Discrimination of stingless bee honey adulteration by cane sugar syrup using chemometric-integrated spectroscopic techniques

Abstract

Stingless bee, a type of bee which is stingless, is one of the bee species which has the ability to produce honey and locally known as kelulut in Malaysia. Increase of demand for its production, has contributed to the scarcity of the pure honey in market and resulted in adulteration of the pure honey for economic gain by unethical individuals. In addressing such an issue, numerous physicochemical and instrumental analyses were developed to detect and discriminate adulterants in honey samples. This study presents the discrimination analyses of adulterated stingless bee honey using rapid, non-destructive, simple, and greener spectrometric methods; Attenuated-Total-Reflectance-Fourier-Transform Infrared (ATR-FTIR) and Raman spectroscopy integrated with Principle Component Analysis (PCA) and Linear Discriminant Analysis (LDA). The pure stingless bee honey was obtained from Skudai, Johor. Adulterated honey samples were attained by adding cane sugar syrup in five different percentages (w/w) namely 5%, 10%, 20%, 40% and 50%. All samples were initially subjected to physical properties (Brix, refractive index and density) analysis. Using one-way ANOVA, density of pure stingless bee honey has no significance differences (p>0.05) between adulterated samples, except between adulterant (p<0.05). Meanwhile, brix value and refractive index of pure honey shows significant difference between adulterant and adulterated honey. Next, characterization of the pure and adulterated stingless bee was done in full region for ATR-FTIR (4000-600 cm-1) and Raman (1430-200 cm-1) spectroscopy. For chemometric analysis, fingerprint region (1700-400 cm-1) for both spectroscopic spectra were analysed. PCA was done to understand the organization of data, while LDA was done to predict the major factors for grouping and prediction. PCA scores for first three components for both ATR-FTIR and Raman were 95.2% and 65.7% respectively. Employment of LDA had improved the groupings of the honey samples when compared with PCA, which resulted in LDA cross-validation for correct classifications rate at 100% for both ATR-FTIR and Raman. The protocol proposed could be an alternative option as opposed to the laborious, time consuming, destructive and involve hazardous chemicals, chromatographic analyses such as GC and HPLC, considering that Raman spectroscopy, like ATR-FTIR is simple, fast, non-destructive and greener analytical techniques.