Sugar Profiling of Honeys for Authentication and Detection Of

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Sugar Profiling of Honeys for Authentication and Detection Of molecules Article Sugar Profiling of Honeys for Authentication and Detection of Adulterants Using High-Performance Thin Layer Chromatography Md Khairul Islam 1,2 , Tomislav Sostaric 2, Lee Yong Lim 2, Katherine Hammer 1,3 and Cornelia Locher 1,2,* 1 Cooperative Research Centre for Honey Bee Products Limited (CRC HBP), University of Western Australia, Perth 6009, Australia; [email protected] (M.K.I.); [email protected] (K.H.) 2 Division of Pharmacy, School of Allied Health, University of Western Australia, Perth 6009, Australia; tom@chromatechscientific.com (T.S.); [email protected] (L.Y.L.) 3 School of Biomedical Sciences, University of Western Australia, Perth 6009, Australia * Correspondence: [email protected] Academic Editor: Soraia I. Falcão Received: 8 October 2020; Accepted: 11 November 2020; Published: 13 November 2020 Abstract: Honey adulteration, where a range of sugar syrups is used to increase bulk volume, is a common problem that has significant negative impacts on the honey industry, both economically and from a consumer confidence perspective. This paper investigates High-Performance Thin Layer Chromatography (HPTLC) for the authentication and detection of sugar adulterants in honey. The sugar composition of various Australian honeys (Manuka, Jarrah, Marri, Karri, Peppermint and White Gum) was first determined to illustrate the variance depending on the floral origin. Two of the honeys (Manuka and Jarrah) were then artificially adulterated with six different sugar syrups (rice, corn, golden, treacle, glucose and maple syrup). The findings demonstrate that HPTLC sugar profiles, in combination with organic extract profiles, can easily detect the sugar adulterants. As major sugars found in honey, the quantification of fructose and glucose, and their concentration ratio can be used to authenticate the honeys. Quantifications of sucrose and maltose can be used to identify the type of syrup adulterant, in particular when used in combination with HPTLC fingerprinting of the organic honey extracts. Keywords: honey; sugar syrup; adulteration; Jarrah; Manuka 1. Introduction Honey has been regarded as nutritious food since ancient times [1,2], and it has also enjoyed increasing recognition for its bioactivities and potential medicinal applications. Monofloral honeys, in particular, have attracted good sale prices due to perceived higher bioactivity levels, which then led to these honeys being the subject of increasingly common adulterations [3–5]. The adulterations involve either the feeding of honeybees with sugar syrups or the deliberate addition of sugar syrups to the honey to increase the bulk weight [6–9]. Typical honey adulterants include sucrose syrup, high fructose corn syrup, maltose syrup, brown rice syrup, corn syrup, golden syrup, treacle syrup, glucose syrup, maple syrup, as well as industrial grade sugars like glucose and fructose. Syrups obtained from starch following enzymatic or acid treatment are also used [10–13]. The detection of these sugar-based adulterations is challenging, as honey is itself a highly concentrated sugar solution, with sugars accounting for about 80–85% of the total solids in most honeys. The main honey sugars are fructose and glucose, alongside much smaller amounts of disaccharides Molecules 2020, 25, 5289; doi:10.3390/molecules25225289 www.mdpi.com/journal/molecules Molecules 2020, 25, 5289 2 of 14 (e.g., sucrose, maltose, trehalose, turanose), trisaccharides (e.g., maltotriose, raffinose, erlose) and oligosaccharides [14]. Various methods are used for honey quality control and the detection of adulterants, such as C12/C13 isotope identification, fluorescence spectroscopy, high performance liquid chromatography (HPLC), ion exchange chromatography, gas chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy and Raman spectroscopy [15–20]. However, these methods are not without challenges [21,22]. Sugars lack a chromophore and are poorly suited for methods that rely on detection by UV. Derivatisation into more easily detectable artefacts is possible; however, these approaches might be hampered by low sensitivity and poor selectivity [23]. Other methods like IR and NMR rely on the nonspecific detection of components and the establishment of adequate reference databases and threshold limits [24,25]. In this paper, we demonstrate the usefulness of a High-Performance Thin Layer Chromatography (HPTLC)-based method for the detection of sugar-based honey adulterants. It is a validated HPTLC method [26] that relies on the qualitative and quantitative analysis of glucose, fructose, maltose and sucrose in potentially adulterated honeys, complemented by HPTLC fingerprinting of the honey’s organic extract [27,28]. Taken together, the two levels of HPTLC analysis detect the natural product-based syrup adulterants (e.g., rice, maple, treacle, corn, golden syrup) and honeys that have been adulterated by adding pure glucose and fructose. The HPTLC analysis is cost-effective and easy to perform, yet also sophisticated as it can detect the presence of sugar-based honey adulteration, identifies the adulterant itself and estimates the level of adulteration. 2. Results and Discussion 2.1. Sugar Analysis of Honeys and Syrups Six honeys (Table 4) were analysed for their HPTLC sugar profile (Figure1). Glucose presented as a green band with an Rf value of 0.32, fructose presented as an orange band with an Rf value of 0.14, sucrose presented as a dark brown band with an Rf value of 0.27, and maltose presented as a grey band with an Rf value of 0.20. Fructose and glucose contents were readily quantifiable in all honey samples (LOD/LOQ for glucose: 33.00/100.00 ng; LOD/LOQ for fructose: 21.98 ng/66.62 ng [26]), but maltose and sucrose, if present, were below the limits of detection and quantification (for sucrose 21.17 ng (LOD) and 64.15 ng (LOQ) and for maltose 63.51 ng (LOD) and 192.45 ng (LOQ) [26]). The fructose to glucose ratio (F:G), which is an important parameter for honey authentication and also a predictor of a honey’s tendency to crystallise [29–31], was calculated from these findings (Table1). Table 1. Fructose and glucose content, and fructose to glucose ratio (F:G) of honeys 1. Honey ID Fructose (mg/g) Glucose (mg/g) F:G Ratio Manuka MAN 392 307 1.3 Jarrah JAR 426 337 1.3 Marri MAR 439 269 1.6 Karri KAR 387 249 1.5 Peppermint PEP 404 257 1.5 White Gum WHG 415 323 1.3 1 Values represent the mean of triplicate data. One-way ANOVA confirmed no significant difference between replicates (p > 0.05). Molecules 2020, 25, 5289 3 of 14 Molecules 2020, 25, x FOR PEER REVIEW 3 of 14 Figure 1. HPTLC images taken at T White light after derivatisation with aniline-diphenylamine- Figure 1. HPTLC images taken at T White light after derivatisation with aniline-diphenylamine- phosphoric acid reagent; Track 1—Standards (fructose, maltose, sucrose and glucose in increasing Rf phosphoric acid reagent; Track 1—Standards (fructose, maltose, sucrose and glucose in increasing Rf values), Track 2—MAN, Track 3—JAR, Track 4—MAR, Track 5—KAR, Track 6—PEP, Track 7—WHG; values), Track 2—MAN, Track 3—JAR, Track 4—MAR, Track 5—KAR, Track 6—PEP, Track 7— Tracks 2–4 were obtained with 3 µL of aqueous methanolic honey solution, while Tracks 5–7 were WHG; Tracks 2–4 were obtained with 3 µL of aqueous methanolic honey solution, while Tracks 5–7 obtained with 2 µL of aqueous methanolic honey solution. were obtained with 2 µL of aqueous methanolic honey solution. Six syrups (Table2) were also analysed for their HPTLC sugar profile (Figure2) and fructose, Six syrups (Table 2) were also analysed for their HPTLC sugar profile (Figure 2) and fructose, glucose, sucrose and maltose contents. All syrups were found to contain one or more of these sugars glucose, sucrose and maltose contents. All syrups were found to contain one or more of these sugars within detectable limits (Table3). As all the syrups contain a sugar other than glucose and fructose within detectable limits (Table 3). As all the syrups contain a sugar other than glucose and fructose (i.e., sucrose or maltose), the adulteration of honey with these syrups should be detectable and the (i.e., sucrose or maltose), the adulteration of honey with these syrups should be detectable and the adulteration levels estimated. adulteration levels estimated. Table 2. Glucose, fructose, sucrose and maltose content of syrups 1. Table 2. Glucose, fructose, sucrose and maltose content of syrups 1. Glucose Fructose Sucrose Maltose Syrup ID Glucose Fructose Sucrose Maltose Remarks Syrup ID (mg/g) (mg/g) (mg/g) (mg/g) Remarks (mg/g) (mg/g) (mg/g) (mg/g) Corn COR - - - 372 Contains additional unidentified compounds Contains additional unidentified RiceCorn RICCOR 250- -- -- 242372 Contains additional unidentified compounds Glucose GLU 178 - - 148 Contains additionalcompounds unidentified compounds Contains additional unidentified GoldenRice GOLRIC 200250 189- 206- -242 - Treacle TRE 191 185 204 -compounds - Maple MAP - - 486 -Contains additional - unidentified Glucose GLU 178 - - 148 1 Values represent the mean of triplicate data. One-way ANOVA confirmed no significantcompounds difference between Goldenreplicates (GOLp > 0.05). 200 189 206 - - Treacle TRE 191 185 204 - - Maple MAP - - 486 - - 1 Values represent the mean of triplicate data. One-way ANOVA confirmed no significant difference between replicates (p > 0.05). Molecules 2020, 25, x FOR PEER REVIEW 4 of 14 Molecules 2020, 25, 5289 4 of 14 Molecules 2020, 25, x FOR PEER REVIEW 4 of 14 Figure 2. HPTLC images taken
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