MATERIALS AND METHODS

Collecting and preparation of honey samples: Three honey samples were collected from different governorates of Egypt, Citrus honey was collected from Giza governorate, while Clover and Marjoram honeys were collected from Al-Fayoum during the active honey bee months, April, May, June and July months in 2018 at different apiaries of different Egyptian governorate (three samples for each) and stored at 4°C until analysis. The weight of each sample was approximately 1 kg.

Physiochemical properties of some Egyptian bee honey samples: Physiochemical analysis was conducted in “Elements Laboratory, Campus of Research Laboratories, FARP”, Faculty of Agriculture, Cairo University Research Park. Determination of water content of bee honey was carried out by measurements of its refractive index value (ABBE WAY-IS) refractometer¹⁷ at 20°C. The quantity of sugars (glucose and fructose) was performed by HPLC according to the method of Bogdanov et al.¹⁸. The electrical conductivity was determined by the method of Bogdanov¹⁹, using EC meter model EN50081-1 at room temperature (2 g of honey sample was dissolved in 10 mL of distilled water and the results were expressed as ppm). The optical density and color of the honey samples were measured by using the relation between optical density and USDA standard as indicated by White²⁰.

The bee honey samples were classified according to their botanical source based on the method of Von Der Ohe²¹. Pollen classification as: Mostly pollen (more than 45%), important minor pollen (10-20%) and minority pollen (less than 3%) by Andrade et al.²².

Rheological properties of some Egyptian bee honey samples: Rheological measurements of bee honey samples (viscosity, shear stress and torque) were carried out in triplicates over temperature of 25°C using a concentric cylinder Brookfield Programmable viscometer (Model DV-II, Brookfield Engineering Laboratories, USA) with UL adaptor and using spindle 7 at several RPM (5-100) in the shear rate range²³ from 6.1-122 s^–1. Win Gather version 1.1, (Brookfield Engineering Laboratories, Inc.) software was used to collect, store and plot the data on a personal computer connected to the viscometer.

Statistical analysis: The two-way statistical analysis of variance (ANOVA), mean separation and correlation required subprogram of MSTAT microcomputer statistical program²⁴ was applied to evaluate physiochemical and rheological parameters of some Egyptian honey samples. Simple and multiple linear regression analysis were applied and the student t-test was used to test mean at p<0.05.

RESULTS

Physiochemical properties of some Egyptian bee honey samples: The physiochemical properties of three different types of Egyptian bee honey “citrus (citrus spp.), clover (Trifolium alexandrenum) and marjoram (Majorana hortensis) were determined. As shown in Table 1 there were clear significant differences in all tested parameters which was conducted on tested Egyptian honey types. The water content (moisture %) of bee honey samples ranged from 18.10% in marjoram honey to 19.74% in citrus honey. Whereas the electrical conductivity (EC) of citrus honey was recorded 0.007% while in clover and marjoram honeys were recorded 0.008 and 0.011%, respectively.

On the other hand pH values of the studied Egyptian honey types ranged between 3.56 in citrus honey and 3.84 in marjoram honey. The obtained data showed that, the glucose content was ranged from 28.00% (marjoram honey) to 30.95% (clover honey) and the fructose content was recorded 39.12% in clover honey, while in marjoram and citrus honeys were detected 37.50 and 37.17%, respectively. The optical density (O.D.) of bee honey samples were measured in Table 1. The O.D. was ranged from 0.25 in marjoram honey to 0.14 in citrus honey.

Rheological properties of some Egyptian bee honey samples: Rheological (viscosity, shear stress and torque) properties were periodically determined at 25°C in cP. Citrus honey scored the lowest values for viscosity 11400 cP, shear stress 1390.8 D\cm² and torque 875.1%, while marjoram honey showed the maximum values 22750 cP, 2775.5 D\cm² and 921.1%, respectively.

In ascending series, citrus honey, clover honey and marjoram honey were represented their values in Fig. 1. Shear stress expressed the force exerted on a specific sample area (1 cm²) followed the same trend as viscosity. The maximum shear stress and torque values were measure in marjoram, clover and citrus bee honeys, respectively as shown in Fig. 1.

Analysis of variance showed significant differences (p<0.001) in viscosity, shear stress and torque as influenced by type of honey and rpm (round per minutes). Statistical analysis of variance, range and regression analysis indicated that type of honey significantly (p<0.001) affected viscosity, shear stress and torque as shown in Table 2. Moreover, LSD confirmed significant differences within honeys Table 2 at α = 0.05 level also, high R² values assured corrected data.

A proportional relationship was found between shear stress torque and the rpm values. Steep rise in shear stress and torque occurred and found that significant differences were found between the honey bee types as a function of rpm. Different shear-dependent fluids behave differently: For some, their viscosity decreased when the shear rate increased as shown in Fig. 2a, b and c.

Fig. 1:	Average viscosity (cP), shear stress (SS values*10) and torque (T, values*10) at 25°C

Table 1:	Physiochemical properties of the tested Egyptian bee honeys
OD: Optical density

Table 2:	Statistical analysis of variance, range and regression analysis of honeys
***p<0.001, **p<0.01 *p<0.05, NS: Not significant

Fig. 2(a-c):	Viscosity, shear stress and torque of (a) Marjoram honey, (b) Clover honey and (c) Citrus honey at different shear rate values

As shown in Fig. 2, the viscosity decrease as a shear rate change by increasing the round per minute (rpm) of the plunger in tested honey as shown in Fig. 2a while torque and the shear stress values increased . The viscosity of each honey type had been measured over a range of shear rates from 6.12-122 sec^–1 the shear stress was proportional to the strain rate in the fluid Fig. 2a, b and c. A thixotropic fluid can be described as a shear thinning system where the viscosity decreases with increasing shear rate as shown in Fig. 2. Honeys were subjected to increasing shear rates. The viscosity of a shear thinning fluid decreases with increasing shear rate. This shear rate dependency of the viscosity can differ substantially between different honeys type depending on concentration of total solid and water contents. The reason for shear thinning flow behavior is that an increased shear rate deforms and rearranges particles, resulting in lower flow resistance and consequently lower viscosity while, shear stress values increased due to need more stress for deformation of particles.

DISCUSSION

Results of the physicochemical analysis of three types of Egyptian bee honey samples suggested that, the floral sources of honey are the most important factor in its variety. The data obtained are presented in Table 1 which were in the range of related standards and were similar to the values found by other researchers²⁵ and showed in general a good quality of the studied honey samples. Higher moisture contents could lead to undesirable honey fermentation during storage, caused by the action of osmotolerant yeasts, The moisture values which obtained in this study were similar to those found with Serem and Bester²⁵ which recorded from 15.3-21.7 g/100 g and with Saxena et al.²⁶ which recorded (from 14.6-21.8 g/100 g).

In general, colour and conductivity are parameters that are inter-correlated and also with the mineral content and the botanical and geographic origin of honey. The E.C. values were in the same range as those reported by other authors in bee honey^27,28. The darker the honey higher was the mineral content and the conductivity^29,30. On the other hand, the sugar content (glucose and fructose) of bee honey samples were similar to those found with Escriche et al.³¹ which detected the levels of glucose (from 27.8-31.9 g/100 g) and fructose (38.3 and 42.7 g/100 g). Honeys with high fructose/glucose ratio would remain liquid for longer periods because of the modification of the saturated level of glucose by the presence of the larger amount of fructose . The pH values of three honey samples were measured and the obtained results confirmed that, all tested samples were acidic, pH values were agree with that obtained by Rateb³².

The high acidity of honey correlates with the fermentation of sugars present in the honey into organic acid, which is responsible for two important characteristics of honey: Flavor and stability against microbial spoilage³³.

Knowing the viscosity of honey is important. Many of the steps involved in extracting and processing honey are difficult to do if the honey is too viscous. Bee honey’s viscosity depends upon the amount of water and the type and amount of sugar it contains. If the concentration of water is increased, bee honey becomes less viscous²⁸. Temperature also changes the viscosity of bee honey and heat is often used to make the honey easier to process.

There are no other studies in the literature regarding the prediction of Egyptian honey rheological parameters using a concentric cylinder Brookfield Programmable viscometer (Model DV-II, Brookfield Engineering Laboratories, USA).

A viscosity function shows the viscosity over the shear rate. For a Newtonian liquid, this function is a straight line but If its viscosity changes with the shear rate, a liquid is non-Newtonian and for exact definition apparent viscosity need to be specified. The viscosity of honey decreases as shear rate increases with respect to water content in the individual samples. In multiple published reports on the rheological properties of honey, there is a common observation that it is in fact a Newtonian fluid but current data observed disagree with them³⁴. The thixotropy (time depended) behavior was observed in honeys sample.

To the author knowledge there are some papers on the rheological behavior of honey bee using a falling ball viscometer based on water content, different temperature and shear rate. It can be observed that all the parameters are influenced primarily by the type of honey bee, followed by the moisture content. This fact is in agreement with other studies, which revealed the high influence of moisture content on the rheological parameters. In the case of glucose and fructose, they had less influence on the rheological parameters than the moisture content.

CONCLUSION

This study investigated the physicochemical characterization and rheological properties of different honey samples to confirm its economical and nutritional quality. Determination of rheological behavior of substances is particularly important for designing of equipments, for transport, pumping, storage of substances and an indirect measure of product consistency and quality. Because of pure honey exhibits thixotropy time dependent flow pattern so outcome of this study can be applied in processing, quality improvement and product storage of honey .it was also established that rheological properties of honey is a newly useful tool for detecting honey adulteration.

SIGNIFICANCE STATEMENT

This study discover a newly and accurate protocol that can be beneficial for assessment the quality improvement and storage of Egyptian honey. Pure honey exhibits thixotropy time dependent flow pattern so this study will help the researcher to uncover the critical areas of honey adulteration that many researchers were not able to explore. Thus a new theory that pure honey is a Non Newtonian fluid may be arrived at.