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. 2024 Apr 25;9(18):20243–20252. doi: 10.1021/acsomega.4c00570

Effect of Temperature and Time on the Physicochemical and Sensory Properties of Crystallized Honey

Taha Rababah †,*, Muhammad Al-U’datt †, Amjad Naqresh †, Sana Gammoh †, Ali Almajwal ‡, Mohammed Saleh §, Sevil Yücel ∥, Yara AL-Rayyan ⊥, Numan AL-Rayyan #,¶,*
PMCID: PMC11079870  PMID: 38737063

Abstract

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This research explores the crystallization process of honey during storage with a focus on its dissolution dynamics and essential characteristics. The investigation includes the examination of the effects of heat treatment at different temperatures (45–90 °C) and durations (23–960 min) on the induced crystallization of honey at 14 °C. Various analyses were conducted, including pH, acidity, color, sugar profile, phenolic and flavonoid contents, DPPH-scavenging activity, hydroxymethylfurfural (HMF), viscosity, and sensory attributes. The results indicated a reduction in the moisture content and pH, an increase in acidity, and higher levels of HMF at elevated temperatures. While the ash content remained relatively unchanged, variables such as color, glucose, fructose, total phenol, flavonoid, and antioxidant content exhibited variations with temperature. Viscosity decreased with an increase in temperature, suggesting Newtonian behavior and implying potential colloidal changes. Consumer sensory tests revealed significant differences among samples, with honey treated at 75 °C demonstrating superior physicochemical and sensory attributes. This study offers valuable insights into the dynamics of crystallized honey, providing information for both production practices and understanding consumer preferences.

1. Introduction

Jordanian honey comes from local sources or is brought in from nearby nations such as Saudi Arabia and Egypt as well as European countries like Germany, Spain, and Türkiye. In the year 2019, around 320 tons of honey were domestically produced in Jordan, while approximately 600 tons were imported.1 Honey has been recognized to have nutritional benefits, unlike plain sugar.2 It is composed mainly of glucose and fructose (65%), water (18%), as well as low lipid and protein contents.3,4 Honey’s chemical composition and physical properties depend on the type of plant, the differences in the type of flora, the geographical region, and climatic conditions that may influence honey’s physical and chemical properties.5 This diversity of honey components has led to it being used for a variety of health and medicinal purposes.6−8 Also, honey is well-known to have antioxidant, anti-inflammatory, and antimicrobial properties.9,10 These effects could be attributed to honey’s high osmolality inhibiting bacterial growth, glucose oxidase antimicrobial effects, and/or the presence of antibacterial substances such as polyphenols.11

Honey is vulnerable to crystallization depending on viscosity, water content, degree of supersaturation, and degree of supersaturation.12 Both the crystalline and liquid phases may coexist at different times when honey crystallizes.13 Water activity rises above that of the honey’s initial liquid phase in the still-liquid state.13 This is brought about by a drop in the solution concentration brought on by the subsequent release of water from the solid phase as a result of crystallization.13 Most commercial honey presents flawed crystallization in contrast to some monofloral kinds of honey that are naturally finely and homogeneously crystallized.12 Spontaneous granulation can lead to undesired coarse crystals and an overall loss in quality.12 Crystallization occurs naturally in honey but can cause problems in the handling and processing of the honey.14 This may reflect consumer preferences that are considered to be unacceptable, and it could create a favorable environment for the growth of yeast that can lead to fermentation.

Processing honey allowed it to be viscous for a long time. Honey is processed using complex technological processes to liquefy, filter, heat, cool, and store under specified time regimes. Thermal treatment is designed to protect against microflora destruction and recrystallization while remaining in a liquid state and reducing viscosity.15 High thermal treatments to inhibit fungi and delay crystallization are not currently accepted as a practice for quality standards. The physical properties of the honey are affected when it is heated, altering its color, aroma, flavor, and biological activity.16 Heating can also decrease diastase activity, a naturally occurring enzyme that is introduced to honey by bees during nectar processing.13 Hydroxymethylfurfural (HMF), a known carcinogen, content of the honey can also increase through heating.13 The primary objective of this study is to identify the best dissolution treatment that maintains the good properties and nutritional values of crystallized honey. This study provides insights into optimizing honey processing methods, ensuring product quality, and meeting consumer preferences. This study delves into the intricate dynamics of crystallized honey with a specific focus on identifying key properties that play a pivotal role in its dissolution process. We achieved that by recrystallizing honey samples at different temperatures, investigating the effect of heat treatment on the physiochemical properties of honey, including moisture, and determining the effect of the heat treatment on the viscosity and sensory attributes of the investigated honey samples.

2. Materials and Methods

The research was conducted in the Food Science Research Laboratory, Department of Nutrition and Food Technology at Jordan University of Science and Technology–Irbid–Jordan.

2.1. Chemicals

Folin-Ciocalteu reagent, sodium carbonate Na2CO3, gallic acid, methanol, sodium nitrite (NaNo2), aluminum chloride (AlCl3), sodium hydroxide (NaOH), 2,2-diphenyl-2-picrylhydrazyl (DPPH), HCL, DNS reagent (Dinitrosalicyclic acid), Hip-His-leu (Hippuryl l-Histidine-leucine), and high-pressure liquid chromatography (HPLC) grade acetonitrile were purchased from Sigma-Aldrich (Sigma-Aldrich, Switzerland).

2.2. Sample Collection

This study was conducted on raw (unprocessed) rape honey collected from a farm located in the northern part of Jordan, in the Shafa Ghuria area in Ajloun. Honey samples were collected in May/June 2020. Fresh honey samples were poured into glass jars with a volume of 250 mL, sealed, and stored at 20 °C. The crystallization process took 46 days, starting from July to September. After 3–5 days of crystallization under these conditions, the honey showed a state of aggregation close to that of a solid substance.

2.3. Heat Treatment

Honey samples were treated at temperatures (45, 60, 75, and 90 °C) using a water bath (as shown in Table 1). Immediately after each liquefaction, the sample jars were placed in a beaker that was inserted on a hot plate at 50 °C so that the jar did not break. Then, samples were placed in the incubator at a temperature of 25 °C and kept in it until all of the tests were carried out.

Table 1. Decrystallization Conditions at Different High Temperatures.

temperature (celsius) time until fully decrystallization (min)
45 °C 960
60 °C 380
75 °C 50
90 °C 23
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2.4. Physiochemical Properties

2.4.1. Ash Determination

Ash content was determined according to the official method of the Association of Official Analytical Chemists (AOAC).16 2 g of each sample was placed in a previously weighed porcelain crucible and dried in an oven at 110 °C for 4 h. This helped remove the moisture that causes honey to foam in the early stages of extracting ash. Once the crucibles were removed from the oven, they were allowed to cool in a desiccator for about 4 more hours before being weighed again to determine the weight of the evaporated sample. The materials were then placed in an electrical furnace at 550 °C for 6 h.17 The materials were then cooled in a desiccator and then weighed. The ash content on a dry basis was calculated according to the following equation

2.4.1. 1

where A = crucible weight, B = weight of crucible and sample after evaporation, and C = crucible and sample weight after ash formation.

2.4.2. Moisture Determination

The indirect refract metric method was used to determine the moisture of samples. All Digital Abbe refractometer was used for all measurements. The Wedmore conversion table and the refractive index were used to obtain the percentage of moisture for the honey sample.18

2.4.3. Acidity and pH

The harmonized methods of the International Honey Commission were used to obtain the pH and free acidity.18 A total of 10 g of the honey samples were dissolved in 75 mL of CO2-free distilled water, and the pH of the solution was measured using a pH meter (CyberScan pH510—Eutech Instruments). The honey CO2-free distilled water solution was titrated using 0.1 M NaOH to pH 8.3 to measure the free acids. The results were expressed in milliequivalents per kilogram. To obtain the mean value, three replicates were created and used.

2.5. Color Measurement

The color of honey samples was measured using a colorimeter (12MM Aperture U 59730 Inc., Pittsford, New York, USA) and recorded in the L*, a*, and b* color systems. According to Rababah et al.,19 this color system consists of a luminance or lightness component (L*), where a* is the component for greenness and redness and the b* component stands for blue to yellow. A standard white ceramic reference (Commission International ale de ÌEclairage L* = 97.91, a* = −0.68, and b* = +2.45) was used to calibrate the calorimeter. Additionally, the chroma and total color difference (ΔE) were calculated by using the following equations

2.5. 2
2.5. 3

A total of three observations were used to calculate the mean value.

2.6. Sugar Profile Analysis

2.6.1. Preparation of Honey Samples

Glucose and fructose were measured according to AOAC20 with some modifications. Each honey sample (1 g) was weighed, transferred to a 100 mL volumetric flask, and dissolved in 25 mL of distilled water. The volume was then brought to the total volume using acetonitrile. By using a syringe and a premounted membrane filter, the final solution was filtered through a 0.45 μm filter and transferred to sample vials.

2.6.2. HPLC Analysis of Honey Sugars

This method is based on AOAC20 with some modifications. To analyze the honey samples, 10 μL of each prepared sample was injected into the HPLC. HPLC equipped with RI-detection [SHIMADZU refractive index (RID-10A)] was used to determine the sugar content of each sample. A separation column [Shim-pack SCR-101N (250 mm L × 4.6 mm I.D., 10 μm)] was used and held at 30 °C. The mobile phase was a mixture of water/acetonitrile (80:20 v/v). The flow rate was found to be 1.3 mL/min. The retention times were used to identify the sugars by comparing them with those of sugar standards. Quantitation is performed according to the external standard method on peak areas or peak heights. The concentration of sugar was calculated using the following equations

2.6.2. 4

where A1 = area for sample, A2 = area for standard, C1 = standard concentration, and C2 = sample concentration.

2.7. Determination of the Hydroxymethylfurfural Content

The HMF content determination was based on the official AOAC.21 In 25 mL of water, 5 g of honey was dissolved and then transferred quantitatively into a 50 mL volumetric flask. In that flask, 0.5 mL of K4Fe(CN)6.3H2 and 0.5 mL of Zn (CH3COO)2 were added and made up to 50 mL with water. The solution was then filtered through paper, and the first 10 mL of the filtrate was discarded. Two test tubes were prepared with 5 mL aliquots of the solution. A total of 5 mL of distilled water were placed in the first test tube and designated to be the sample solution, and 5 mL of sodium bisulfite solution was added to the second test tube and designated to be the reference solution. The absorbance of the solutions was measured at 284 and 336 nm and determined using a spectrophotometer (Varian Cary, model 1E UV/visible spectrophotometer). The HMF content was calculated using the following equation

2.7. 5

where A284 is the absorbance at 284 nm, A336 is the absorbance at 336 nm, and 14.97 is a factor calculated by the molecular weight of HMF.

2.8. Phytochemical Determinations

2.8.1. Honey Extraction

The homogenized honey samples were diluted using distilled water to achieve a ratio of 1:10 (w/v). The solution was then filtered through Whatman no. 1 filter paper, and the obtained filtrate was used for physiochemical and color analysis in triplicate.22

2.8.2. Determination of the Total Phenolic Content

The total phenolic content of previous honey extracts was measured using the Folin-Ciocalteu reagent according to Singleton and Rossi,23 with some modifications. 0.2 mL of the honey extracts (observations) was transferred into test tubes, then mixed with 2.5 mL of the Folin-Ciocalteu reagent, and allowed to stand for 5 min at room temperature. A total of 2 mL of 10% Na2CO3 were added, and then the mixture was allowed to stand in the dark for 2 h at room temperature. The absorbance was measured at 760 nm by using a spectrophotometer (Varian Cary, model 1E UV/visible spectrophotometer) against a methanol blank. Gallic acid was used as the calibration standard, and the results were expressed as milligrams of gallic acid equivalent (mg of GAE/100 g of honey). The total phenolic content of previous honey extracts was measured using the Folin-Ciocalteu reagent according to Singleton and Rossi,23 with some modifications. 0.2 mL of the honey extracts (observations) was transferred into test tubes, then mixed with 2.5 mL of the Folin-Ciocalteu reagent, and allowed to stand for 5 min at room temperature. A total of 2 mL of 10% Na2CO3 were added, and then the mixture was allowed to stand in the dark for 2 h at room temperature. The absorbance was measured at 760 nm using a spectrophotometer (Varian Cary, model 1E UV/visible spectrophotometer) against a methanol blank. Gallic acid was used as the calibration standard, and the results were expressed as milligrams of gallic acid equivalent (mg GAE/100 g of honey).

2.8.3. Determination of Radical Scavenging Activity

The antioxidant activity of honey samples was determined using a procedure described by Turkmen et al.,24 with some modifications. A total of 5 g of honey were dissolved in 50 mL of methanol, centrifuged at 4350g, and then filtered through Whatman filter paper no. 1. A total of 0.2 mL of the methanol solution of DPPH (50 mg/100 mL) were mixed with the filtrated sample. The resulting mixture was then brought to a volume of 4.0 mL, mixed completely, and allowed to stand for 60 min in a dark place. Absorbance (A) was then measured at 517 nm against the blank using a spectrophotometer (Varian Cary, model 1E UV/visible spectrophotometer). The radical scavenging activity of the samples was expressed as the inhibition % of the free radical according to the following equation

2.8.3. 6

where Abs control and Abs sample are the absorbances of the control and sample, respectively.

2.8.4. Determination of Total Flavonoids

The colorimetric method was used to determine the total flavonoid content, as described by Zhishen et al.25 Briefly, 0.5 mL from each previous extract sample was mixed with 2 mL of distilled water, followed by 150 μL of a 15% NaNO2 solution. The mixture was allowed to sit for 6 min before adding 150 μL of 10% AlCl3 solution, and the mixture was allowed to stand for 6 min at ambient temperature. Afterward, 2 mL of a 4% NaOH solution was added to the mixture. The volume was made up of 5 mL, and the mixture was mixed very well and allowed to stand for 15 min. Absorbance was determined at 510 nm against a water blank using a spectrophotometer (Varian Cary, model 1E UV/visible spectrophotometer). Results were expressed as catechin equivalents per gram of the honey sample. The total flavonoid content was calculated using a standard curve for (+)-catechin hydrate solutions. All measurements were made in triplicate.

2.9. Viscosity Determination

The viscosity of honey was determined according to Ereifej et al.26 at 25 °C. A Haake Falling Ball viscometer (Haake Mess Technik, “Falling Ball Viscometer” Manual, Dieselstr. 6-7500 Karlsuhe 41, Germany) was used to determine the viscosity of honey at 25 °C. 5 mL from each sample was used to measure the viscosity. The viscosity was calculated as

2.9.

where viscosity is in Pa·s, A = ball constant, K1 = ball density (kg/m3), K2 = sample density (kg/m3), and t = time (s). Nominal size of balls: 1/16-in., 3/32-in. Duran borosilicate glass specification: length: 362 mm. Inner diameter: 50 mm. Outer diameter: 53 mm.

2.10. Sensory Evaluation

The consumer sample population was selected from consumers in the Food and Drug Administration, who were 18–45 years of age and of various socioeconomic backgrounds. Only individuals who commonly consumed honey were selected to participate in the study. A total sample of 50 participants were selected for the evaluation, and consumer testing was conducted at the Food and Drug Administration Laboratories (Irbid, Jordan). Participants were directed to taste the samples at individual tables. Participants were asked to evaluate the samples using written instructions and ballots. Samples were identifiable using three random digit numbers and presented in numerical order. Participants were provided with five cups, individually and separately, containing honey samples treated with different heat treatments. Participants were also provided with water and green apples at room temperature to cleanse their mouths between samples. Participants were asked to record their intensity and acceptability scores for their impressions of each sample. The overall aroma, flavor, taste, viscosity, and color were scored on a 9-point scale, with 9 = like extremely and 1 = dislike extremely. The flavor was scored on a 5-point flavor scale, with 5 = much too cooked, and 1 = much too weak. The color was scored on a 5-point scale with 5 = much too light and 1 = much too dark, and astringency was recorded on a 5-point scale with 5 = very low acidic and 1 = much too acidic.

2.11. Statistical Analysis

Data were analyzed using the general linear model procedure with the JMP statistical package (JMP Institute Inc., Cary, NC, USA). Means were separated by LSD analysis at the least significant difference of P ≤ 0.05 values.

3. Results and Discussion

3.1. Physicochemical Properties

3.1.1. Ash Content

The effect of heat treatment at different temperatures and times of crystallized honey on the ash content is shown in Table 2. The ash values varied in honey samples that were exposed to different high temperatures. There was no significant difference in the values as the temperature increased. It ranged between 1.14 and 1.17%. The higher ash content was found at 75 °C (1.17%), followed by the control sample (1.16%), 45 °C (1.15%), and 60 and 90 °C (1.14%). The obtained range is compatible with what was reported by Kędzierska-Matysek et al.27 This pioneering study reported that the values of the ash content for honey samples vary from 0.7 to 1.9%. Several researchers, including Missio da Silva et al.,28 Minhas et al.,29 and Chuttong et al.,30 have documented the absence of substantial modifications in the ash content of honey subjected to varying storage temperatures, including both low and high conditions. While most of the studies noted a concurrent lack of significant change in the ash content, Minhas et al. (2016) and Chuttong et al.30 specifically identified a diminishing pattern in the ash content when honey was stored at room temperature, addressing the temporal aspect of ash content alterations, and Salin et al.31 suggested that the reduction in the ash content over time is attributed to the physiological activities of honey. The ash content represents the mineral content of the food and is a part of proximate analysis for nutritional evaluation, making it an important characteristic of food quality.32 A standard value for this parameter is not provided by the Codex Alimentarius Committee on sugars.33 However, studies have shown that the average ash content in honey is 0.17% (w/w) and ranges between 0.02 and 1.03% (w/w).34 The low ash content is normal in honey but may differ between samples since it primarily depends on what material bees collect during foraging.35,36 Additionally, the ash content depends on the foraging preference of the bees.37

Table 2. Ash, Moisture, pH, Acidity, and TSS Measurements of Crystallized Honey Samples Treated at Different Temperatures.
temperature moisture (%) ash pH acidity (meq/kg) TSS (°Brix)
control 18.53 ± 0.12a 1.16 ± 0.08a 4.02 ± 0.03a 13.50 ± 0.002e 80.45 ± 0.96b
45 °C 17.00 ± 0.20b 1.15 ± 0.03a 4.01 ± 0.01a 13.82 ± 0.001d 81.20 ± 0.20b
60 °C 16.47 ± 0.12c 1.14 ± 0.07a 4.00 ± 0.01a 14.32 ± 0.005c 81.13 ± 0.58b
75 °C 15.80 ± 0.20d 1.17 ± 0.04a 4.00 ± 0.01a 14.90 ± 0.001b 84.20 ± 0.40a
90 °C 15.06 ± 0.12e 1.14 ± 0.09a 3.99 ± 0.03a 16.00 ± 0.001a 84.96 ± 0.16a
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All values are means of three observations and calculated on a wet basis; *means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05); and **no significant differences between all the treatments.

3.1.2. Moisture Content

The effect of heat treatment at different temperatures and times of crystallized honey on the moisture content is shown in Table 2. The moisture content in all honey samples varied between 15.06 and 18.53%. The highest moisture content was found in the control sample (18.53%), followed by 45 (17.00%), 60 (16.47%), 75 (15.80%), and 90 °C (15.06%). All the moisture content values were below the standard value of 20–21% reported by Codex Alimentarius33 and the Jordan Standard Specification. The water activity values were expected to change in the same proportion with an increasing temperature. Our results agree with Kretavičius et al.38 that the moisture content in honey samples treated with high temperatures ranged between 16.2 and 18.4%. This limited range is used to control maturity and quality since climate conditions, the season in which the honey is harvested, and the quality of beekeeping practices all affect the moisture content of the honey.39

3.1.3. Effect of Temperature on pH

The effect of heat treatment at different temperatures and times for crystallized honey on the acidity and pH is shown in Table 2. The pH of honey samples treated at different temperatures decreases slightly from 3.99 to 4.02. The higher pH value was found in the control sample (4.02), followed by 45 (4.01), 60 (4.00), 75 (4.00), and 90 °C (3.99). The obtained pH value is similar to what was found by Stojković et al.,40 who reported a reduction in all honey samples after heat treatment. Furthermore, Karabagias et al.41 found that the increase in heating temperature was found to be a key parameter that contributes to the decrease in pH. The pH is a measurement of the concentration of hydrogen ions, and a change in the temperature of a solution will impact the pH. At higher temperatures, hydrogen ions form fewer hydrogen bonds, thereby increasing the concentration of hydrogen ions and decreasing the pH.42 Moreover, the release of organic acids from the pollens of honey during ultrasonic treatments or heat could also affect the pH decline in the final samples.43 The acidity in all treatments is noticed to increase from 13.5 to 16.00 mequiv/kg, indicating that all values were below the standard value (50 mequiv acid/kg) reported by Codex Alimentarius33 and Jordan Standard Specification (not to exceed 50 mequiv acid/kg). The higher value of the acidity was found at 90 °C (16.00), followed by 75 (14.90), 60 (14.32), 45 (13.82), and control samples (13.5). Stan et al.44 studied the effects of different temperatures (at 40, 60, 80, and 100 °C) on honey characteristics and investigated the changes that occurred in the acidity value. It is found that acidity increases as the temperature increases (5.75 to 8.25 mequiv/kg). Moreover, the obtained value of the acidity is consistent with the findings of Kędzierska-Matysek et al.27 The study indicates that the acidity of honey samples at various temperatures (30, 40, 50, 60, 70, and 80 °C) exhibits values between 11.50 and 24.00 mequiv/kg. The obtained results of high acidity are explained by Dranca and Oroian.45 They demonstrated that it could be a result of fructose decomposition at high temperatures in formic and levulinic acid, which leads to an increase in honey’s acidity. Also, the total acidity of honey shows a polynomial increase with the length of exposure.

3.2. Fructose and Glucose Content

The effect of heat treatment at different temperatures and times on the reducing sugar of crystallized honey is presented in Table 3. The glucose content significantly decreases as the temperature increases. Its value varies from 20.65 to 33.36%. The higher glucose content was found in the control sample (33.36%), followed significantly by 45 (32.25%), 60 (30.15), 75 (26.18%), and 90 (20.65%). The fructose contents significantly decrease with increasing temperature. Its value varies from 28.30 to 38.87%. The highest fructose content was found in the control sample (38.87%), followed by 45 (38.19%), 60 (36.51%), 75 (31.97%), and 90 °C (28.30%). The sum of the glucose and fructose contents was below the standard value in the control sample. For 45, 60 °C, while 75 and 90 °C temperatures, the sum was not below the standard value (not less than 60 g/100 g) reported by Codex Alimentarius33 and not less than 65% according to Jordan Standard Specification. Regarding glucose results, Samira conducted the only similar study focused on the changes that occur in the glucose content in honey samples treated with different temperatures and found that glucose content decreased.46 The study reported that this reduction may be interpreted in terms of the degradation of glucose with the generation of another derivative of HMF with regard to the sugar content. Moreover, the reduction in the fructose content could be related to the instability of fructose in acid solution and to the Maillard reaction, which occurs when carbohydrates are heated in the absence of amino groups. In caramelization, the carbohydrate undergoes 1,2-enolization, and the resulting enol is susceptible to β-elimination of water, forming anhydro-rings or other reactive intermediates. Typical products of this include furans, which, in the case of fructose, is predominantly HMF.47

Table 3. Glucose, Fructose, and Total Sugar of Crystallized Honey Samples Treated at Different Temperatures.

temperature glucose (%) fructose (%) total sugar
control 33.36 ± 0.20a 38.87 ± 0.12a 72.72
45 °C 32.25 ± 0.25b 38.19 ± 0.30b 72.36
60 °C 30.15 ± 0.10c 36.51 ± 0.30c 70.09
75 °C 26.18 ± 0.43d 31.97 ± 0.15d 62.45
90 °C 20.65 ± 0.30e 28.30 ± 0.40e 53.73
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All values are means of three observations and calculated on a wet basis. **Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05).

3.3. Hydroxymethylfurfural Content

The effects of elevated temperatures and increasing exposure times on the HMF content of crystallized honey are shown in Table 4. The HMF contents of all samples were not higher than 40 mg/kg for 50, 70, and 85 °C during the first 5 h. However, a dramatic increase in HMF formations was observed after 5 h of incubation at 70 and 80 °C. It is found that the HMF content significantly increases with temperature. Its values vary from 6.79 to 55.11 mg/kg. The higher HMF content was found for the 90 °C case (55.11 mg/kg), followed by 75 (27.50 mg/kg), 60 (24.85 mg/kg), 45 °C (9.89 mg/kg), and the control sample (6.79 mg/kg). HMF contents were below the standard value (not to be more than 40 mg/kg) reported by Codex Alimentarius33 and according to Jordan Standard Specification, except for the 90 °C that exceeds the standard value. The obtained range is compatible with what was reported by Turhan et al.48 and Al-Diab and Jarkas.49 The two studies reported that honey samples contain HMF values ranging from 0.62 to 193.81 mg/kg and 29.9 to 36.1 mg/kg, respectively. These results are also compatible with Turkut et al. (2018), who found that HMF values did not exceed 40 mg/kg when the honey was treated for less than 5 h at 50, 70, and 85 °C, but these values increased dramatically after 5 h of the incubation period at 70 and 80 °C.50 Another study conducted by Kędzierska-Matysek et al.27 reported a low HMF content of honey samples with different temperatures ranging between 3.07 and 4.97 mg/kg. However, the increase in HMF could be explained by the increased concentration of fructose, which surmounted the energy barrier and activated the Maillard reaction to form HMF compounds, as reported by Hasan.51

Table 4. Hydroxymethylfurfural (HMF) Content in Crystallized Honey Samples Treated With Different Temperatures.

temperature HMF (mg/kg)
control 6.79 ± 1.50e
45 °C 9.89 ± 0.40d
60 °C 24.85 ± 0.46c
75 °C 27.50 ± 1.47b
90 °C 55.11 ± 1.04a
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All values are means of three observations and calculated on a wet basis. **Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05).

3.4. Color Measurement

The results on the color of the crystallized honey subjected to different heat temperatures and times are shown in Table 5. The results can be expressed as L* for darkness/lightness (0 black, 100 white), a* (−a greenness, +a redness), and b* (−b blueness, +b yellowness).

Table 5. Color Measurements (L*, a*, b*, ΔE, and Chroma) of Crystallized Honey Samples Treated with Different Temperatures.

temp L* a* b* ΔE Chroma
control 41.61 ± 0.49a 9.42 ± 0.02e 47.75 ± 0.12c 63.74 ± 0.39a 48.67 ± 0.12d
45 °C 36.36 ± 0.31b 9.84 ± 0.03d 49.71 ± 0.05b 61.11 ± 0.04c 50.67 ± 0.04c
60 °C 33.58 ± 0.12c 11.59 ± 0.10c 49.73 ± 0.03b 62.37 ± 0.15b 51.06 ± 0.04b
75 °C 32.44 ± 0.92d 12.25 ± 0.02b 49.76 ± 0.10b 60.65 ± 0.56c 51.25 ± 0.09b
90 °C 31.84 ± 0.01d 12.54 ± 0.06a 52.84 ± 0.4a 62.95 ± 0.35b 54.30 ± 0.40a
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All values are means of three replicates and are calculated on a wet basis. *Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05).

The L values of the honey ranged from 31.84 to 41.61, with a low L value expressing darkness. Lightness decreases as the temperature increases. Also, a* increases, and its value ranges between 9.42 and 12.54, b* values between 47.75 and 52.84, ΔE values vary from 60.65 to 63.74, and chroma values vary from 48.67 to 54.30. The lightness (L) of the honey samples follows an increasing order: in the control sample (41.61) > 45 (36.36) > 60 (33.58) >75 (32.44) > 90 °C (31.84). Furthermore, redness follows an increasing order: 90 (12.54) > 75 (12.25) > 60 (11.59) > 45 °C (9.84) > control sample (9.42). The yellowness exhibits the following: 90 (52.48) > 75 (49.76) > 60 (49.73) > 45 °C (49.71) > control sample (47.75). A pioneering study conducted by Karabagias et al.41 found that different temperature treatments affect the color of honey samples. The color variations reported in this work agree well with Karabagias’s study and with what was reported by Kędzierska-Matysek et al.27 The later study reported changes in the lightness reduction (L*), an increase in yellowness (b*), and the insignificant rise in redness (a*) values observed in honey samples heated at 20 °C and those treated at 50 °C. Furthermore, the study reported that the darkening observed for samples heated between 20 and 50 °C could be attributed to Maillard reactions or fructose caramelization. The variations in the browning rate of honey can also be explained in terms of the decreased sugar content and the differences in its amino acids. Additional factors that could affect the kinetics of Maillard browning can also include reducing sugars that participate in the reaction and the type and thermal stability of amino.24 In addition, Abu-Jdayil et al.52 confirmed that the stability of color after heating is an indication of no protein precipitation but only protein denaturation in solution.

3.5. Phytochemical Contents

3.5.1. Total Phenolic Content

Table 6 shows the effect of heat treatment at different temperatures and times of crystallized honey on the phenol content. It shows that the total phenolic compounds for control, 45, 60, 75, and 90 °C are 48.25, 51.04, 51.11, 51.56, and 54.18, respectively. The results indicate that all investigated samples exhibit a high content of total phenols. These results are compatible with what was found by Bucekova et al.53 who found that the values of the total phenolic content of the honey samples were 17 to 44 mg GAE/100 g. Another study conducted by Stojković et al.40 reported that the total phenolic content of honey samples was as low as 1.361 mg GAE/g. On the other hand, Kowalski54 reported that certain types of honey increased the total phenolic compounds after thermal processing. Wang et al.55 observed a different effect of the heat treatment on the total phenols. Pimentel-González et al.56 showed a decrease in the total phenolic content of Buckwheat honey subjected to thermal processing and practically no effect for clover honey. These contradictory results could probably be due to the phenolic compounds, which are known to be susceptible to thermal degradation, taking into consideration the floral origin of honey samples.57

Table 6. Total Phenols, Antioxidant, and Flavonoid Contents of Crystallized Honey Samples Treated at Different Temperatures.
temperature total phenols (mg GAE/100 g) antioxidant (%) flavonoid content (mg/100 g)
control 48.25 ± 0.88c 31.17 ± 0.69a 12.18 ± 0.36c
45 °C 51.04 ± 1.79b 31.02 ± 0.91a 12.40 ± 0.47b
60 °C 51.11 ± 1.79b 29.31 ± 0.75b 12.53 ± 0.15b
75 °C 51.56 ± 0.76b 29.15 ± 0.79b 12.80 ± 0.10a
90 °C 54.18 ± 0.90a 27.67 ± 0.73c 12.93 ± 0.32a
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All values are means of three observations and calculated on a wet basis. **Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05).

3.5.2. Antioxidant Activity

The effect of heat treatment at different temperatures and times of crystallized honey on its antioxidant activity is shown in Table 6. The antioxidant content varied among all honey samples, and it was found to range from 27.67 to 31.17%. The antioxidant content was found in the control sample (31.17%) and (31.02%) for samples heated at 45 °C. For samples heated at 60, 75, and 90 °C, it is found to be 29.31, 29.15, and 28.67%, respectively. It was observed that as the treatment temperature increased, the antioxidant activity exhibited a decline. In the control samples, it was recorded at 31.17%, and with heat treatment, it gradually decreased, reaching 28.67% at 90 °C. This reduction in antioxidant activity correlates with the increase in the percentages of total phenol and flavonoid contents. This is likely attributed to the presence of other compounds at elevated temperatures, distinct from flavonoids or other phenolic compounds, known for their susceptibility to thermal degradation.58 The obtained values agree with what was reported by Karabagias et al.41 for samples subjected to similar thermal conditions. The antioxidant activity of honey samples analyzed and subjected to different heating temperatures differs according to the heating temperature. It decreases as heat treatment increases. However, Turkut et al. (2018) observed an increase in the antioxidant activity, and they found that the DPPH increased with increasing temperature during the heating process.50 Moreover, Chaikham et al.43 found that the antioxidant contents of honey samples treated at high temperatures were high. Their values varied from 45.14 to 53.58%. Other studies conducted by Karabagias et al.41 and Pimentel-González et al.56 found that the antioxidant activity of honey was also affected by mild thermal treatment.

Surprisingly, Wang et al.55 reported that the antioxidant activity of honey samples was not significantly impacted by heat treatment. However, Turkmen et al.24 found that the antioxidant content could increase after thermal processing. Saric et al.58 found that the antioxidant activity of food could increase after heat treatments but sometimes does not cause any modifications. The Maillard reaction products (MRPs) produced during the heat treatments could explain the antioxidant changes observed.55 Manzocco et al.59 showed that the formation of non-nutrient antioxidants, such as MRPs, could compensate for the loss of natural antioxidants during heating.

3.5.3. Flavonoid Content

Table 6 also summarizes the effect of heat treatment at different temperatures and times of crystallized honey on the flavonoid content. The flavonoid content in all honey samples was significantly different, and their values varied from 12.18 mg/100 g to 12.93 mg/100 g. The total flavonoid content for samples treated at 90, 75, 60, and 45 °C was found to be 12.93.00 12.80, 12.53, and 12.18 mg/100 g, respectively, as compared with the control sample, which exhibited a value of 12.40 mg/100 g. These results agree with Turkut et al. (2018), who found that flavonoids increased with increasing temperature during heat treatment50 and agree fairly well with what was reported by Pimentel-Gonzalez et al.,56 but with lower values (3.5 mg/100 g to 6.4 mg/100 g), and with Elamine et al.57 results (14.52 mg/100 g to 23.70 mg/100 g). The explanation of the flavonoid content could be due to the positive correlation between polyphenols and flavonoids, where honey samples with low polyphenol content also yield also low flavonoid levels.60

3.6. Viscosity

One of the most important characteristics of honey is its viscosity because it affects honey quality and the design of processing equipment.61 Chemical constitution, temperature, and moisture content are all factors that impact the viscosity of honey.62,63 Viscosity is a parameter used in all stages of honey production, from extraction to packing.

Table 7 indicates the effect of heat treatment at different temperatures and times of crystallized honey on the viscosity. It is observed that the viscosity decreases as the temperature increases. The differences were significant and exhibited values from 0.48 to 0.62 (Pa·s). The value of viscosity in the investigated honey samples exhibits a value of 0.62 in control, 0.61, 0.57, 0.52, and 0.48 for samples heated at 45, 60, 75, and 90 °C, respectively. Kędzierska-Matysek et al.27 studied the effect of different temperatures on the viscosity of honey and found that dynamic viscosity decreases significantly with the increase in temperature, as expected. There is excellent agreement between the values of viscosity obtained from this work and those reported previously for honey samples heated at 10–40, 25–45, and 90 °C by Dobre et al.,64 Yanniotis et al.,62 and Chaikham et al.,43 respectively. Al-Mahasneh et al.65 found that wild-flower Jordanian honey exhibited non-Newtonian behavior. Other studies have observed Newtonian behavior in Jordanian honey. However, the Newtonian behavior observed in previous studies56,66 was most probably a result of honey-preheating treatments. Elevated temperatures beyond a certain threshold enhance the solubility of glucose, thereby impeding the crystallization process in honey, as explained by Zaizuliana et al. (2017). Paradoxically, however, these heightened temperatures possess the dual capacity to simultaneously reduce honey viscosity, facilitating the movement of crystal nuclei and thereby fostering the overall crystallization of honey. Consequently, it becomes apparent that an optimum temperature range exists, strategically poised to effectively promote the desired crystallization dynamics in honey.14

Table 7. Relationship between Temperature and Viscosity for Crystallized Honey Samples Treated with Different Temperatures.

treatment viscosity (mPa·s)
control 0.62 × 104 ± 0.01a
45 °C 0.61 × 104 ± 0.01b
60 °C 0.57 × 104 ± 0.01c
75 °C 0.52 × 104 ± 0.01d
90 °C 0.48 × 104 ± 0.01e
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All values are means of three observations and calculated on a wet basis. **Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05).

The presence of a structured network between honey and solid particles such as colloidal and crystalline compounds found in honey could explain the non-Newtonian behavior. Higher shear rates can break the network, resulting in a lower viscosity. Therefore, a shear-thinning non-Newtonian behavior is observed.65 The inverse relationship between viscosity and temperature is due to hydrodynamic forces associated with temperature increase and the decrease of molecular friction.67

3.7. Consumer Testing

Consumers’ opinions of honey samples are summarized in Table 8. Hedonic scale results showed some significant differences (P < 0.05) between all honey samples for overall impression, overall flavor, overall acidity, taste, viscosity, and crystallization. The results of consumer testing showed that higher values of overall impression were found in the control sample, followed by samples heated at 45, 60, 75, and 90 °C. The highest values of overall aroma are found in the control sample. Its value for samples heated at 45, 60, 75, and 90 °C is considerably less. The highest value of overall flavor is found in the control sample. Its value for samples treated at 45, 60, 75, and 90 °C is relatively lower. The highest value of color is found in the control sample. Samples heated at 45, 60, 75, and 90 °C exhibit lower values. Furthermore, the highest value of viscosity is found in the control sample, and samples treated at 45, 60, 75, and 90 °C exhibit lower values. Table 9 shows just about the right scale of the effect of heat treatment on the honey samples. The highest value of flavor is found in samples treated at 90 and 75 °C, and its values are statistically significant when compared to the flavor score at 60, 45 °C, and the control sample. The higher value of color was found at 90 and 75 °C, followed by significant decreases for samples heated at 60 and 45 °C, as well as for the control sample. Turkut et al. (2018) found similar results when they used color measurements relying on the Hunter L*, a*, and b* scales. The L*, a*, and b* values consistently decreased across all examined samples, suggesting that the color of honey was progressively shifting toward black, green, and blue as a result of the heating process.50 Moreover, the highest value of astringency is found in the control sample. It decreases for samples heated at 45, 60, 75, and 90 °C. Inan et al. (2012) studied the sensory properties of thermally treated honey samples. Their sensory analysis showed that the samples differed significantly in color, odor, taste, and consistency. Honey samples heated at 75 °C had the highest scores for all attributes, followed by samples treated at 65 °C.68

Table 8. Consumer Scores of Effect of Heat Treatment on Crystallized Honey Samples.

temperature overall impression overall aroma overall flavor color viscosity
control 8.23 ± 0.03a 8.86 ± 0.12a 8.73 ± 0.20a 8.82 ± 0.30a 7.25 ± 0.20a
45 °C 7.88 ± 0.08b 8.70 ± 0.30ab 8.71 ± 0.40a 8.75 ± 0.10ab 7.33 ± 0.30a
60 °C 7.75 ± 0.05b 8.67 ± 0.13b 7.86 ± 0.10b 8.13 ± 0.60bc 7.55 ± 0.50a
75 °C 7.20 ± 0.20c 7.30 ± 0.10c 7.53 ± 0.20b 7.93 ± 0.40c 7.11 ± 0.40a
90 °C 6.20 ± 0.10d 6.38 ± 0.20d 6.25 ± 0.40c 7.23 ± 0.30d 6.13 ± 0.50b
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**Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05). Hedonic scales: 1 = dislike extremely, 2 = dislike very much, 3 = dislike moderately, 4 = dislike slightly, 5 = neither like nor dislike, 6 = like slightly, 7 = like moderately, 8 = like very much, and 9 = like extremely.

Table 9. Just about the Right Scale of the Effect of Heat Treatment on Crystallized Honey Samples.

temperature flavor color astringency
control 2.95 ± 0.40b 2.96 ± 0.20b 3.57 ± 0.10a
45 °C 2.96 ± 0.28b 3.15 ± 0.15b 3.26 ± 0.26ab
60 °C 3.16 ± 0.50b 3.22 ± 0.20b 3.13 ± 0.30ab
75 °C 3.80 ± 0.20a 3.49 ± 0.50ab 2.89 ± 0.20b
90 °C 4.23 ± 0.20a 3.86 ± 0.40a 2.71 ± 0.50b
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All values are means of three replicates and are calculated on a wet basis. **Means ± SD in the same column with the same letter are not significantly different (P ≤ 0.05). Hedonic scales for flavor: 1 = too much weak cooked, 2 = too weak cooked, 3 = just about right, 4 = too cooked, and 5 = much too cooked. Hedonic scales for color: 1 = too much light, 2 = too light, 3 = just about right, 4 = too dark, 5 = much too dark. Hedonic scales for astringency: 1 = much too acidic, 2 = too acidic, 3 = just about right, 4 = too acidic, and 5 = much too acidic.

4. Conclusions

Results of this study demonstrate that the amounts of pH, ash, moisture, HMF, color, glucose, and fructose content, total phenol, antioxidant activity, total flavonoid, viscosity, and sensory evaluation are affected by heat treatment at different temperatures. The moisture content is found to decrease significantly in honey samples treated under different thermal conditions. However, the ash content shows no variation for different honey samples. The pH values are found to slightly decrease, while acidity increases as the temperature increases. Other key parameters, such as the HMF content, fructose, and glucose content, are found to decrease in honey samples. In addition, color measurements show significant variation among honey samples treated at different temperatures. Total phenols and flavonoid content of honey samples are found to increase, while antioxidant activity decreases as the temperature varies. Viscosity exhibits Newtonian behavior and is found to decrease as the temperature is increased. Interestingly, sensory attributes were found to vary significantly among honey samples treated at different temperatures. Strikingly, the honey samples treated at 75 °C showed the best physicochemical and sensory properties. The main recommendation of this study is based on honey samples treated at 75 °C since they are found to exhibit optimized physical and chemical properties.

Acknowledgments

The support provided by the Deanship of Research (552-2020) at Jordan University of Science and Technology is appreciated. The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R502), King Saud University, Riyadh, Saudi Arabia for funding this project.

Data Availability Statement

Data are available online at the following link: https://data.mendeley.com/datasets/j4dmwtw5by/1.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data are available online at the following link: https://data.mendeley.com/datasets/j4dmwtw5by/1.

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