Honey Adulterated with Glucose Syrup: Rheological Modeling

The power-law equation is given as:

Where, η is the consistency coefficient or dynamic viscosity (Pa.s, applicable as mPa.s); τ is the shear stress (Pa or N/m²), γ is the shear rate of the fluids (s⁻¹); and n (dimensionless) is the power-law index or behaviour index of the fluid. If the power-law index, n=1, the power-law equation simplifies to the Newtonian model; if n<1, the fluid is shear thinning and finally if the value of n is >1, the fluid is shear thickening. The simplest empiricism of the two-parameter power-law expression is mathematically represented as:

Where η_o is the zero shear viscosity of the fluid measured in Pa.s

This four-parameter model describes the non-Newtonian flow with asymptotic viscosities at zero (η_o) and infinite (η_∞) shear rates, and with no yield stress, power-law index n, and consistency constant k₁.

For this work, the control honey sample, Sample A, was cultivated, harvested, and processed in 2013 at the AOT-Mafe Farm, Imuwen, Ogun State, to ensure that it is pure because of incessant adulteration of honey in Nigeria. The adulterating material, glucose syrup (GS) was purchased from an open market in Lagos, Nigeria in 2013. The water content of the glucose syrup used in this work is the same as the control honey sample A. It was then used to adulterate honey. The mass levels of adulteration obtained from mixtures of glucose syrup and control honey sample were AG₁₀, AG₅₀, AG₇₀ and AG₉₀ at 10%, 50%, 70% and 90%, respectively. The mixtures of adulterated samples were homogenised completely, the control sample was analysed within twenty-four hours of harvest, the different percentages of glucose adulterants were within twenty-four hours of the formulation and samples picked at random from the open market within twenty-four hours of procurement. During the chromatographic analysis for the determination of hydroxymethylfurfural in honey, the mobile phase was water-methanol (90:10 by volume), both of High-Performance Liquid Chromatography (HPLC) qualities. The Standard Solution of 5-hydroxyl methyl-furan-2-carbaldehyde (H40807) from Sigma-Aldrich, Milan was used. All other reagents used were of HPLC standard.

The rheology of the samples was studied with the use of the Brookfield DV-III Ultra Programmable Rheometer. The principle of operating the DV-III Ultra is to drive a spindle, which is immersed in the test sample, through a calibrated spring. The spring deflection measures the viscous drag of the fluid against the spindle. Spring deflection is measured with a rotary transducer. The rotational speed of the spindle determines the viscosity measurement range of the Rheometer, size and shape of the spindle. The container in which the spindle is rotated and the full-scale torque of the calibrated spring determines the full-scale viscosity range. All measurements were done with a cone radius of 24 mm and with a 0.8 cone angle; this gives a gap height of 0.1 mm at the circumference of the cone. The effect of glucose syrup adulteration on honey rheology was investigated at a room temperature of 27°C. The experiment was conducted at room temperature (27°C) because honey is eaten and commercially stored at room temperature (Kamboj & Mishra, 2014; Anidiobu, 2014). The samples were made to stand in the container for thirty minutes before the analysis order, to allow for the complete structural buildup from shear-induced flow into the container. Each experimental determination was repeated after 24 hours to verify the reproducibility of the results. Before being used the samples were warmed up to 40°C to dissolve any crystals and kept in flasks at 30°C to remove air bubbles which could interfere in rheological studies as Mossel et al. (2000) recommended.

The rheograms of control honey sample A and other samples purchased from the open market, B, D₁, D₂, and D₃ were given in Fig. 1. Sample A was cultivated, harvested and processed for this study. The rheological signature (profile) of Market sample D₂ is similar to that of pure sample A, which indicates that the Market sample was unadulterated. Other market samples B, D₁, and D₃ (having similar water content as A, 18.2±0.4%) exhibited varying discrepancies from the rheological signature of pure sample A, which suggests some levels of adulteration. Anidiobu (2016) and Anidiobu (2014) observed that wild honey samples from three different geographical locations, though sharing similar climatic and floral conditions, in Nigeria exhibited similar rheological properties. The observed rheograms were traced to a similar balance of oligomers and monosaccharides and water in the samples. Fig. 2 rheogram shows the effect of glucose syrup adulteration on honey. The colloidal materials, polymerised monosaccharides, melezitose, raffinose, and some oligosaccharides have been suggested to be responsible for honey's non-Newtonian behaviour (Anidiobu, 2014). The substitution of these high molecular weight materials with the lower molecular weight adulterants (glucose syrup) leads to a sequential decrease in the viscosity of the resulting adulterated samples observed in Fig. 2 (Sopade et al., 2004). This is consistent with the findings of Bakier et al. (2007) and Bera et al. (2008) that rheology is dependent on composition.

In this section, the rheological curve fit of Nigerian honey was carried out using Malcolm Cross and Ostwald-de Waele Power-Law models, as shown in figures 3–12. The Ostwald-de Waele Power-Law model was first used to obtain the flow behaviour index and the zero shear viscosity with large sums of errors using the least square analysis. The summary of the results obtained is presented in Tab. 2. The values of power-law index or flow behaviour index, n, and zero shear viscosity, η_o are fundamental rheological parameters since they have a direct correlation with the composition, structural and molecular orientation of the sample (Nwalor et al., 2018). The flow behaviour index of honey was found to increase with the level of adulteration of honey with glucose syrup in Tab. 2. The flow behaviour indices of the control honey sample A compared with other market samples suggests that sample D₂ (0.144) deviates the least from control sample A (0.250) as shown in Tab. 2 confirming that sample D₂ is pure. These results were used as the first guess during the rheological modelling using the Malcolm Cross model. The Malcolm Cross model describes the non-Newtonian flow with asymptotic viscosities at zero (η_o) and infinite (η_∞) shear rates. The flow behaviour index value of 0.210 obtained from pure honey A suggests a shear thinning behaviour. The flow behaviour index in the Malcolm Cross model also increased with increasing adulteration of honey with glucose syrup. The summary of parameters obtained from the curve fit is shown in Tab. 3, where there are importantly four adjustable variables. The adjustable parameters include the flow behaviour index and zero shear viscosity from the Power-Law defined above, infinite shear viscosity, η_∞, and constant k₁.

In summary, based on the respective curve-fits as well as the values of Error Sum of Square (ESS) it can be inferred that the Malcolm Cross model better correlated the rheology of the samples compared to the Power-Law model. When The ESS value is closer to zero, the fitted rheological values are more dependable (Morrison, 2005).

This section seeks to establish a possible correlation of chromatographic characterization with conclusions from the rheological quality assessment of honey. The HMF content of honey was determined using reverse-phase HPLC equipped with a Diode Array Detector (DAD) set at 285 nm. The DAD spectrophotometer reads the absorbance of HMF at 285 nm. The HMF concentration was 0.001 mg/100 ml of the mobile phase. The mobile phase used was 90% by volume of double distilled water and 10% by volume of methanol. The HMF elutes from the HPLC at a retention time of 4.073 minutes. The peak area was found to be 5,483,162 under this concentration. This chromatograph in Fig. 13 was generated as part of the calibration data for the experiment.

Next chromatograph of HMF content of sample A, pure honey from Imuwen, Ijubu Mushin in Ogun State was generated. The HMF contents of the samples were quantified by comparison of areas under the HMF peak and those of the other samples with corrections for sample dilutions. The result of 0.04 mg/kg obtained on Imuwen Ijebu Mushin honey (sample A) is an indication of the freshness and purity of the standard honey. Its value is also below the Codex Alimentary threshold of 80 mg/kg indicating compliance with the HMF standard for pure honey (Bogdanov, 2008). The 5-hydroxymethyl furfuraldehyde (HMF) content of honey samples is a measure of freshness, adulteration or heat treatment of honey (Anidiobu, 2016). HMF is formed in honey during acid-catalyzed thermal dehydration of hexoses (fructose and glucose) occasioned by disequilibrium introduced by the adulterating material, storage or heat treatment of honey (Belitz & Grosch, 1999). Thus, sample A is established as unadulterated pure honey.

As shown in Tab. 4, sample D₂ is established chromatographically as pure honey. The HMF value of 22.90 mg/kg obtained (are below the Codex Alimentary Standard) confirmed the earlier rheological characterization results. Though sample D₁'s HMF content of 55.49 mg/kg passed the Codex Alimentary standard, it failed the European Union Standard of 40 mg/kg. This result indicates a mild level of adulteration. Sample D₃ earlier was predicted rheologically as an adulterated sample but expectedly failed the HMF test. It then concludes that the chromatographic analysis of market samples correlated with conclusions from rheological characterisation.

Similarly, glucose syrup (GS) yielded a high furfural content of 180.23 mg/kg. When the reference pure honey (A) was adulterated to 10% glucose (sample AGS₁₀), a furfural content of 80.89 mg/kg resulted which is just above the Codex Standard of 80 mg/kg. Likewise, at 50% adulteration of honey (AGS₅₀), the furfural content increased to 93.44 mg/kg. Similarly, at 70% (AGS₇₀) and 90% adulteration of honey with glucose syrup (AGS₉₀), HMF contents of 111.77 and 135.19 mg/kg were obtained. This most likely came from the rich HMF content of glucose syrup, employed in diluting sample A. The high content of the furfural in the syrup adulterant could be attributed to the enzyme invertase (saccharase and α-glucosidase) which leads to the decomposition of glucose, thereby releasing HMF as a by-product in the presence of good acidic medium and high temperature (Zappala et al., 2005).

Pure honey has a characteristic rheogram at a fixed condition of temperature. The viscosity generally decreases as the shear rate increases. Still, a peculiar shear thickening behaviour is observed at low rates of shear (0.01 to 0.1 s⁻¹) and temperatures around 27°C leading to maxima in the rheogram. Glucose syrup often fraudulently employed to enhance net product mass was found to drag the samples towards Newtonian behaviour even in the low shear rate zone. The degrees of honey adulteration (mass % of adulterant in the sample) reflected on the rheograms. Rheological data from market samples suggests that sample D₂ is pure and other samples exhibited varying degrees of adulteration. A correlation was found between the rheological characterization of honey and the chromatographic assay of hydroxymethylfurfural in honey. The furfural was found to increase with the increased adulteration of honey with glucose syrup.