Protein extraction and identification as a honey adulteration detection method.

Honey is a nutritious and healthy natural food produced by honey bees from plant nectars: it consists mostly of the sugars glucose and fructose, as well as maltose, sucrose, water, pollen and other minor components. Food authentication is an international issue in quality control and food safety. Regulatory authorities, food processors, retailers, and consumers are interested in knowing the origin and quality of foods; for these reasons the deliberate mislabeling and adulteration of foods, particularly honeys, are matters of increasing global concern. Many studies have reported on the chemical constituents of honey such as sugars and flavonoids, and the analysis of many of those components is applicable to adulteration detection. Another approach is based on analysis of proteins, the honey contains many different proteins in minute quantities and several enzymes, such as amylase, α-glucosidase, β-glucosidase and glucose oxidase. Since they originate from Apis mellifera, the identification of a protein or an enzyme of a different origin could be interpreted as a proof of adulteration. We have focused on the detection of an α-amylase from Aspergillus niger, an enzyme that can be employed to produce glucose syrup, used for honey adulteration. This enzyme was added to honey samples in different concentration, ranging from 0,1 to 1 nmol/ml, and different isolation and analysis techniques were tested. Honey samples were dialyzed against ultrapure water for 3 days using a cellulose membrane with a 12KDa cutoff, then lyophilized to concentrate the residue protein. Passivation of the membrane to avoid loss of protein during the dialysis was also tested, but no significative difference was found after determination with Bradford assay. A separation using disposable C18 SPE column was also tested. Samples obtained after isolation were analyzed using both MALDI-TOF MS and HPLC CHIP Q-TOF MS. The sensitivity of MALDI-TOF resulted much lower than the one achieved with HPLC-MS, which was able to detect concentration as low as 0,1 nmol/ml for dialyzed samples. The separation obtained with the SPE columns was not satisfactory, probably because amylase was strongly bound to the C18 column and elution was not efficient.