Physico-chemical properties (moisture content, pH, free acidity, diastase number, electrical conductivity, HMF, proline, colour) of blossom honey samples (H1 and H2), adulterants (MS and GFS syrups), and adulterated honey samples (H1GFS, H1MS, H2GFS, and H2MS) are presented in Table 3. HPLC-RID sugar profile and IR-MS C4 sugar analysis are given in Table 4 and Table 5 respectively.
3.1 Moisture Content
In terms of honey quality, moisture content is one of the most important parameters that affect physicochemical properties of honey and will help to determine the storage conditions and shelf life of the product. Moisture content in honey above 20% can trigger yeast growth which subsequently causes fermentation process (Codex Alimentarius, 2001). The initial moisture content of H1 and H2 honey samples and GFS and MS adulterants were found as 14.96%, 14.56%, 17.16%, and 13.76%, respectively (see Table 3). There were linear downward with addition of GFS syrup and linear upward trends with addition of MS syrup (see Fig. 1). Since both sugar syrups have lower than 20% of moisture content, all adulterated samples were ranged below 20% which do not exceed the maximum limit of moisture specified in Turkish food codex communiqué on honey (2020). Among the adulterated samples, the highest moisture content was seen in H1GFS50 sample with the amount of 15.96%. In a study, different honey samples were adulterated with sucrose beet syrup at the ratio of 10% − 50% and moisture content of honeys were exceed the 20% limit at 40% and 50% adulteration levels (Tosun and Keles, 2021). In another study conducted by Kamboj and Mishra (2015), it was determined that the moisture content of honey adulterated with palm sugar syrup in the range of 5% − 30% decreased from 19.7–21.6% with the increase of the added syrup level. By examining the data obtained from this study and current literature, it can be said that it is not always possible to detect adulterated honeys according to their moisture content.
3.2 pH
pH value is another parameter that has impact on the characterization of honey. Furthermore, in the presence of low pH, microbial growths as well as microbial reproduction are inhibited Ribeiro et al. (2014). The early pH values of H1 and H2 honey samples and GFS and MS syrups were observed as 3.70, 3.76, 5.42, and 5.19 respectively (see Table 3). In general, pH values of the adulterated samples were increased with the increase in syrup addition levels. Since MS syrup has a higher pH value than GFS syrup, increase in pH levels of adulterated samples in the samples prepared with MS syrup were higher than samples prepared with GFS syrup. Among the adulterated samples, the highest pH value was examined in B2MS50 sample as 3.80. Considering the pH values of the syrup and honey samples, changes in pH values of adulterated samples were found lower than expected (Fig. 2). This could be due to the buffering property of honey matrix that keeping the pH values of the adulterated samples at lower levels. Similar results reported by Ribeiro et al. (2014). In their study, adding of HFCS at different ratio (10%, 25%, 50% and, 75%) to pure honey sample increased honey’s pH value. Initially honey and HFCS had 3.10 and 4.70 pH values respectively. Increase in addition level of HFCS, increased pure honey’s pH rate. In the adulterated honey samples, the pH rates were ranged between 3.30 and 3.98.
3.3 Free acidity
The main source of free acidity is the presence organic acids. Although organic acids are around 0.5% of the total honey composition, they have important roles on the organoleptic, physical and chemical properties of honey (Karabagias et al., 2014). Free acidity value is also seen as a fermentation indicator. Bad storage conditions, exposure to direct heat, microbial contamination and/or components that are decomposed by the naturally occurring osmophilic yeasts in honey cause increase in the free acidity value (Cavia et al., 2007; Ajlouni and Sujirapinyokul, 2010). Free acidity of H1 and H2 honey samples were measured as 27.00 mEq/kg and 18.5 mEq/kg respectively, while the value for GFS and MS syrup were found as 1.63 mEq/kg and 1.40 mEq/kg respectively (see Table 3). It was determined that there were high negative correlations between the free acidity values and the amount of syrup added to the samples. As the syrup addition level was increased, the free acidity values of the adulterated samples were decreased linearly (Fig. 3). Free acidity values of samples ranged between 24.17 mEq/kg and 17.17 mEq/kg depending on adulteration levels. In a study, glucose, hydrolysed inulin syrup, malt wort and inverted sugar were used for adulteration of different authentic honeys at ratio of 5%, 10%, 20%, 30%, 40%, and 50% respectively. It was observed that, glucose, hydrolysed inulin syrup, malt wort and inverted sugar syrups increased amount of free acidity, although fructose did not alter much the free acidity values of the samples. Average free acidity of authentic honeys went up from 19.44 mEq/kg to 162.88 mEq/kg with addition of 50% sugar syrups (Oroian et al., 2018a).
According to Turkish food codex communiqué on honey (2020) and Council Directive (2001), honeys should not have more than 50 mEq/kg free acidity; however, there is no minimum tolerance level for free acidity. In this study, due to all the adulterated samples were remained lower than 50 mEq/kg, no adulterations were detected among the samples (Fig. 3).
3.4 Diastase number (DN)
Diastase is considered as an indicator of the freshness and purity of honey (Da Silva et al., 2016). The initial DN of the H1 and H2 honey samples were determined as 28.93 and 32.74 while, diastase activity was not found in both GFS and MS syrups. There were negative correlations between the amount of syrup added and the diastase numbers of the adulterated samples. DN of the adulterated honey samples were generally gradually decreased with the increasing of syrups added. The DN of the adulterated honey samples ranged between 31.25–17.89. DN number values of all adulterated samples were found over 8.00 which are in acceptable range stated in EC Council Directive (2001) and Turkish food codex communiqué on honey (2020) (Fig. 4). Pure honey samples adulterated with sucrose syrup at the range of between 10% − 50%. Due to absence of diastase activity in sucrose syrup, average DN of the pure honey samples were gradually decreased from 14.6 to 7.5 with increasing adulteration level (Tosun and Keles, 2021). Czipa et al. (2019) directly added different type of sugar syrups (glucose, invert and frucrose-glucose syrup) at a ratio of 30% and 40% to pure acacia honey samples. They reported that, average DN of samples was 28.8 and depending on type of sugar syrup added, the value decreased down to 15.4. In a study conducted by Ozcan et al. (2006), the diastase activities of honey obtained from bees fed with sucrose syrup and invert syrup were compared with honey obtained from bees not fed with sugar. The highest diastase value was found in pure honey with 10.9 and the lowest diastase value was determined in honey obtained from bees fed with invert syrup. The diastase activity of honey produced by bees fed with sucrose syrup was found to be 8.30. It can be seen from our study and the literature that, direct or indirect addition of sugar syrups decrease the diastase activity of honeys but the DN value of the adulterated samples mostly remain within safe limits which is amount of 8 set by Turkish Honey Communiqué (2020) and Council Directive (2001) as minimum requirement.
3.5 Electrical conductivity
The amount of electrical conductivity (EC) varies directly with the presence of mineral substances, organic acids and other organic compounds (Kropf et al., 2008). The EC values of H1 and H2 honeys were determined as 307.00 µS/cm and 242.00 µS/cm, respectively, while the EC values of GFS and MS syrups were found to be 5.20 µS/cm and 3.00 µS/cm, respectively (see Table 3). In the measurements of adulterated samples, high negative correlations were detected between the added syrup level and the EC of adulterated samples. It was determined that the amount of electrical conductivity decreased proportionally as the amount of added sugar syrups increased (see Fig. 5). Oroian et al., (2018b) found that EC values of different types of honeys (acacia, tilia, and polifloral) were altered by adulteration with fructose and hydrolysed inulin syrups. While addition of fructose syrup decreased, hydrolysed inulin increased EC value of the samples as the adulteration rate increased. EC values of the adulterated samples were found between 24.30–2920 µS/cm. According to Turkish Honey Communiqué regulation (2020), EC value for blossom honey should be less than 800.00 µS/cm. In our study, blossom honeys and all the adulterated honey samples showed complete conformity to this regulation. The highest and the lowest conductivity values in adulterated samples were determined as 295.00 µS /cm and 149.87 µS/cm in B1GFS5 and B2MS50 samples, respectively (see Table 3). These results show that, according to regulations, addition of sugar syrups to blossom honeys affected positively the electrical conductivity value so there should also be a minimum requirement for electrical conductivity requirement of blossom honey in the regulation.
3.6 HMF
HMF is an indicator of honey freshness (Bettar et al., 2019). It is known that sugar syrups added to honey generally increase the HMF content due to the higher presence of HMF in sugar syrups (Swallow et al., 1994). Furthermore, in order for the added sugars to disperse homogeneously in the honey-syrup mixture and gain a uniform appearance, the mixture is heated after adding the syrup, and this process causes an increase in the amount of HMF. While HMF content of H1 and H2 honey were determined as 7.12 mg/kg and 5.21 mg/kg respectively, in GFS and MS syrups HMF contents were found as 16.96 mg/kg and 10.53 mg/kg respectively (see Table 3). As the syrup addition level increased in the adulterated samples, the amount of HMF increased linearly showing high positive correlations (see Fig. 6). HMF content of adulterated honey samples were ranged between 5.68 mg/kg and 11.94 mg/kg. In a study conducted by Craciun et al. (2020), honey samples were adulterated by adding three different sugar syrups directly to authentic honey and indirectly feeding the bees with sugar syrups. While the average HMF content of authentic honeys was 1.21 mg/kg, the average of HMF of directly syrup-added honeys was found to be 21.2 mg/kg. Furthermore, the average HMF value of adulterated honey obtained from bees fed with sugar syrups was found as 29.9 mg/kg. In another study, monofloral acacia, tilia, and sunflower honeys were adulterated with maple, inverted sugar, agave, rice and corn syrup in concentration of 5%, 10%, and 20%. Initially honey samples had average of 3.5 mg/kg HMF content with addition of sugar syrups average HMF values of the samples increased gradually in the range of 10.1 to 35.1 mg/kg (Ciursa et al., 2021). Although sugar syrups used for adulteration of honeys had higher content of HMF, adulterated honeys mostly did not exceed limit of 40 mg/kg set by Turkish food codex communiqué on honey (2020).
3.7 Proline content
Salivary glands of honey bees and plants are main sources of amino acids of honey counted as an indicator in determining honey fraud whether it has been imitated or adulterated. Proline is the dominant amino acid in honey and it is seen as an indicator of protein amount in honey, since it constitutes 50–85% of the total amino acid content (Iglesias et al., 2004; White, 1978). In H1 and H2 honey samples, proline contents were found as 965.54 mg/kg and 587.37 mg/kg respectively. Proline values of GFS and MS syrups were calculated as 53.13 mg/kg and 85.88 mg/kg respectively (see Table 3). High linear negative correlations were determined between the addition rate of syrups and the proline content of adulterated samples (see Fig. 6). The highest and the lowest amount proline were found in H1MS5 and H2GFS50 as 902.53 mg/kg and 314.96 mg/kg respectively. In the study conducted by Czipa et al. (2019), a pure honey sample was adulterated with fructose, glucose and two different invert sugar syrups at the rates of 30% and 40%. While the proline value of pure honey was determined as 284 mg/kg, it was also determined that the proline values of the syrup added honey samples decreased and the proline values of the samples ranged from 179 mg/kg to 274 mg/kg. In another study, proline values of honey obtained from bees fed with sucrose syrup at different rates were compared. Proline amounts of 416.4 mg/kg, 501.6 mg/kg and 630 mg/kg were determined in honey obtained from bees fed with sugar syrup continuously, fed only with sugar syrup in spring and not fed with sugar syrup, respectively (Guler et al., 2007).
3.8 Colour
Colour variability in honey depends on the nectar of plants and the plant origin because honeybees directly collect nectar and pollen from plants. It plays an important role in determining the market price of honey as it is one of the most important physical parameters affecting the preferences of consumers in many countries (Krell, 1996). As a result of the colour test, the L, a, and b values were obtained. And colour differences, ΔE, were generated from L, a, and b values of the samples. Raising the percentage of adulterants, GFS and MS, in the adulterated samples caused gradually increase in the L and ΔE values (see Fig. 8 and Fig. 11) while, gradually decrease in a and b values (see Fig. 9 and Fig. 10). The highest L and ΔE values were detected in the B2GFS50 and B1MS50 samples. And the lowest a and b values were found in the B2GFS50 and B2MS40 respectively (see Table 3).
A study carried out by Ribeiro et al. (2014), honey samples were adulterated with high fructose corn syrup at a rate of 10%-75%. It was found that, while the L value of honeys with syrup added increased and b value decreased proportionally with the syrup addition rate. Yılmaz et al. (2014) in their study, detected the changes in L, a, and b values by adulterating a honey sample with fructose and sucrose syrups in the range of 5%-50%. With the increase in the syrup ratio added to the honey sample, an increase in L value and a decrease in a and b values were determined. While the highest L value was detected in the sample with 50% sucrose syrup added, the lowest a and b values were determined in the samples including 50% sucrose syrup and 50% fructose syrup, respectively.
3.9 Sugar Profile by HPLC
In order to prevent imitation and adulteration, there are certain criteria set by national and international standards for sugar content of honey. According to Turkish food codex communiqué on honey (2020), maltose and sucrose can be found at a maximum of 4 (g/100g) and 5(g/100g) respectively, and the F + G value should be at least 60%, and the fructose / glucose ratio should be in the range of 0.9–1.4. Honey that does not meet these criteria is considered as a fraudulent. In the adulterated honey samples, fructose + glucose (g/100g) values and F/G ratios were decreased gradually (Fig. 12 and Fig. 13 respectively), while maltose (g/100g) value progressively increased with increasing addition of GFS and MS syrups (Fig. 14). Except H1MS50 and H2MS50, all the adulterated samples contain more than 60% of F + G. Furthermore F / G ratio of all adulterated samples ranged between 0.9–1.4. Sucrose sugar was not found in H1 and H2 honey samples and in both GFS and MS syrups. Results of F + G (g/100g), F/G ratio and maltose (g/100g) are exhibited in Table 4. These results indicate that adulterations detected at the level of 5% − 50% depending on sugar syrup and honey type. In the samples of H1GFS, H1MS, H2GFS, and H2MS at ratio of starting from 20%, 10%, 20%, and 5% respectively (see Table 4).
Oroian et al. (2018c) adulterated a honeydew honey with glucose, fructose, inverted sugar, hydrolysed inulin syrup, and malt wort at the rate of 5% − 50%. According to in EC Council Directive (2001), adulterations could be detected in samples which were adulterated with glucose, fructose, and malt worth additions at the rate of between 20% − 50%, 5% − 50%, and 20% − 50% respectively. Adulterations were discovered by considering F/G ratio and maltose ratio of the adulterated samples. F / G ratio of adulterated samples were ranged between 1.39–3.68 and 1.12–0.37, respectively. Furthermore, maltose value of adulterated with malt worth ranged between 3.0–24.92 (g/100 g). In a similar study carried out by Tosun (2004), three different blossom honey samples were adulterated with glucose, sucrose, and HFCS at the range of between 10% – 50%. According to his results, F + G ratio of three honey samples went below 0.9 with the addition of glucose syrup at a rate of 40% and 50%. F / G ratio of samples ranged between 0.54–0.77. F + G value of adulterated samples were found within safe limits which were more than 60 g/100 g.
3.10 C₄ Sugars by IR-MS
According to Turkish food codex communiqué on honey (2020), ratio of C4 should not be greater than 7%, δ13protein - δ13honey (‰) value should not be greater than ‰1 and honey δ13 C value should be – 23 (‰) or more negative (Table 2). δ13 C honey (‰) values of H1 and H2 pure honey samples were found as -25.80 and − 26.13 respectively, while the value for GFS and MS syrups were detected as -15.86 and − 16.14 respectively. C4 sugar adulteration levels (%) of the samples were increased with increasing ratio of sugar syrups in the samples. As it can be seen from Table 5, adulterations were detected at the level of ranged between 5–50% depending on honey and sugar syrup type by using EA-IRMS.
Honey samples were adulterated with high fructose corn syrup, glucose syrup, and sucrose syrup by the addition level of 10%, 20%, 30%, 40%, 50%. In this study, Although adulterations could not detected in samples adulterated with glucose syrup and sucrose syrup, adulterations made with high fructose corn syrup were detected at the rate of 20%, 30%, 40%, 50% as 14.30%, 32.80% and 41.6 5% respectively (Tosun, 2013). In a similar study, honey sample adulterated with high fructose corn syrup at the rate of 20%, 60%, 90% and detection of adulteration levels discovered as 11.2%, 30.6%, and 48.2% respectively (Padovan, 2003).