Physicochemical and bioactive properties of six honey samples from various floral origins from Tunisia

2.1 Chemicals and reagents

Glucose, fructose, sucrose, maltose, Folin–Ciocalteu reagent, ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl, (+)-catechin, l-proline and methanol were purchased from Sigma Chemical Co. (St. Louis, USA). Ultrapure water was made using a Milli-Q water purification system (Millipore, Bedforde, MA, USA). All the other used chemicals were high purity analytical grade reagents.

2.2 Samples

There are six honey samples coming from different botanical origins and produced in Tunisia. They were collected throughout a whole year in different regions in Tunisia (see Fig. 1). Table 1 shows different origins, and type of the examined honeys. Obviously, all honey samples were directly obtained from beekeepers. The beekeepers directly extracted the samples into 250 mL sterilized glass sample bottles with glass caps. The samples were then stored in a dry and dark place at a temperature of 20 °C. Subsequently, the honey samples were classified into six categories as they belong to the following diverse floral origin: eucalyptus, orange, thyme, mint, rosemary, and horehound.

Close

Figure 1

Map of North Tunisia indicating the regions where honey samples were collected.

Export to PPT

2.3 Proximate composition analyses

Water content was measured by a Carl Zeiss 16531 refractometer at 20 °C and the corresponding water content was calculated using the Association of Official Analytical Chemists method (AOAC, 1990). While the total nitrogen (TN) was determined using the Kjeldahl method from which the crude protein could be calculated as % N × 6.25. The ash content was determined by burning the samples in a muffle furnace at 500 °C for 6 h. The proline content was determined according to the Bogdanov method (Bogdanov et al., 1997). The pH, however, was measured using a Mettler Toledo pH meter (California, USA). The pH probe was immersed in a 250 mL beaker that contained a solution of 4 grams of honey dissolved in 30 mL of ultrapure water. Free acidity was determined according to the AOAC method (AOAC, 2000), by the titrimetric method. To make things clear, the electrode of the pH meter was immersed in the solution, stirred with a magnetic stirrer and titrated to pH 8.5 by adding a 0.05 N of NaOH solution. The Electrical conductivity (EC) of a honey solution, at 20% (w/v) (dry matter basis) in ultrapure water, was measured at 20 °C using a Consort conductometer (Consort C830, Belgium). The results were expressed as milli Siemens per centimeter (mS/cm). The water activity (a_w) was determined at 25 ± 0.02 °C using an AquaLab water activity meter (AquaLab CX2T, Decagon Devices, USA). Viscosity was determined by an Ostwald viscometer at 25 °C and at a shear rate of 5 rpm.

Note that all the tests were performed in triplicate and were expressed as mean ± standard deviation.

2.4 Invertase activity

Invertase activity was spectrophotometrically measured with 4-nitrophenyl-a-d-glucopyranoside and the results are expressed in international units (IU).

2.5 Mineral analyses

Mineral elements were analyzed using a Varian Spectra Atomic Absorption Spectrophotometer (Model 220, Varian, USA) after ash drying. The sodium (Na) and potassium (K) contents were determined by emission photometry (AOAC, 1990). The contents of magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn) and copper (Cu) were determined by flame atomic absorption spectrophotometry. Lead (Pb), cadmium (Cd) and chromium (Cr) contents were determined by electrothermal atomic absorption spectrometry (23). Phosphorus (P) content was measured by a UV–visible spectrophotometer (Model U2001, Hitachi Co., Tokyo, Japan) according to the method described by the AOAC (AOAC, 1990).

2.6 Color measurement

CIELAB L^∗ a^∗ b^∗ color parameters were measured in a Minolta colorimeter (Minolta, Model CM-3600 d, UK) controlled by a computer that calculated color from the reflectance spectrum. Samples were placed in Petri dishes and filled to the brim. Each Petri dish was placed directly on the colorimeter sensor (3.5 cm in diameter) and in order to be measured there were five measurements taken per treatment every week. The instrument was calibrated using a white tile (L^∗ = 94.52, a^∗ = −0.36 and b^∗ = 1.04) as standard. The L^∗ parameter (lightness index scale) ranges from 0 (black) to 100 (white). However, the a^∗ parameter indicates the degree of red (+a^∗) or green (−a^∗) colors, whereas the b^∗ parameter measures the degree of the yellow (+b^∗) or blue (−b^∗) colors. The results were expressed in accordance with the CIELAB system with reference to illuminant D65 and with a visual angle of 10°.

2.7 Sugar composition determination

The sugar composition was determined by an HPLC (Shimadzu, Tokyo, Japan) equipped with a refractive index detector (HPLC-RI) at 30 °C. A honey sample (1 g) was dissolved in 19 mL Milli-Q water, filtered through a 0.22 mL nylon filter into an HPLC vial, capped and injected (20 μl) into the HPLC. The HPLC column was Supelcogel C-610H, 30 cm, 7.8 mm I.D. fitted with a guard column Supelguard C-610H 5 cm,4.6 mm I.D. Col: H + 11,855. The mobile phase was 0.005 M sulfuric acid at a flow rate of 0.75 mL/min. The external calibration curves produced by standard solutions were used to quantify the amount of sugars in the samples (Bonta et al., 2007). Honey sugars are identified and quantified by the method of external etalonanage by comparing their retention times and their peak areas with those of standard sugars. The results were expressed as gram sugar per 100 g of honey.

2.8 Hydroxymethylfurfural (HMF) analysis

HMF was determined according to the European commission honey method (Bogdanov et al., 1997). Briefly, 20 g of honey samples was dissolved in 100 ml Milli-Q water, filtered through a 0.22 mL nylon filter into an HPLC vial, capped and injected (20 μl) into the HPLC. HMF concentration in the honey is calculated by comparing the peak area to that of the standards taking into account the dilutions. There are a linear relationship between the concentration and the peak area and the results were expressed as mg/kg honey.

2.9 Biochemical analyses

2.10 DPPH radical scavenging activity

The DPPH radical scavenging activity was determined by the method described by Ferreira et al. (2009), with slight modification. That is to say, 0.30 mL of a honey sample (0.5 g/mL) was added to 2.7 mL of 0.20 mM DPPH-methanol solution, and the absorbance of the sample solution (A_sample) was measured at 517 nm against a blank after 60 min of incubation period at room temperature (≈22 °C). The sample concentration required to scavenge 50 % of free radicals (IC₅₀) was calculated on the basis of the regression equation. $$\text{Scavenging effect}(\%)=\left(1-\left[\frac{{\text{A}}_1-{\text{A}}_2}{{\text{A}}_0}\right]\right)\times 100$$ where A₀ is the absorbance of the control (water instead of the sample test solution), A₁ is the absorbance of the sample. A₂ blank stands for the absorbance of the samples under identical conditions as A₁ and with water instead of DPPH^- solution.

Honey sample concentration providing 50% inhibition (IC₅₀) was calculated according to the linear regression algorithm of the plotted inhibition graph percentage in comparison to the honey sample concentration. For the calculation of these values, a Sigma plot (version 10) was used.

2.11 Statistical analysis

All the tests were performed in triplicate using the SPSS statistical package (SPSS Inc., Chicago, IL), version 18.0. The result was expressed as mean ± standard deviation. The Duncan’s multiple-range test was used to evaluate significance of difference (p < 0.05) between results.

3.1 Physicochemical properties

The physicochemical parameters of the six Tunisian honeys namely, rosemary, horehound, orange, thyme, eucalyptus, and mint were analyzed as shown in Table 2. The water content found in the above honey samples ranged from 17.27% to 19.80%. Evidently, these samples are within the range of the required standards (⩽20%) according to the International Regulations of Quality (Codex Alimentarius Commission, 2001). However, it is prominent to state that the highest water content value was found in mint and orange honeys (p < 0.05). Nevertheless, there are no significant differences (p > 0.05) between horehound and thyme honeys in terms of water content. These results were similar to those previously reported for this monofloral honey and whose corresponding values ranged from 15.20% to 20.60% (Manzanares et al., 2011; Feás et al., 2010; Vanhanen et al., 2011). It has been reported that higher water content might cause undesirable honey fermentation during storage and formation of acetic acid (Chirifie et al., 2006). Conspicuously, honey’s water content depends on various factors, such as the harvesting season, the degree of maturity reached in the hive, and the geographic and environmental factors (Acquarone et al., 2007; Kahraman et al., 2010).

Similarly, protein contents of most of the Tunisian honeys were also convergent (0.13 and 0.16 g/100 g honey) but higher than the rosemary honey. This variation may be attributed to the type of flora. Hence, there are no significant differences (p < 0.05) in terms of protein content of all the examined honeys. As previously demonstrated, honey contains about 0.20% protein, as α-amylase, invertase, catalase, glucose oxidase, and phosphatase. This is related to the plant origin as well as pollens and nectars (Anklam, 1998). It is important to remark now that the results are in total conformity with the results obtained by Azeredo et al. (2003). The proline content in the honey samples varied from 39.62 to 102.60 mg/kg and the highest was observed in mint and eucalyptus honeys all of which were not significant at 95%. However, rosemary honey had a significantly lower (p < 0.05) of proline content than the other observed honeys samples.

Ash content is also another parameter used for the determination of the botanical origin such as flora mix (White, 1978). In the present study, the ash content of Tunisian honeys ranged from 0.08% to 0.69%. Note that the results are within the average percentage for floral honeys (0.60%). Hence, there are no significant differences between mint, horehound and eucalyptus in terms of ash.

The electrical conductivity (EC) of honey is closely related to the concentrations of minerals or total ash, salts, organic acids and proteins, and is a parameter that shows great variation according to the honey’s floral origin (Terrab et al., 2003; León-Ruiz et al., 2011). The results obtained after examining the six Tunisian honey samples show that the values for EC ranged from 0.39 to 0.89 mS cm⁻¹. There is a positive correlation between electrical conductivity and ash (r = 0.971). Hence, the above results demonstrate a large variation that depends on the floral origin. The orange honey sample has the highest values for EC (0.89 mS cm⁻¹), compared with mint, horehound, rosemary, thyme and eucalyptus honeys. These results conform with previous studies (Kaškonienė et al., 2010).

The water activity values obtained in this study ranged between 0.56 and 0.64. There are no significant differences between honey samples in terms of water activity (p < 0.05). In addition, the viscosity of honeys was also examined, and the highest viscosity was observed in thyme, followed by eucalyptus, rosemary, horehound, orange, and mint. These results showed that thyme and eucalyptus honeys are not significantly different (p > 0.05) in terms of viscosity. Tunisian honeys have high viscosities in comparison to other honeys (El-Bialee and Sorour, 2011).

Another important parameter during the extraction and storage of honey is the pH value. It influences honey texture, stability and shelf life (Terrab et al., 2002). As shown in Table 2, all the examined honeys had an acidic character. Their pH values ranged from 3.67 to 4.11. These values were similar to those previously reported in Algerian, Spanish and Portugal honeys that have been found to vary between pH 3.50 and pH 4.58 (Rashed and Soltan, 2004; Feás et al., 2010; Ouchemoukh et al., 2007). In general, a low pH of honey inhibits the growth and proliferation of microorganisms.

In the six Tunisian honey samples, the total acidity of honey ranged from 7.11 to 27.20 meq/kg honeys (Table 2). No significant difference (p > 0.05) was observed in terms of acidity value in mint and horehound honeys. But it must be noted that the lowest acidity value was observed in rosemary honey (7.11 meq/kg honey). These data illustrate the significant influence of floral type on the total acidity of honey. According to the obtained results of free acidity, none of the analyzed samples exceeded the limit of 40 meq kg⁻¹ as required by the international regulations (Bogandov et al., 1999). Thus, all samples were in compliance with the standards. Variation in free acidity among different honeys could be due to the different floral origins or to the variation in harvest seasons (Perez-Arquillué et al., 1994).

It has been reported that high free acidity values may indicate the fermentation of honey sugar by yeasts. It is well known that during fermentation, glucose and fructose are converted into carbon dioxide and alcohol. Alcohol is further hydrolyzed in the presence of oxygen and converted into acetic acid. This, obviously, greatly contributes to the level of free acidity in honey (Ajlouni and Sujirapinyokul, 2010).

3.2 HMF content

HMF is, undoubtedly, an excellent indicator of honey freshness and purity (Codex Alimentarius, 2000). High concentrations of HMF in honey indicate overheating or poor storage conditions. According to the International Trade Guidelines (European Economic Commity, 2002), the honey’s HMF content should not exceed 40 mg/kg. In the present study, the HMF of the six examined Tunisian honeys ranged from 12.07 to 27.43 mg/kg. The highest HMF was observed in eucalyptus followed by thyme, orange, horehound and rosemary honeys. There are no significant differences (p > 0.05) between mint and rosemary. All honey samples had an HMF value lower than the above limit, and none showed values higher than 40 mg/kg. Hence, the honey samples herein investigated are to be deemed as fresh honeys. It has been demonstrated that the HMF parameter is related to the honey’s quality and its heat processing but not to its origin (Anklam, 1998). Several factors influence the formation of HMF, such as storage conditions (e.g. temperature) and floral sources (Fallico et al., 2004; Terrab et al., 2002; Meda et al., 2005). In addition to these facts, the HMF level in honey also depends on the sugar type present in honey itself like fructose: glucose ratio (Doner, 1979). Similarly, it is well known that honey heating results in the formation of HMF, which is produced during acid-catalyzed dehydration of hexoses, such as fructose and glucose (Fallico et al., 2004; Tosi et al., 2008).

3.3 Invertase activity

Invertase is a natural honey enzyme which is commonly used in Europe as a determinant of freshness. Its level depends on the geographic and floral origins of the product, as well as on its freshness. Table 2 illustrates that invertase activity values ranged from 46.25 to 184.68 unit/kg honey. Besides, all honey samples contained a significant invertase activity. The highest value was found in mint honey while the lowest was detected in eucalyptus. Note that all investigated samples herein conform to the required standards (40 unit/kg honey) (Codex Alimentarius Commission, 2001). Obviously, this suggests optimal storage or processing conditions.

3.4 Mineral composition

The mineral composition of the six Tunisian honeys is shown in Table 3. Generally, the most abundant elements found in the investigated honey samples were potassium, sodium and calcium, and which ranged between 976.75 and 172.48; 521.22 and 251.34 and 221.07 and 113.85 mg/kg honey, respectively. Other minerals were meagerly present such as sulfur, copper, iron and zinc. These results are in total agreement with the previous studies (Vanhanen et al., 2011; Rodriguez-Otero et al., 1994). On the other hand, lead, nickel and chromium levels ranged from 0.01 to 0.05, 0.04 to 0.40 and 0.04 to 0.32 mg/kg in the Tunisian honey samples, respectively. The highest lead content was observed in the mint honey sample. Cadmium was not detected in all honey samples. It has been reported that cadmium and lead are non-essential elements in plant nutrition and are one of the most toxic substances that accumulate in biological systems (Mena et al., 1996).

3.5 Color

Color is the physical property that is immediately perceived by the consumer. The determination of the color of honeys is a useful criterion of classification for unifloral honeys. The color parameters of the six honeys are illustrated in Table 4. The lightness values (L^∗) of the six honey samples ranged from 36.64 to 51.37. Mint and thyme honeys testify lower lightness values (36.64 and 42.49, respectively). The lightness of ingredients plays a major role in the honey assessment due to consumer preferences. It is clear that the analyzed honey samples had orange, yellow and green components. The green components (negative a^∗ values) were present in rosemary honey, whereas mint honey showed the highest redness and then comes eucalyptus honey second in rank. The brownish values attested a great disparity among the different honey samples. The orange honey had the lowest value and the highest value was observed in the mint honey. The yellow hue (b^∗) had a great variation, but two main groups could be distinguished. The honeys that had b^∗ values higher than ten (between 10 and 20) are thyme, orange, eucalyptus, horehound and mint, but rosemary honey had a value lower than 10.

3.6 Major sugar composition

The sugar composition of the six Tunisian honeys was analyzed as shown in Table 4. This study affirms that in all the examined samples the percentage of fructose and glucose ranged from 35.78% to 37.84 % and from 31.07% to 36.58 %, respectively. Yet, eucalyptus and rosemary honeys specifically contained a significant amount of fructose and glucose. The predominant sugar of the six investigated honeys was fructose followed by glucose, while sucrose and maltose were present in low amounts in all other samples. Obviously, a high sucrose concentration usually means a premature harvest of honey as sucrose has not been fully developed to glucose and fructose by the action of invertase (Özcan et al., 2006). Note that the sucrose amount of all the honey samples was below the maximum conventional limit of 5% recommended by the European Community (European Economic Commity, 2002). In addition, the fructose/glucose ratio was calculated for all the six honey samples. This ratio tells about the crystallization state of honey, i.e. when fructose is higher than glucose the honey is fluid (Ouchemoukh et al., 2010; Venir et al., 2010). It has also been reported that the fructose/glucose ratio may also have an impact on the honey flavor since fructose is much sweeter than glucose (Alvarez-Suarez et al., 2010). Subsequently, all the examined Tunisian honey samples were fluid (ratio great than 1). This result is asserted by the viscosity value of the above samples.

3.7 Biochemical characteristics

3.8 Antioxidant activity

The DPPH free radical is a stable free radical, which has been widely used as a tool to determine the free radical-scavenging activity of antioxidants. Antioxidants, when interacting with DPPH, transfer either electrons or hydrogen atoms to DPPH, thus neutralizing the free radical character (Devasagayam et al., 1995). The color of the reaction mixture changes from purple to yellow, and its absorbance decreases at 517 nm. Note that the DPPH radical scavenging activities of the Tunisian honey samples (expressed by IC₅₀) are shown in Table 4. Note that lower IC₅₀ value indicates a higher DPPH free radical scavenging activity. Therefore, the present study demonstrates that the DPPH radical scavenging activity of mint honey is significantly (p < 0.05) higher (11.08 mg/mL) than all other honey samples (rosemary, horehound, orange, thyme, and eucalyptus). Note that a significant difference (p < 0.05) between all honey samples was found. Yet, higher correlations were observed between the DPPH radical scavenging activity and the total polyphenol (r = −0.945, p < 0.01), antioxidant activities and total flavonoids (r = −0.866, p < 0.01), and between total flavonoids and total polyphenols (r = 0.957, p < 0.01). These results are in perfect accordance with the works of Alvarez-Suarez et al. (2010), Sant’Ana et al. (2012), and Ferreira et al. (2009) who found that there is a positive correlation between DPPH radical scavenging activity and total polyphenols, and total flavonoids.