Abstract
Owing to the globalization of trade in food products and extensive growth in technology there has been a growing interest in the research of food authenticity and security. The promotion of a healthy lifestyle increased the demand for honey due to its documented health benefits to the human body and the high demand for honey increases its adulteration chances. Natural honey consumption is higher in developed countries such that the market demand is not always met by home production. South Africa’s honey production used to be sufficient for the local demand, but as the demand drastically increased and the supply fell, cheap imports filled the gap.
Therefore, this study aims to address the issue of honey adulteration in South Africa by developing analytical techniques for the detection of sugar adulterants in honey products. Honey adulteration procedures employed in this study included mixing commercial honey with different sugar syrups such as agave, beet sugar, golden, glucose liquid, and inverted sugar. Commercial honey and purposely adulterated honey were investigated by inspecting physicochemical parameters such as Lugol test, water content, Lund reaction, ash content, pH, and acidity and honey quality by determining hydroxymethylfurfural (HMF) content. To accomplish this, crystals from the samples were removed and the aqueous honey solution was obtained, water content was investigated using the freeze-drying method, ash content using the calcination method, and tannic acid and the Lugol reagent were used for the Lund and Lugol test, respectively. In addition to the physicochemical parameters study, different techniques such as nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies were used for the identification and detection of different sugars (fructose, glucose, sucrose, and maltose) and possible added ingredients in honey. High-performance liquid chromatography (HPLC) equipped with refractive index (RI) detector was used to quantify the total sugar content of honey. The HPLC quantitative analysis method was validated through the evaluation of analytical figures of merit. To this effect, limit of detection (LOD) and limit of quantification (LOQ) were determined by analysing ten reagent blanks. Percentage relative standard deviation was calculated to evaluate the precision of quantification method.
The obtained results showed that adulterated honey samples did not contain albuminoids and pure honey did not contain starch, which shows negative tests for
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Lund and Lugol tests, respectively. The pH of pure honey decreased from 4.43 to 3.10 in the presence of inverted sugar syrup and increased to 5.14 in the presence of glucose liquid. Ash content was increased from 0.001% in pure honey to 2.30% in adulterated honey. The FTIR spectra of adulterated honey samples showed a peak at 2361.4 cm-1 and double peaks near the maltose characteristic peak (1029.17 to 1052.1 cm-1) and carbonyl peaks (1652.4 and 1738.2 cm-1), which were not observed in commercial honey. Proton (1H) NMR showed anomeric carbons at the 4.5 to 6.0 ppm region and carbon (13C) NMR showed additional signals for adulterated honey at the anomeric region (90 to 110 ppm). Commercial honey had an HMF value of ˂20.0 mg/kg while adulterated honey samples had HMF values up to 248 mg/kg. Commercial honey, honey adulterated with agave syrup, and honey adulterated with 20% golden syrup all had sugar contents of less than 5%, whereas all other adulterated honey and their respective adulterants had sucrose contents of greater than 5%. The LOD and LOQ were found to be 2.51 mg/L and 8.35 mg/L for fructose, 2.45 mg/L and 8.18 mg/L for glucose, and 2.24 mg/L and 7.48 mg/L for sucrose, respectively. Quantification of sugar standards was achieved using HPLC-RI, and the fructose/glucose ratio was calculated. The fructose/glucose ratio for honey samples ranged from 1.51 to 1.85 and for adulterated honey samples the fructose/glucose ratio (F/G) ranged from 1.30 to 5.54.
The FTIR and NMR spectroscopic analysis results confirm the significant variable composition of sugar in the honey samples. They both proved to be suitable methods for accurate and direct determination of individual sugar. The physicochemical parameters of commercial honey samples were found to be within the acceptable values by agricultural products standard act 119; however, the adulterated honey samples did not adhere to the requirements. The HMF is affected by heating, storage, increased temperature, pH, and adulteration. The HMF content increased with increasing levels of adulteration. The HPLC analysis showed successful quantification of the sugar content and the obtained F/G allowed for the discrimination between honeydew and floral honey. The obtained biplot results showed a successful differentiation between authentic and counterfeit honey. These results indicate the importance of multivariate statistical analysis (MVA) in honey authentication determination. The use of FTIR and NMR results in MVA is recommended.