Elucidating the false positive tendency at AOAC 998.12 C-4 sugar test for pine honey samples: Modified sample preparation method for accurate δ13C measurement of honey proteome

Abstract

The majority of the authentic pine honey samples fail from C-4% sugar test. Pine honey is generally characterized by small-sized insoluble honeydew elements, and these can form an appreciable amount of precipitate during protein flocculation. These substances may shift the protein-centric δ13C (13C/12C) values inaccurately. In this study, honey proteins were isolated, enriched, and cleaned-up prior to flocculation by applying the optimized and validated ultrafiltration method. Authentic honeys were analyzed along with adulterated samples using both AOAC 998.12 and novel protocol. When the interfering substances were eliminated, most of the samples, which were interpreted as adulterated according to the AOAC's method previously, were identified as authentic. It has been proven that inconsistent protein measurements may cause false positivity. These findings revealed that the developed method could correct the artificially high C-4% results and the proposed modification will pave the way for increasing the overall reliability of the test.