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3COMPREHENSIVE METHODOLOGY FOR ANALYZING PHYSICOCHEMICAL AND BIOFUNCTIONAL PROPERTIES OF HONEY FROM DIVERSE BOTANICAL ORIGINS
1Hyeonjeong Jang, 2Sukjun Sun, 3Sampat Ghosh, 4Chuleui Jung
1,2Department of Plant Medicals, Andong National University, Republic of Korea
3,4Agriculture Science and Technology Research Institute, Andong National University,
Republic of Korea https://doi.org/10.5281/zenodo.11428629 Abstract. Honey, a natural food produced by honey bees from nectar, has chemical properties that are primarily influenced by botanical origins. This paper aims to detail the main methodologies to analyze the physicochemical and biofunctional properties of honey from various floral sources. The methods employed are based on established standards from AOAC, Codex Alimentarius and Honey Commission, among others. In this study, we present the methodologies and findings for measuring honey's moisture content, sugar composition, HMF levels, carbon isotope ratio, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities using DPPH and ABTS assays.
Key words: hydroxymethylfurfural, antioxidant activity, radical, scavenging activity.
1. Introduction. Honey (mainly blossom honey) is produced by honey bees (Apis mellifera) from the nectar of flowers (Pita-Calvo et al., 2017). Bees collect and convert the nectar, then deposit, dehydrate and store it in honeycombs to ripen and mature (Solayman et al. 2016). This organic product contains more than 180 biochemical substances, including sugars, water, minerals, phenolic compounds, amino acids, vitamins, organic acids and enzymes (Scripca and Amariei, 2021). Its chemical properties, both physicochemical and biofunctional, are mainly influenced by pre-harvest factors such as geographical and botanical origin (Kang et al., 2023).
Honey produced by bees predominantly from the nectar of a single type of flower is called monofloral honey and has several distinct characteristics (Machado et al., 2020). The consumer demand of monofloral honey has increased due to its taste and pharmacological features, raising its market value in recent decades (Soares et al., 2017). Korean beekeepers produce, process, and sell primarily acacia, chestnut, jujube, citrus, rapeseed, buckwheat, clover, and snowbell honey (Kim et al., 2014). Numerous domestic studies have reported on the chemical composition and functional properties of these honey (Kim et al., 2014; Jung and Chon, 2016; Jung et al., 2017; Sung et al., 2018).
However, it is crucial to analyze honey using standardized methodologies to accurately assess its physicochemical and biofunctional properties. Therefore, this paper aims to describe the standard methodologies adopted for physicochemical and biofunctional analysis and to review these characteristics of honey from different floral origins based on available scientific literature.
2. Physicochemical analysis
2.1.1. Methodology. The most commonly used method for measuring honey moisture is the refractometric method, which relies on the increase in refractive index with solid content. Moisture content is measured using a refractometer without diluting the honey. The moisture content is expressed as g/100g (Machado et al., 2020).
2.1.2. Reviewed paper. Moisture content is a critical physicochemical parameter of honey. Honey with more than 18% of its moisture is likely to ferment, while honey with less than 15%
has a higher chance of granulating over time (Singh and Singh, 2018). Several factors affect moisture content, including edaphic and climatic conditions, season of harvesting, manipulation by beekeepers, processing/storage conditions, and geographical and botanical origin etc. (Macchado De-Melo et al., 2018). Table 1 (adopted from Jung and Chon, 2016) presents the moisture contents of Korean honey samples from three floral origins: acacia, chestnut, and multiflora. Acacia honey had the highest moisture content (21.4%), followed by chestnut (19.9%) and multiflora honey (19.4%).
2.2. Sugar
2.2.1. Methodology. There are several methods for determining sugar content in honey, including HPLC (high performance liquid chromatography), GC (gas chromatography), CE (capillary electrophoresis), and FTIR (Fourier transform infrared spectroscopy) etc. However, HPLC with RID (refractive index detection) is most commonly used method (Machado et al., 2020).
For sample preparation, 1 g of pure honey is dissolved in 100 mL of water and vortexed until fully diluted. The diluted honey is then filtered using a syringe filter. Sugar content in honey is estimated using an HPLC equipped with a column containing amine-modified silica gel (250 x 4.6 mm, 0.5 p,m particle size). The mobile phase consists of acetonitrile and water in a 75:25 ratio, with a flow rate maintained at 1.0 mL/min. The sample injection volume is 10 ^L, and the column temperature is set to 40 °C. A stock solution is prepared by mixing 1 g each of fructose, glucose, and sucrose in 100 mL of distilled water (10,000 ppm). The calibration curve is generated within the concentration range of the samples by appropriate dilution from the stock solution. Sugar content is expressed as g/100g (Jalaludin and Kim, 2021).
2.2.2. Reviewed paper
Sugars in honey are produced by the honey bees through the enzymatic conversion of sucrose in nectar of flower. Honey consists of about 80% sugars, of which 70% are monosaccharides and 10% is disaccharides (Ouchemoukh et al., 2010). Table 1 (adopted from Jung and Chon, 2016), reports the contents of fructose, glucose, inverted sugar, F/G ratio, and sucrose. Significant differences were observed in the amounts of fructose, glucose and sucrose in honey among the different floral origins. The F/G ratio were 1.49, 1.75, 1.34 for acacia, chestnut and multiflora honey respectively. The F/G ratio is an indicator of honey crystallization, as fructose is more soluble in water than glucose (Sohaimy et al., 2015).
2.3. HMF (Hydroxymethylfurfural)
2.3.1. Methodology. The most commonly used method for HMF determination is HPLC with DAD (diode array detector). Before injection into HPLC, 5 g of honey is mixed with 50 mL water and vortexed for complete dilution. The diluted honey is then filtered using a syringe filter. HMF content is determined using an HPLC equipped with reverse-phase column (250 x 4.6 mm, 0.5 p,m particle size). The mobile phase consists of water and methanol in a 9:1 ratio, with a flow rate of 1.0 mL/min. The detector wavelength is set at 280 nm. The injection volume is 20 ^L, and the column temperature is set to 40 °C. For a stock solution, 100 mg of HMF is mixed with 100 mL of distilled water (1,000 ppm). The calibration curve is generated by appropriate dilution of the stock solution within the concentration range of the samples. HMF content is expressed as mg/kg (Khalil et al., 2010).
2.3.2. Reviewed paper. HMF is formed by the acid-catalysed dehydration of hexoses. Its content in honey increases during heating and storage, making HMF a parameter for assessing honey freshness and detecting adulteration (Zappala et al., 2005). HMF content is listed in Table
1 (adopted from Jung and Chon, 2016). Significant difference was observed among the honey samples. Multiflora honey had a higher HMF content (7.4 mg/kg) compared to chestnut (4.7 mg/kg) and acacia honey (4.9 mg/kg) .
2.4. Stable carbon isotope ratio
2.4.1. Methodology. According to AOAC Official Method 998.12 (1999), 1 mg of honey is wrapped in a tin capsule with minimal air and placed in the automatic sampler of an EA-IRMS (elemental analyzer-stable isotope ratio mass spectrometer). After complete combustion of the samples at 1020 °C in the oxidation/reduction reactor, detection is conducted for 300 seconds. The flow rates of gases are 90 mL/min for helium (carrier), and 250 mL/min for oxygen (Vasic et al., 2020).
2.4.2. Reviewed paper. 13C/12C isotope ratio distinguishes natural honey from sugar-contaminated honey. Sugars in honey are synthesized through the C3 cycle, which first fixate CO2 into a 3-carbon compound during photosynthesis. In contrast, sugars in corn and sugarcane are biosynthesized via the C4 cycle, which fixes CO2 into a 4-carbon compound. The origin of sugars in honey can be determined by their stable carbon isotope ratios: -32 and -21 %% for C3 plants and -19 and -12 % for C4 plants (£inar et al., 2014). Carbon isotope ratio of different honey is depicted in Table 1 (adopted from Jung and Chon, 2016). The carbon isotope ratio of chestnut honey was lower (-24.4 %) compared to acacia (-23.4 %) and multiflora honey (-23.2 %), consistent with C3 plant characteristics.
3. Biofunctional analysis
3.1. TPC (total phenolic content) and TFC (totalflavonoid content)
3.1.1. Methodology. Quantitative estimation of TPC and TFC in honey can be performed using colorimetric assays. TPC is determined by the reaction with Folin-Ciocalteu reagent, and TFC is evaluated using AlCh.
For total phenolic content, 5 g of honey is dissolved in 50 mL of distilled water and shaken for complete dilution. 100 ^L of the solution (corresponding to 10 mg of raw honey) is added to 1 mL of 10% Folin-Ciocalteu reagent, and vortexed for 2 min. After 20 min incubation at room temperature, 200 ^L of the mixture is placed in a 96-well microplate, and absorbance is measured at 750 nm using spectrophotometer. Distilled water is used as a control. For a stock solution, 0.1 g of gallic acid is added to 100 mL of water (1,000 ppm). The calibration curve is generated by appropriate dilution of the stock solution within the concentration range of the samples. Total phenolic content is expressed as g gallic acid equivalents/100g of honey (mg GAE/100 g) (Bertoncelj et al., 2007).
For total flavonoid content, 500 p,L of the solution is mixed with 500 ^L of 2% AlCh. After 1 h at room temperature, 200 ^L of the mixture is put in a 96-well microplate, and absorbance is measured at 420 nm. Distilled water is used as a control. For stock solution, 100 mg of quercetin is mixed with 100 mL of 70% ethanol (1,000 ppm). The calibration curve is generated by appropriate dilution from the stock solution within the concentration range of the samples. Total flavonoid content is expressed as g quercetin equivalents/100g of honey (g QE/100g) (Kumazawa et al., 2004).
3.1.2. Reviewed paper. Phenolic compounds, including phenolic acids (non-flavonoids) and flavonoids, contribute to the antioxidant capacity of honey. There are 16 classes of phenolic compounds based on their carbon chain structure. Flavonoids, a class of low molecular weight phenolic compounds, are mainly present in honey as flavonols, flavanone, and flavones (Zawawi et al., 2021). The antioxidant capacity of honey is correlated with its polyphenol content (Alvarez-
Suarez et al., 2009). Table 2 (adopted from Sung et al., 2018) illustrates the TPC and TFC of Korean honey from 4 botanical origins: acacia, chestnut, multiflora and jujube. Chestnut honey contained the highest TPC (77.7 mg/100g), and acacia honey contained the lowest TPC (9.5 mg/100g). There was no significant difference in TFC among the different honey. Several studies report that the phenolic composition and the antioxidant activity in honey vary widely depending on the season, environmental conditions and floral sources (Al-Mamary et al., 2002; Yao et al., 2003; Dong et al., 2013).
3.2. Assay of DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,20-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid diammonium salt]
3.2.1. Methodology. For determination of free radical scavenging activity by DPPH assay, 750 p,L of the solution is added to 1.5 mL of 0.002% DPPH* solution. After 15-minute incubation at room temperature, 200 ^L of the mixture is places in a 96-well microplate, and absorbance is measured at 517 nm. Distilled water is used as a control. For a stock solution, 0.1 g of trolox is added to 100 mL of distilled water (1,000 ppm). The calibration curve is generated by appropriate dilution of the stock solution within the concentration range of the samples. The DPPH scavenging activity is calculated using following equation:
DPPH scavenging activity (%) As**™;
where Asample and Acontrol are the absorbance of sample and control. Results are expressed as |iM of trolox equivalents/g of honey (^mol TE/g of honey) (Chua et al., 2013). To estimate radical scavenging activity using ABTS assay, ABTS^+ solution is prepared by mixing 7mM ABTS solution and 2.5mM K2S2O8 in 1:1, and keeping it in the dark condition for 16 h at room temperature. ABTS^+ solution is diluted with distilled water to achieve an absorbance of 0.70 to 0.80 at 734 nm. 10 ^L of the honey solution is mixed with 990 p,L of diluted ABTS^+ solution. After immediate mixing, 200 p,L of the mixture is placed in a 96-well microplate, and absorbance is measured at 734 nm. Distilled water is used as a control. The preparation of the standard curve for trolox is the same as for the DPPH assay (Machado et al., 2020).
3.2.2. Reviewed paper. Antioxidant capacity can be explained by radical scavenging capacity. This is a process by which an oxidant obtains an electron from an antioxidant. The DPPH assay estimates that the antioxidants in honey decrease the free radical DPPHV ABTS assay determines based on the ability of a compound to scavenge the stable ABTS radical cation (ABTS^+). ABTS^+ is generated by reacting ABTS with K2S2O8. The two assays are so simple and efficient that it has become the most commonly used for antioxidant capacity (Alvarez-Suarez et al., 2009). Table 2 (adopted from Sung et al., 2018) shows scavenging activity of DPPH and ABTS in honey samples from different sources. In chestnut honey, scavenging activity of DPPH and ABTS were higher than other honey. ABTS scavenging activity had significantly higher value than DPPH scavenging activity. Since, DPPH radical reacts only with lipophilic antioxidants, but ABTS^+ radical reacts with hydrophilic and lipophilic antioxidants (Prior et al., 2005).
Conclusion. This study highlights the significant variations in physicochemical and biofunctional properties of honey from different botanical origins. Using standard methodologies, we found notable differences in moisture content, sugar composition, HMF levels, carbon isotope ratios, and antioxidant properties among the honey samples. Acacia honey had the highest moisture content, while chestnut honey exhibited the highest total phenolic content and antioxidant capacity. The carbon isotope analysis effectively detected adulteration, differentiating between C3 and C4 plant origins. These findings underscore the importance of botanical origin in determining
honey's quality and therapeutic potential, offering valuable insights for quality control and
commercial valuation of honey.
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Table 1. Physicochemical properties of Korean honey from 3 floral origins. All data were
adopted from Jung and Chon, 2016
n Moisture (%) Fructose (%) Glucose (%) Inverted sugar (%) F/G Sucrose (%) HMF (mg/kg) Carbon isotpe ratio (%»)
Acacia 2987 21.4±2.4 41.9±2.5 28.1±1.8 70 1.49 2.4±1.0 4.9 23.4±2.7
Chestnut 97 19.9±1.7 41.9±3.4 24.0±2.2 65.9 1.75 2.7±1.0 4.7 24.4±0.5
Multiflora 712 19.4±1.7 38.7±2.9 28.8±2.1 67.5 1.34 2.7±0.7 7.4 23.2±2.8
Table 2. Biofunctional properties of Korean honey from 3 floral origins. All data were adopted from Sung et al., 2018. * SA means Scavenging Activity
n TPC (mg/100g) TFC (mg/100g) DPPH SA* (%) ABTS SA (%)
Acacia 47 9.5±5.4 1.8±1.6 3.2±2.1 9.2±2.3
Chestnut 17 77.1±16.9 8.4±3.0 10.5±2.7 32.9±3.9
Multiflora 42 41.3±24.8 6.7±6.4 5.5±4.7 19.6±9.3
Jujube 5 55.1±15.7 8.7±7.3 8.1±3.0 25.3±5.7
