salivary α-amylase activity. To establish an inhibitor-resistant blue starch amylase test, we applied bovine serum albumin (BSA) and calcium chloride (CaCl2) to the reaction system. The results show that salivary α-amylase inhibition of the forensic mock samples occurred under normal test conditions (i.e., 300 ng/µL BSA, 0 mM CaCl2), and that inhibition was significantly relieved under the BSA + CaCl2 conditions (i.e., 1000 ng/µL BSA, 5 mM CaCl2). Therefore, the results of this study demonstrate that both BSA and CaCl2 can be utilized as reaction stabilizers in forensic saliva screening.