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Effect of heat treatment and storage conditions on mead composition
Food Chemistry 219 (2017) 357–363
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Effect of heat treatment and storage conditions on mead composition David Kahoun a,⇑, Sonˇa Rˇezková b, Josef Královsky´ b a b
Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia in Cˇeské Budeˇjovice, Branišovská 1760, 37005 Cˇeské Budeˇjovice, Czech Republic Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic
a r t i c l e
i n f o
Article history: Received 22 April 2016 Received in revised form 23 September 2016 Accepted 26 September 2016 Available online 28 September 2016 Keywords: Mead Phenolic compounds 5-Hydroxymethylfurfural Antioxidant activity Heat treatment Storage conditions HPLC Electrochemical detection
a b s t r a c t The effects of heat treatment and storage conditions on the composition of pure mead (honey wine) made from only honey and water were investigated. Heat treatment experiments were performed at 7 different temperatures ranging from 40 °C to 100 °C with 10 °C increments for 60 min. Storage condition experiments were performed at room temperature (20–25 °C) in daylight without direct sunlight and in darkness in a refrigerator at 4 °C for 1, 2, 4 and 12 weeks. The parameters evaluated were phenolic compounds, peak area of unidentified compounds, 5-hydroxymethylfurfural content and antioxidant capacity. Significant changes in compound content were observed in the case of 6 identified compounds and 9 unidentified compounds. However, the antioxidant activity was not affected by the heat treatments or storage at room temperature. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Mead, or honey wine, is a fermented alcoholic beverage made from bee honey and water. The traditional production process includes the heating or boiling of honey must (unfermented solution of honey and water) before its fermentation for the purpose of protein removal and microorganism elimination (Ramalhosa, Gomes, Pereira, Dias, & Estevinho, 2011). This step reduces the risk of uncontrolled fermentation but it may also cause the degradation of thermolabile compounds e.g. some phenolic acids and flavonoids originate from honey (Escriche, Kadar, Juan-Borrás, & Domenech, 2014). Another negative consequence of these treatments is the potential increase of 5-hydroxymethylfurfural content, as is known in the case of the heat treatment of honey (Tosi, Ciappini, Ré, & Lucero, 2002). With respect to these facts, new, gentle mead production processes without heat treatment have been developed. The chemical composition of various types of mead was evaluˇ ezková, Veškrnová, ated in a comprehensive study of Kahoun, R Královsky´, and Holcˇapek (2008) focused on the content of 25 phenolic compounds and 5-hydroxymethylfurfural in 50 meads obtained from 14 producers. The results of the study confirmed the strong effect of ingredients added to mead (fruit juices, herbal ⇑ Corresponding author. E-mail address: [email protected] (D. Kahoun). http://dx.doi.org/10.1016/j.foodchem.2016.09.161 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
extracts, spices etc.) on their phenolic compound profile and big differences in 5-hydroxymethylfurfural content ranging from 2.74 to 157 mg/L. Similar results were also published by Švecová, ˇ ová and Hájek Bordovská, Kalvachová, and Hájek (2015) and Ben (2010); in their studies they showed the benefits of HPLC with coulometric array detection for the determination of phenolic compounds in wines, meads, and Japanese knotweeds. Socha, Paja˛k, Fortuna, and Buksa (2015) evaluated the influence of mead type and the effect of various mead ingredients on phenolic compound profile and antioxidant activity. A principal component analysis (PCA) statistical method was used in order to differentiate meads in terms of phenolic compounds content. It is very likely that the composition and content of phenolic compounds in meads are influenced not only by the ingredients added, but also by technological processes, such as the means of honey must preparation (Wintersteen, Andrae, & Engeseth, 2005), fermentation, maturing, storage in bottles, warming just before consumption etc. because these effects have been observed in the case of other similar fermented beverages such as wines (Fernández de Simón et al., 2014; García-Falcón, Pérez-Lamela, Martínez-Carballo, & Simal-Gándara, 2007; Mateˇjícˇek, Mikeš, ˇ , 2005) or beers (Vanbeneden, Gils, Klejdus, Šteˇrbová, & Kubán Delvaux, & Delvaux, 2008). The presumption made for this research is that meads made under cold conditions contain different number of phenolic and other electroactive compounds than meads made in the traditional
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way, and that some of these compounds may be damaged by the unsuitable storage of mead in glass bottles or by its warming just before consumption (Ioannou & Ghoul, 2012). Potential changes in the chemical composition of meads may be accompanied by the formation or elimination of a compound, which could then be used as a marker of these treatments. The objective of this study was to evaluate the effects of heat treatment and storage conditions on the composition of cold-made mead using HPLC with coulometric-array detection (for electroactive compounds), HPLC with UV detection (for 5-hydroxymethylfurfural) and UV/VIS spectrophotometry (for antioxidant activity). No study has been published so far on this topic.
2. Experimental 2.1. Chemicals Standard phenolic compounds (HPLC purity) were obtained from the 2 following sources: Fluka (Buchs, Switzerland): gallic acid (3,4,5-trihydroxybenzoic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), a-resorcylic acid (3,5-dihydroxybenzoic acid), homoprotocatechuic acid (3,4-dihydrophenylacetic acid), protocatechuicaldehyde (3,4dihydroxybenzaldehyde), b-resorcylic acid (2,4-dihydroxybenzoic acid), 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, vanillic acid (4-hydroxy-3methoxybenzoic acid), esculetin (6,7-dihydroxycoumarin), caffeic acid (3,4-dihydroxycinnamic acid), (+)-catechin hydrate (trans3,30 ,40 ,5,7-pentahydroxyflavane), vanillin (4-hydroxy-3-methoxybenzaldehyde), isovanillin (3-hydroxy-4-methoxybenzaldehyde), chlorogenic acid hemihydrate (3-O-(3,4-dihydroxycinnamoyl)-Dquinic acid), p-coumaric acid (trans-4-hydroxycinnamic acid), m-coumaric acid (trans-3-hydroxycinnamic acid), ethylvanillin (3-ethoxy-4-hydroxybenzaldehyde), sinapic acid (3,5-dimethoxy4-hydroxycinnamic acid) and o-coumaric acid (trans-2-hydroxycinnamic acid). Sigma-Aldrich (St. Louis, MO, USA): gentisic acid (2,5-dihydroxybenzoic acid), syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid), ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid) and isoferulic acid (3-hydroxy-4-methoxycinnamic acid). Standards of 5-hydroxymethylfurfural and Trolox ((±)-6-Hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid), both HPLC purity, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol, LiChrosolv gradient grade, were obtained from Merck (Darmstadt, Germany). Formic acid (98–100%), sodium acetate trihydrate (99%), acetic acid (98%), potassium dichromate (99%) and ABTS (2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) tablets were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate (99.995%) was obtained from Fluka (Buchs, Switzerland). Water was distilled in glass and purified using a Mili-Q water purification system (Bedford, MA, USA). 2.2. Standard solutions The standards of the phenolic compounds were dissolved in a mixture of water and methanol (1:1, v/v) to obtain 100 mg/L stock solutions and filtered through the 0.2 lm PTFE filter. The stock solutions were stored in dark brown glass vials at 4 °C in darkness for a maximum of one week. Calibration solutions ranging from 0.025 mg/L to 2.5 mg/L were prepared by diluting the stock solutions with the mobile phase A in the desired volume ratios and 10 lL of each prepared calibration solution was immediately injected to HPLC-ECD.
The standard of 5-hydroxymethylfurfural was dissolved in a mixture of water and methanol (9:1, v/v) to obtain 400 mg/L stock solution and filtered through the 0.2 lm PTFE filter. The stock solution was stored in dark brown glass vials at 4 °C in darkness for a maximum of one week. Calibration solutions ranging from 0.5 mg/L to 18 mg/L were prepared by diluting the stock solutions with aqueous methanol (9:1, v/v) in the desired volume ratios and 10 lL of each prepared calibration solution was immediately injected to HPLC-UV. The standard of Trolox stock solution (0.004 lmol/lL) was prepared freshly before any analysis by dissolution in methanol. Calibration solutions ranging from 0.005 lmol/25 lL to 0.05 lmol/25 lL were prepared by dilution of stock solutions with the methanol in the desired volume ratios and 25 lL of each prepared calibration solution was immediately measured using a UV/Vis spectrophotometer. 2.3. Mead samples 2.3.1. Production process The production process for pure mead, cold-made (without heating or boiling of honey must) from bee honey and water only, is the trade secret of JANKAR PROFI company, and thus can be described in general terms only. Honey (approx. 2200 kg) was gently preheated using a hot-air dryer at max. 40–42 °C and then mixed with water (approx. 3000 dm3) in a 5000 dm3 stainless steel fermentation tank until the appropriate value of refractometric dry solids was reached. This honey solution was inoculated with 2.5 kg of the lyophilized yeast Saccharomyces cerevisiae and fermented at 18–20 °C for 5–6 weeks until the appropriate value of refractometric dry solids was reached. During the turbulent fast fermentation, the mead was cooled to keep the temperature below 18–20 °C. Fermentation continued to completion at 16 °C for 4 weeks. The mead was then removed by suction and matured at 12–14 °C for 1 year. After maturing, primary filtration was done using a diatomaceous earth filter followed by fine filtration using a membrane filter with a 0.45 lm pore size. Finally, the mead was bottled under nitrogen atmosphere, the bottles closed with corks, and the bottle necks covered with molten wax. 2.3.2. Mead samples The samples of fresh pure mead were obtained in two clear glass bottles from JANKAR PROFI company. Both samples (bottles) were made in the same batch. One bottle of mead was used for the evaluation of the effect of heat treatment and the other for the evaluation of the effect of storage conditions. The bottles were stored at 4 °C in darkness for a maximum of one week. The effect of heat treatment was tested using a screw Duran Premium bottle (100 mL) with 25 mL of mead sample. The bottle was placed into a water bath at 7 different temperatures ranging from 40 °C to 100 °C with 10 °C increments for 60 min. Then, the bottle was taken out and cooled using a stream of cold water. The effect of storage conditions was tested using an Erlenmeyer flask (250 mL) with 200 mL of mead sample at room temperature (20–25 °C) in daylight (without direct sunlight) and in darkness in a refrigerator at 4 °C for 1, 2, 4 and 12 weeks. The 12 weeks’ cut-off time was chosen on the basis of the manufacturer’s recommendation because this is the average period from bottling by the producer to purchase by the customer. 2.4. Instrumentation An HPLC-ECD system for phenolic compound analysis was set up. It consisted of a vacuum degasser DG 3014 (Ecom, Prague, Czech Republic), two chromatographic pumps model 582
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(ESA, Chelmsford, MA, USA) a CoulArray thermostatic organizer (ESA, Chelmsford, MA, USA) containing: a pulse damper, a gradient mixer, a manual injector with 10-lL sampling loop (Rheodyne, Cottati, CA, USA); an electrochemical 8-channel CoulArray 5600A detector (ESA, Chelmsford, MA, USA) and a PC with a CoulArray software for data acquisition, processing and analysis (ESA, Chelmsford, MA, USA). Chromatographic column Gemini C18 (150 3 mm I.D., 3 lm particle size) was obtained from Phenomenex (Torrence, CA, USA). Hold-up volume (0.59 mL) was determined using uracil as the non-retained marker solute. 5-Hydroxymethylfurfural analysis was carried out using a HPLC-UV system Hewlett-Packard model HP 1090M (Waldbronn, Germany) equipped with a diode array detector and an autosampler. Chromatographic column LiChrospher 60 RP-select B (250 4 mm I.D., 5 lm particle size) was obtained from Merck (Darmstadt, Germany). Hold-up volume (3.18 mL) was determined using uracil as the non-retained marker solute. Antioxidant activity was measured using single beam VIS spectrophotometer Thermo model Spectronic Helios d (Madison, WI, USA) equipped with 10-mm optical path length glass cuvettes. 2.5. HPLC analysis of phenolic compounds All chromatographic separations were carried out at 35 ± 0.1 °C using a gradient elution with mobile phases A and B. The mobile phase A was 5 mM ammonium acetate in water, the pH value of which was adjusted to 3.0 by adding formic acid (1000 lL/L). The mobile phase B was a mixture of mobile phase A and acetonitrile at 1:2 (v/v) and pH value was adjusted to 3.0 by the addition of formic acid (600 lL/150 mL). The mobile phases were filtered through the 0.45 lm filter and degassed (for 10 min) by ultrasonication before use. The applied gradient programme was: 0–27 min, 2% B isocratic; 27–72 min, linear gradient from 2% to 7% B; 72–108 min, linear gradient from 7% to 25% B; and finally, washing and reconditioning of the column was done. The flow rate was 0.45 mL/min and the injection volume was 10 lL. Electroactive compounds were monitored and quantified using eight electrochemical cells with applied potentials from 200 to 900 mV (100 mV increment). Mead samples were diluted with the initial mobile phase at 1:1, filtered through the 0.2 lm PTFE filter only and 10 lL was immediately injected to HPLC. No other sample pre-treatments were used. Results were expressed as an amount (mg) of an analyte per volume (L) of a sample. 2.6. HPLC analysis of 5-hydroxymethylfurfural All chromatographic separations of 5-hydroxymethylfurfural were carried out at laboratory temperature using the isocratic elution with the mobile phase consisting of water and methanol (9:1 v/v). The mobile phase was filtered through the 0.45 lm filter and degassed (for 10 min) by ultrasonication before use. The flow rate was 1.0 mL/min and the injection volume was 10 lL. 5-Hydroxymethylfurfural was determined using a diode array UV detector at 285 nm. Mead samples were diluted with the initial mobile phase at 1:1, filtered through the 0.2 lm PTFE filter only and 10 lL was immediately injected on to the HPLC. No other sample pre-treatments were used. Results were expressed as an amount (mg) of 5-hydroxymethylfurfural in a volume (L) of a sample. 2.7. Determination of antioxidant activity Antioxidant activity was determined using the ABTS method. A tablet of ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) containing 10 mg of ABTS was dissolved in a 10 mL
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volumetric flask with 5 mL of water and then 100 lL of aqueous solution of 17.24 mg/mL potassium dichromate was added. A volumetric flask was wrapped with aluminium foil and kept at laboratory temperature in darkness for 16 h. Before analysis, this ABTS stock solution was diluted with the acetate buffer (pH 4.3) in a 10 mL volumetric flask at 1:39 (v/v) and the ABTS working solution was immediately used for measurement. At first, the absorbance (AABTS) of this ABTS working solution at 734 nm was measured. Afterwards, 2 mL of ABTS working solution and 25 lL of a sample solution (or a Trolox calibration solution) were mixed in a vial, and then the absorbance (Asample) at 734 nm was measured. The decrease in absorbance (DA, %) was calculated according to the equation: DA = 100 ((AABTS Asample)/AABTS). Results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC, lmol/mL). This index describes the amount (lmol) of Trolox in which the antioxidant capacity is equivalent to the antioxidant capacity of a volume (mL) of a sample. 2.8. Methods validation All chromatographic measurements were done using our optimized and validated analytical methods (Kahoun et al., 2008). Validation of the ABTS method used for the determination of antioxidant activity includes the assessment of the following validation parameters. Linearity was assessed from the calibration curves obtained at 7 concentration levels. Decreases in absorbance (DA, %) were plotted versus theoretical amounts of Trolox (x, lmol) and the calibration curve DA = (1658 ± 17)x + (2.50 ± 0.47) was obtained from a least-squares regression analysis. The limit of detection (0.0043 lmol Trolox) and limit of quantification (0.0067 lmol Trolox) were determined according to ISO 11843-2 by statistical software QC Expert. All measurements were performed in triplicate and results are expressed as an arithmetic mean ± 2 standard deviation. 3. Results and discussion 3.1. Effect of heat treatment 3.1.1. Effect of heat treatment on electroactive compounds content Electroactive compound content was evaluated by comparing the peak areas of all detected compounds in chromatograms of the fresh mead and the mead after 100 °C/60 min heat treatment (Fig. 1). Changes in peak areas were registered in 16 peaks and the trend of each change is marked with appropriate arrows. All 7 identified phenolic compounds (gallic acid, protocatechuic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, syringic acid and p-coumaric acid) and two unidentified compounds (B and X) were chosen for more detailed evaluation. Concentrations of phenolic compounds in fresh mead ranged from 0.159 ± 0.002 to 0.466 ± 0.007 mg/L which is consistent with previously reported ˇ ová & Hájek, 2010; Kahoun et al., 2008; Švecová results (Ben et al., 2015). Gallic acid content increased by 32%, changes in protocatechuic acid content were almost negligible (decrease of 5%), 4-hydroxyphenylacetic acid content after 100 °C/60 min heat treatment could not be evaluated because of coelution with an unidentified compound, hereafter refer to as compound X, vanillic acid content decreased by 16%, caffeic acid content decreased by 22%, p-coumaric acid content decreased by 30%. p-Coumaric acid was also evaluated within research focused on the effect of 2 different heat treatments (60 °C and boiling) of honey must on mead composition (Wintersteen et al., 2005). The influence of hightemperature heat treatment was observed only in the case of buckwheat mead (concentration increased from 1.94 ± 0.20 mg/L to 2.49 ± 0.16 mg/L), but in the case of soya mead concentration
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at 100 °C for 1 h and at 100 °C for 8 h and the peak areas of compound X were compared. Both analyses gave almost the same peak area (difference 6%) and this result increased the probability that compound X is a degradation product of compound B. On the condition that compound B is a naturally occurring compound always present in honey, or that this compound is always formed during mead production, it could potentially be used as a marker of the heat treatment (temperature over 70 °C) of mead. However, since the chemical composition of a honey, even from the same floral source, depends on the year of its production, and on the region, due to different climatic and geographical factors, respectively (Al et al., 2009; Juan-Borrás, Domenech, Hellebrandova, & ˇ eksteryte˙, 2010), the Escriche, 2014; Kaškoniene˙, Venskutonis, & C suitability of the marker needs to be proven using either fresh, cold made, pure mead from one producer from different years, or using mead obtained from various producers.
Fig. 1. HPLC-ECD chromatogram of the fresh mead (A) and the mead after 100 °C/60 min heat treatment (B). Symbol " represents content increase and symbol ; represents content decrease.
1.06 ± 0.03 mg/L was not changed (1.06 ± 0.08 mg/L). The results of the measurements of the fresh mead and the mead after all 7 heat treatments are listed in Table 1. A detailed evaluation of heat treatment on two unidentified compounds B and X showed interesting findings. Heat treatment with the range 90–100 °C/60 min caused total degradation of the unidentified compound marked as B and the formation of the new unidentified compound marked as X, so only these two compounds could be hypothesized to be markers of heat treatment. Changes in the content of these two compounds start at 70 °C/60 min heat treatment. At 90 °C/60 min and 100 °C/60 min heat treatment compound B was totally eliminated and the peak area of compound X achieved invariable maximum values (Fig. 2). We hypothesized that compound X is a degradation product of compound B. To confirm this fact, the mead was treated
3.1.2. Effect of heat treatment on 5-hydroxymethylfurfural content 5-Hydroxymethylfurfural is formed during the heat treatment and long-term storage of carbohydrate rich foodstuffs and this compound is used as one of the most important quality parameter of honey in particular. Its maximum allowed amount in honey is normalised at 40 mg/kg (Council Directive, 2001), but no regulation for mead has yet been established. However, in view of the fact that 5-hydroxymethylfurfural is used for honey quality evaluation, it could also be used as a quality parameter of mead because its amount in mead depends on honey quality, mead production technology, and storage conditions etc. 5-Hydroxymethylfurfural content in the mead used for all experiments was 6.40 ± 0.38 mg/L, which is consistent with the study of Kahoun et al. (2008) who found contents ranging from 2.74 to 157 mg/L (Kahoun et al., 2008). In another study, the concentration range started at a 10 times higher concentration (27 mg) (Švecová et al., 2015). An extraordinary low content of 5-hydroxymethylfurfural in some mead samples in the study of Kahoun et al. (2008) may have been caused by two factors – the honey used for mead production was of a very good quality and/ or the meads were produced using cold-made technology (without the heating or boiling of honey must). The two papers mentioned above provide extensive overview of the occurrence of 5-hydroxymethylfurfural in various types of mead, so further research was focused on the effect of heat treatment and storage conditions, because this topic had not previously been studied. No significant changes in 5-hydroxymethylfurfural content were observed until the 80 °C/60 min heat treatment (increase of only 10% from 6.40 ± 0.38 mg/L to 7.06 ± 0.36 mg/L). At 90 °C/60 min heat treatment 5-hydroxymethylfurfural content was 8.73 ± 0.36 mg/L (+36%) and finally, at 100 °C/60 min heat treatment, 5-hydroxymethylfurfural content increased to up to 12.4 mg/L (+93%). With regard to these results it is evident that the maximum
Table 1 Effect of heat treatment on phenolic compounds content. No.
1 2 8 11 13 18 19
Electroactive compound
Gallic acid Protocatechuic acid 4-Hydroxyphenylacetic acid Vanillic acid Caffeic acid Syringic acid p-Coumaric acid
Phenolic compounds content [mg/L] Fresh mead
40 °C/60 min
50 °C/60 min
60 °C/60 min
70 °C/60 min
80 °C/60 min
90 °C/60 min
100 °C/60 min
0.455 ± 0.004 0.182 ± 0.006 0.159 ± 0.002 0.220 ± 0.023 0.466 ± 0.007
0.452 ± 0.012 0.170 ± 0.010 0.158 ± 0.003 0.210 ± 0.009 0.474 ± 0.009
0.445 ± 0.018 0.166 ± 0.008 0.151 ± 0.003 0.212 ± 0.010 0.452 ± 0.032
0.451 ± 0.017 0.164 ± 0.011 0.153 ± 0.002 0.205 ± 0.007 0.448 ± 0.032
0.447 ± 0.021 0.164 ± 0.009 Coel.a 0.202 ± 0.005 0.438 ± 0.026
0.443 ± 0.042 0.161 ± 0.009 Coel.a 0.193 ± 0.009 0.419 ± 0.019
0.499 ± 0.014 0.171 ± 0.002 Coel.a 0.192 ± 0.006 0.383 ± 0.019
0.603 ± 0.021 0.173 ± 0.005 Coel.a 0.184 ± 0.012 0.365 ± 0.023
All measurements were performed in triplicate and results are expressed as arithmetic mean ± 2 standard deviation. a Not evaluated due to coelution with unidentified compound X. b Detected, but not quantified.
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Fig. 2. Effect of heat treatment on two unknown electroactive compounds content.
temperature for warming up mead (e.g. served as a drink) should not exceed 80 °C. 3.1.3. Effect of heat treatment on antioxidant activity Antioxidant activity expressed as TEAC measured by ABTS method is a complex parameter often used for the characterization of the antioxidant properties of beverages e.g. beer (Zhao, Chen, Lu, & Zhao, 2010), wine (Garaguso & Nardini, 2015)or fruit juices (Moreno-Montoro, Olalla-Herrera, Gimenez-Martinez, NavarroAlarcon, & Rufián-Henares, 2015). The antioxidant activity of mead was studied by Wintersteen et al. (2005) using the Oxygen Radical Absorbance Capacity (ORAC) method. Antioxidant activity was measured in 2 home-brewed meads from soya honey (3.53 ± 0.56 lmol TEAC/mL and 7.12 ± 1.11 lmol TEAC/mL), 3 experimental meads (1.11 ± 0.25 lmol TEAC/mL; 3.47 ± 0.05 lmol TEAC/mL and 3.79 ± 0.16 lmol TEAC/mL) and 2 mead analogs (0.54 ± 0.13 lmol TEAC/mL and 0.74 ± 0.09 lmol TEAC/mL), but no significant differences in the antioxidant capacity of experimental meads were found due to the heat treatment of its honey must. Unfortunately, research of Tafulo, Queriós, Delerue-Matos, and Sales (2010) focused on the control and comparison of the antioxidant capacity of beers showed that the results of the ORAC method provide multiple higher values of antioxidant activity than the ABTS method and so these methods are not mutually comparable. Changes in the antioxidant activity of the tested mead, ranging from 0.567 ± 0.012 lmol TEAC/mL (fresh mead) to 0.552 ± 0.020 lmol TEAC/mL (100 °C/60 min heat treatment), were not significant at any heat treatment. These results are in harmony with the results of Wintersteen et al. (2005) and with the results of our HPLC-ECD analyses of electroactive compounds, because changes were registered in only a few compounds. 3.2. Effect of storage conditions 3.2.1. Effect of storage conditions on electroactive compounds content Electroactive compound content was evaluated comparing peak areas of all detected compounds in the chromatograms of the fresh mead, the mead stored for 12 weeks in a refrigerator and the mead stored for 12 weeks at room temperature (Fig. 3). Changes in peaks areas were determined for 16 compounds and the trend of each change was marked with appropriate arrows. Detailed evaluations were again focused on all identified phenolic compounds (gallic acid, protocatechuic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, syringic acid and p-coumaric acid) and two unidentified compounds B and X. The most significant differences were observed in gallic acid content. Storage in a refrigerator caused an increase of 10% only, but storage at room temperature caused an increase of up to
Fig. 3. HPLC-ECD chromatogram of the fresh mead (A), the mead stored for 12 weeks in a refrigerator at 4 °C in darkness (B) and the mead stored for 12 weeks at room temperature (20–25 °C) on daylight without direct sunlight (C). Symbol " represents content increase and symbol ; represents content decrease.
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72%. Protocatechuic acid content was not changed during storage in a refrigerator but storage at room temperature caused an increase of by 26%. The content of 4-hydroxyphenylacetic acid was evaluated for samples stored in a refrigerator only because samples stored at room temperature provided chromatograms wherein this compound again coelutes with unidentified compound X (as already mentioned in Section 3.1.1) and due to this fact, it could not be evaluated. Changes of 4-hydroxyphenylacetic acid content evaluated under storage in a refrigerator were not significant. Vanillic acid content evaluated under storage in a refrigerator was also without significant change and storage at room temperature caused only a slight increase of 20%. The content of caffeic acid and p-coumaric acid decreased under both means of storage. Caffeic acid content decreased under storage in a refrigerator by 6%, p-coumaric acid content decreased under the same conditions by 20%. More significant changes were found under storage at room temperature caffeic acid content decreased by 22%, p-coumaric acid content decreased under the same conditions by 33%. All the results of measurements of the fresh mead and meads after all storage conditions are listed in Table 2. All identified phenolic compounds mentioned above were evaluated within research focused on the maturing of barrique red wines, but instead of a trend (increase or decrease) a co-sinusoidal timedependence of concentration was found (Mateˇjícˇek et al., 2005). This could be caused by the fact that the wooden barrels used for wine maturing also contain some of these phenolic compound representatives (gallic acid, 4-hydroxyphenylacetic acid, vanillic acid and syringic acid) which influence the chemical composition of wines. Obvious trends were presented in a study of García-Falcón et al. (2007) which was focused on the influence of bottle storage of young red wines on their evolution. A decrease in levels or else no change was observed in the case of gallic acid, protocatechuic acid 4-hydroxyphenylacetic acid and syringic acid. An increase in levels or else no change was observed in the case of vanillic acid only. In comparison to our results, the same trends were achieved only in the case of vanillic acid and 4-hydroxyphenylacetic acid. An important output of these measurements is the total degradation of the unidentified compound marked as B and the formation of the new unidentified compound marked as X during 12 weeks of storage at room temperature (Fig. 4), but only 20% degradation of the unidentified compound marked as B during 12 weeks of storage in darkness in a refrigerator at 4 °C. With respect to this findings, these two compounds could be also hypothesized to be markers of storage at room temperature. Comparing results from heat treatment and storage condition evaluation highlights another interesting fact. The peak area of the unidentified compound marked as X from the chromatogram of the mead sample after 100 °C/60 min (1993 ± 93 nC) did not differ from the chromatogram of the mead sample after 12 weeks’ storage at room
Fig. 4. Effect of storage conditions on two unknown electroactive compounds content.
temperature (1965 ± 30 nC). Because of this insignificant difference (only 1.4%) it could be stated that 90–100 °C/60 min heat treatment causes the same effect as 12 weeks’ storage at room temperature. 3.2.2. Effect of storage conditions on 5-hydroxymethylfurfural content 5-Hydroxymethylfurfural content increases not only with heat treatment, but also during storage at improper conditions (da Silva, Gauche, Gonzaga, Costa, & Fett, 2016). Taking into consideration the fact that the absolute majority of meads are stored for months or years at room temperatures, we assumed a significant influence for this means of storage. Thus, mead was stored under 2 different storage conditions – in a refrigerator and at room temperature. No significant changes in 5-hydroxymethylfurfural content were observed during 12 weeks of storage in darkness in a refrigerator at 4 °C (increase from 6.40 ± 0.38 to 6.45 ± 0.36 mg/L), but 12 weeks of storage at room temperature in daylight caused a gradual increase of up to 8.92 ± 0.36 mg/L (+46%). With regard to these results it is evident that only storage at room temperature influences hydroxymethylfurfural content and this effect is almost the same as 90 °C/60 min heat treatment (8.73 ± 0.36 mg/L). 3.2.3. Effect of storage conditions on antioxidant activity Changes in the antioxidant activity of mead stored under both storage conditions were, again, not significant. Results ranging from 0.585 ± 0.020 lmol TEAC/mL (fresh mead) to 0.573 ± 0.014 lmol TEAC/mL (12 weeks’ storage in a refrigerator at 4 °C in darkness) and 0.571 ± 0.022 lmol TEAC/mL (12 weeks’ storage at room temperature in daylight). These results are also
Table 2 Effect of storage conditions on phenolic compounds content. No. Electroactive compound
Phenolic compounds content [mg/L]
1 2 8
0.429 ± 0.029 0.428 ± 0.025 0.429 ± 0.032 0.426 ± 0.025 0.474 ± 0.004 0.494 ± 0.009 0.508 ± 0.011 0.530 ± 0.030 0.737 ± 0.007 0.170 ± 0.007 0.166 ± 0.005 0.165 ± 0.014 0.160 ± 0.007 0.166 ± 0.004 0.180 ± 0.007 0.188 ± 0.004 0.190 ± 0.011 0.215 ± 0.009 0.162 ± 0.005 0.154 ± 0.007 0.153 ± 0.011 0.153 ± 0.006 0.153 ± 0.005 Coel.a Coel.a Coel.a Coel.a
Fresh mead
Refrigerator (4 °C, darkness) Week 1
11 13 18 19
Gallic acid Protocatechuic acid 4-Hydroxyphenylacetic acid Vanillic acid Caffeic acid Syringic acid p-Coumaric acid
0.216 ± 0.005 0.475 ± 0.008
0.219 ± 0.004 0.468 ± 0.020
Week 2
0.222 ± 0.009 0.457 ± 0.010
Room temperature (20–25 °C, daylight) Week 4
0.215 ± 0.014 0.454 ± 0.015
Week 12
0.218 ± 0.021 0.449 ± 0.034
Week 1
0.227 ± 0.005 0.496 ± 0.041
All measurements were performed in triplicate and results are expressed as arithmetic mean ± 2 standard deviation. a Not evaluated due to coelution with unidentified compound X. b Detected, but not quantified.
Week 2
0.230 ± 0.008 0.465 ± 0.011
Week 4
0.232 ± 0.007 0.418 ± 0.010
Week 12
0.259 ± 0.011 0.372 ± 0.009
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in agreement with the results of HPLC-ECD analyses of electroactive compounds, because only a minority of changes in a few compounds were found again. 4. Conclusions The HPLC method with coulometric-array detection provides very good selectivity and sensitivity well suited to the evaluation of small changes in electroactive compound content. The phenolic compound profile of meads is not significantly influenced by heat treatment or storage conditions in general, but an unknown electroactive compound was found to be useful as a possible marker of heat treatment and/or storage conditions. Identification of this unknown electroactive compound is an objective for further research. Considerable changes in 5-hydroxymethylfurfural content were observed at 90–100 °C/60 min heat treatment (an increase of +36% and +93% respectively), so it is evident that maximum temperature for the warming up of mead (e.g. served as a drink) should not exceed 80 °C. Storage at room temperature in daylight also causes a significant increase of 5-hydroxymethylfurfural content (an increase of +46%), thus we recommend the storage of bottles of mead in dark places at lower temperatures (the manufacturer recommends 11–15 °C) if possible, especially in the case of long-term storage. Antioxidant activity was not significantly influenced by either heat treatment or the storage conditions tested. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the grant project No. MSM0021627502 sponsored by the Ministry of Education, Youth and Sports of the Czech Republic and project 203/08/1536 sponsored by the Czech Science Foundation. Authors thank to Dr. Karel Bojda (owner of JANKAR PROFI company) for valuable advice and mead samples. References Al, M. L., Dezmirean, D., Moise, A., Bobis, O., Laslo, L., & Bogdanov, S. (2009). Physicochemical and bioactive properties of different floral origin honeys from Romania. Food Chemistry, 112, 863–867. http://dx.doi.org/10.1016/j.foodchem. 2008.06.055. ˇ Benová, B., & Hájek, T. (2010). Utilization of coulometric array detection in analysis of beverages and plant extracts. Procedia Chemistry, 2, 92–100. http://dx.doi.org/ 10.1016/j.proche.2009.12.015. Council Directive (2001). Council Directive of 20 December relating to honey 2001/ 110/EC. Off. J. Eur. Commun. da Silva, P. M., Gauche, C., Gonzaga, L. V., Costa, A. C., & Fett, R. (2016). Honey: Chemical composition, stability and authenticity. Food Chemistry, 196, 309–323. http://dx.doi.org/10.1016/j.foodchem.2015.09.051.
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Escriche, I., Kadar, M., Juan-Borrás, M., & Domenech, E. (2014). Suitability of antioxidant capacity, flavonoids and phenolic acids for floral authentication of honey. Impact of industrial thermal treatment. Food Chemistry, 142, 135–143. http://dx.doi.org/10.1016/j.foodchem.2013.07.033. Fernández de Simón, B., Sanz, M., Cadahía, E., Martínez, J., Esteruelas, E., & Muñoz, A. M. (2014). Polyphenolic compounds as chemical markers of wine ageing in contact with cherry, chestnut, false acacia, ash and oak wood. Food Chemistry, 143, 66–76. http://dx.doi.org/10.1016/j.foodchem.2013.07.096. Garaguso, I., & Nardini, M. (2015). Polyphenols content, phenolics profile and antioxidant activity of organic red wines produced without sulfur dioxide/sulfites addition in comparison to conventional red wines. Food Chemistry, 179, 336–342. http://dx.doi.org/10.1016/j.foodchem.2015.01.144. García-Falcón, M. S., Pérez-Lamela, C., Martínez-Carballo, E., & Simal-Gándara, J. (2007). Determination of phenolic compounds in wines: Influence of bottle storage of young red wines on their evolution. Food Chemistry, 105, 248–259. http://dx.doi.org/10.1016/j.foodchem.2006.11.006. Ioannou, I., & Ghoul, M. (2012). Biological activities and effects of food processing on flavonoids as phenolic antioxidants. In M. Petre (Ed.), Advances in applied biotechnology (pp. 111–118). Rijeka, Croatia: InTech. Retrieved September 23, 2016, from. Juan-Borrás, M., Domenech, E., Hellebrandova, M., & Escriche, I. (2014). Effect of country origin on physicochemical, sugar and volatile composition of acacia, sunflower and tilia honeys. Food Research International, 60, 86–94. http://dx.doi. org/10.1016/j.foodres.2013.11.045. ˇ ezková, S., Veškrnová, K., Královsky´, J., & Holcˇapek, M. (2008). Kahoun, D., R Determination of phenolic compounds and hydroxymethylfurfural in meads using high performance liquid chromatography with coulometric-array and UV detection. Journal of Chromatography A, 1202, 19–33. http://dx.doi.org/10.1016/ j.chroma.2008.06.016. ˇ eksteryte˙, V. (2010). Carbohydrate Kaškoniene˙, V., Venskutonis, P. R., & C composition and electrical conductivity of different origin honeys. LWT – Food Science and Technology, 43, 801–807. http://dx.doi.org/10.1016/j.lwt.2010.01. 007. Mateˇjícˇek, D., Mikeš, O., Klejdus, B., Šteˇrbová, D., & Kubánˇ, V. (2005). Changes in contents of phenolic compounds during maturing of barrique red wines. Food Chemistry, 90, 791–800. http://dx.doi.org/10.1016/j.foodchem.2004.05.057. Moreno-Montoro, M., Olalla-Herrera, M., Gimenez-Martinez, R., Navarro-Alarcon, M., & Rufián-Henares, J. A. (2015). Phenolic compounds and antioxidant activity of Spanish commercial grape juices. Journal of Food Composition and Analysis, 38, 19–26. http://dx.doi.org/10.1016/j.jfca.2014.10.001. Ramalhosa, E., Gomes, T., Pereira, A. P., Dias, T., & Estevinho, L. M. (2011). Mead production: Tradition versus modernity. In R. S. Jackson (Ed.). Advances in food and nutrition research (Vol. 63, pp. 101–118). Burligton: Elsevier Inc.. http://dx. doi.org/10.1016/B978-0-12-384927-4.00004-X. Socha, R., Paja˛k, P., Fortuna, T., & Buksa, K. (2015). Phenolic profile and antioxidant activity of polish meads. International Journal of Food Properties, 18, 2713–2725. http://dx.doi.org/10.1080/10942912.2015.1004588. Švecová, B., Bordovská, M., Kalvachová, D., & Hájek, T. (2015). Analysis of Czech meads: Sugar content, organic acids content and selected phenolic compounds content. Journal of Food Composition and Analysis, 38, 80–88. http://dx.doi.org/ 10.1016/j.jfca.2014.11.002. Tafulo, P. A., Queriós, R. B., Delerue-Matos, C. M., & Sales, M. G. (2010). Control and comparison of the antioxidant capacity of beers. Food Research International, 43, 1702–1709. http://dx.doi.org/10.1016/j.foodres.2010.05.014. Tosi, E., Ciappini, M., Ré, E., & Lucero, H. (2002). Honey thermal treatment effects on hydroxymethylfurfural content. Food Chemistry, 77, 71–74. http://dx.doi.org/ 10.1016/S0308-8146(01)00325-9. Vanbeneden, N., Gils, F., Delvaux, F., & Delvaux, F. R. (2008). Formation of 4-vinyl and 4-ethyl derivatives from hydroxycinnamic acids: Occurrence of volatile phenolic flavour compounds in beer and distribution of Pad1-activity among brewing yeasts. Food Chemistry, 107, 221–230. http://dx.doi.org/10.1016/ j.foodchem.2007.08.008. Wintersteen, C. L., Andrae, L. M., & Engeseth, N. J. (2005). Effect of heat treatment on antioxidant capacity and flavor volatiles of mead. Food Chemistry and Toxicology, 70, C119–C126. http://dx.doi.org/10.1111/j.1365-2621.2005.tb07071.x. Zhao, H., Chen, W., Lu, J., & Zhao, M. (2010). Phenolic profiles and antioxidant activities of commercial beers. Food Chemistry, 119, 1150–1158. http://dx.doi. org/10.1016/j.foodchem.2009.08.028.
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Effect of heat treatment and storage conditions on mead composition David Kahoun a,⇑, Sonˇa Rˇezková b, Josef Královsky´ b a b
Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia in Cˇeské Budeˇjovice, Branišovská 1760, 37005 Cˇeské Budeˇjovice, Czech Republic Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic
a r t i c l e
i n f o
Article history: Received 22 April 2016 Received in revised form 23 September 2016 Accepted 26 September 2016 Available online 28 September 2016 Keywords: Mead Phenolic compounds 5-Hydroxymethylfurfural Antioxidant activity Heat treatment Storage conditions HPLC Electrochemical detection
a b s t r a c t The effects of heat treatment and storage conditions on the composition of pure mead (honey wine) made from only honey and water were investigated. Heat treatment experiments were performed at 7 different temperatures ranging from 40 °C to 100 °C with 10 °C increments for 60 min. Storage condition experiments were performed at room temperature (20–25 °C) in daylight without direct sunlight and in darkness in a refrigerator at 4 °C for 1, 2, 4 and 12 weeks. The parameters evaluated were phenolic compounds, peak area of unidentified compounds, 5-hydroxymethylfurfural content and antioxidant capacity. Significant changes in compound content were observed in the case of 6 identified compounds and 9 unidentified compounds. However, the antioxidant activity was not affected by the heat treatments or storage at room temperature. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Mead, or honey wine, is a fermented alcoholic beverage made from bee honey and water. The traditional production process includes the heating or boiling of honey must (unfermented solution of honey and water) before its fermentation for the purpose of protein removal and microorganism elimination (Ramalhosa, Gomes, Pereira, Dias, & Estevinho, 2011). This step reduces the risk of uncontrolled fermentation but it may also cause the degradation of thermolabile compounds e.g. some phenolic acids and flavonoids originate from honey (Escriche, Kadar, Juan-Borrás, & Domenech, 2014). Another negative consequence of these treatments is the potential increase of 5-hydroxymethylfurfural content, as is known in the case of the heat treatment of honey (Tosi, Ciappini, Ré, & Lucero, 2002). With respect to these facts, new, gentle mead production processes without heat treatment have been developed. The chemical composition of various types of mead was evaluˇ ezková, Veškrnová, ated in a comprehensive study of Kahoun, R Královsky´, and Holcˇapek (2008) focused on the content of 25 phenolic compounds and 5-hydroxymethylfurfural in 50 meads obtained from 14 producers. The results of the study confirmed the strong effect of ingredients added to mead (fruit juices, herbal ⇑ Corresponding author. E-mail address: [email protected] (D. Kahoun). http://dx.doi.org/10.1016/j.foodchem.2016.09.161 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
extracts, spices etc.) on their phenolic compound profile and big differences in 5-hydroxymethylfurfural content ranging from 2.74 to 157 mg/L. Similar results were also published by Švecová, ˇ ová and Hájek Bordovská, Kalvachová, and Hájek (2015) and Ben (2010); in their studies they showed the benefits of HPLC with coulometric array detection for the determination of phenolic compounds in wines, meads, and Japanese knotweeds. Socha, Paja˛k, Fortuna, and Buksa (2015) evaluated the influence of mead type and the effect of various mead ingredients on phenolic compound profile and antioxidant activity. A principal component analysis (PCA) statistical method was used in order to differentiate meads in terms of phenolic compounds content. It is very likely that the composition and content of phenolic compounds in meads are influenced not only by the ingredients added, but also by technological processes, such as the means of honey must preparation (Wintersteen, Andrae, & Engeseth, 2005), fermentation, maturing, storage in bottles, warming just before consumption etc. because these effects have been observed in the case of other similar fermented beverages such as wines (Fernández de Simón et al., 2014; García-Falcón, Pérez-Lamela, Martínez-Carballo, & Simal-Gándara, 2007; Mateˇjícˇek, Mikeš, ˇ , 2005) or beers (Vanbeneden, Gils, Klejdus, Šteˇrbová, & Kubán Delvaux, & Delvaux, 2008). The presumption made for this research is that meads made under cold conditions contain different number of phenolic and other electroactive compounds than meads made in the traditional
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way, and that some of these compounds may be damaged by the unsuitable storage of mead in glass bottles or by its warming just before consumption (Ioannou & Ghoul, 2012). Potential changes in the chemical composition of meads may be accompanied by the formation or elimination of a compound, which could then be used as a marker of these treatments. The objective of this study was to evaluate the effects of heat treatment and storage conditions on the composition of cold-made mead using HPLC with coulometric-array detection (for electroactive compounds), HPLC with UV detection (for 5-hydroxymethylfurfural) and UV/VIS spectrophotometry (for antioxidant activity). No study has been published so far on this topic.
2. Experimental 2.1. Chemicals Standard phenolic compounds (HPLC purity) were obtained from the 2 following sources: Fluka (Buchs, Switzerland): gallic acid (3,4,5-trihydroxybenzoic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), a-resorcylic acid (3,5-dihydroxybenzoic acid), homoprotocatechuic acid (3,4-dihydrophenylacetic acid), protocatechuicaldehyde (3,4dihydroxybenzaldehyde), b-resorcylic acid (2,4-dihydroxybenzoic acid), 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, vanillic acid (4-hydroxy-3methoxybenzoic acid), esculetin (6,7-dihydroxycoumarin), caffeic acid (3,4-dihydroxycinnamic acid), (+)-catechin hydrate (trans3,30 ,40 ,5,7-pentahydroxyflavane), vanillin (4-hydroxy-3-methoxybenzaldehyde), isovanillin (3-hydroxy-4-methoxybenzaldehyde), chlorogenic acid hemihydrate (3-O-(3,4-dihydroxycinnamoyl)-Dquinic acid), p-coumaric acid (trans-4-hydroxycinnamic acid), m-coumaric acid (trans-3-hydroxycinnamic acid), ethylvanillin (3-ethoxy-4-hydroxybenzaldehyde), sinapic acid (3,5-dimethoxy4-hydroxycinnamic acid) and o-coumaric acid (trans-2-hydroxycinnamic acid). Sigma-Aldrich (St. Louis, MO, USA): gentisic acid (2,5-dihydroxybenzoic acid), syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid), ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid) and isoferulic acid (3-hydroxy-4-methoxycinnamic acid). Standards of 5-hydroxymethylfurfural and Trolox ((±)-6-Hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid), both HPLC purity, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol, LiChrosolv gradient grade, were obtained from Merck (Darmstadt, Germany). Formic acid (98–100%), sodium acetate trihydrate (99%), acetic acid (98%), potassium dichromate (99%) and ABTS (2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) tablets were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate (99.995%) was obtained from Fluka (Buchs, Switzerland). Water was distilled in glass and purified using a Mili-Q water purification system (Bedford, MA, USA). 2.2. Standard solutions The standards of the phenolic compounds were dissolved in a mixture of water and methanol (1:1, v/v) to obtain 100 mg/L stock solutions and filtered through the 0.2 lm PTFE filter. The stock solutions were stored in dark brown glass vials at 4 °C in darkness for a maximum of one week. Calibration solutions ranging from 0.025 mg/L to 2.5 mg/L were prepared by diluting the stock solutions with the mobile phase A in the desired volume ratios and 10 lL of each prepared calibration solution was immediately injected to HPLC-ECD.
The standard of 5-hydroxymethylfurfural was dissolved in a mixture of water and methanol (9:1, v/v) to obtain 400 mg/L stock solution and filtered through the 0.2 lm PTFE filter. The stock solution was stored in dark brown glass vials at 4 °C in darkness for a maximum of one week. Calibration solutions ranging from 0.5 mg/L to 18 mg/L were prepared by diluting the stock solutions with aqueous methanol (9:1, v/v) in the desired volume ratios and 10 lL of each prepared calibration solution was immediately injected to HPLC-UV. The standard of Trolox stock solution (0.004 lmol/lL) was prepared freshly before any analysis by dissolution in methanol. Calibration solutions ranging from 0.005 lmol/25 lL to 0.05 lmol/25 lL were prepared by dilution of stock solutions with the methanol in the desired volume ratios and 25 lL of each prepared calibration solution was immediately measured using a UV/Vis spectrophotometer. 2.3. Mead samples 2.3.1. Production process The production process for pure mead, cold-made (without heating or boiling of honey must) from bee honey and water only, is the trade secret of JANKAR PROFI company, and thus can be described in general terms only. Honey (approx. 2200 kg) was gently preheated using a hot-air dryer at max. 40–42 °C and then mixed with water (approx. 3000 dm3) in a 5000 dm3 stainless steel fermentation tank until the appropriate value of refractometric dry solids was reached. This honey solution was inoculated with 2.5 kg of the lyophilized yeast Saccharomyces cerevisiae and fermented at 18–20 °C for 5–6 weeks until the appropriate value of refractometric dry solids was reached. During the turbulent fast fermentation, the mead was cooled to keep the temperature below 18–20 °C. Fermentation continued to completion at 16 °C for 4 weeks. The mead was then removed by suction and matured at 12–14 °C for 1 year. After maturing, primary filtration was done using a diatomaceous earth filter followed by fine filtration using a membrane filter with a 0.45 lm pore size. Finally, the mead was bottled under nitrogen atmosphere, the bottles closed with corks, and the bottle necks covered with molten wax. 2.3.2. Mead samples The samples of fresh pure mead were obtained in two clear glass bottles from JANKAR PROFI company. Both samples (bottles) were made in the same batch. One bottle of mead was used for the evaluation of the effect of heat treatment and the other for the evaluation of the effect of storage conditions. The bottles were stored at 4 °C in darkness for a maximum of one week. The effect of heat treatment was tested using a screw Duran Premium bottle (100 mL) with 25 mL of mead sample. The bottle was placed into a water bath at 7 different temperatures ranging from 40 °C to 100 °C with 10 °C increments for 60 min. Then, the bottle was taken out and cooled using a stream of cold water. The effect of storage conditions was tested using an Erlenmeyer flask (250 mL) with 200 mL of mead sample at room temperature (20–25 °C) in daylight (without direct sunlight) and in darkness in a refrigerator at 4 °C for 1, 2, 4 and 12 weeks. The 12 weeks’ cut-off time was chosen on the basis of the manufacturer’s recommendation because this is the average period from bottling by the producer to purchase by the customer. 2.4. Instrumentation An HPLC-ECD system for phenolic compound analysis was set up. It consisted of a vacuum degasser DG 3014 (Ecom, Prague, Czech Republic), two chromatographic pumps model 582
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(ESA, Chelmsford, MA, USA) a CoulArray thermostatic organizer (ESA, Chelmsford, MA, USA) containing: a pulse damper, a gradient mixer, a manual injector with 10-lL sampling loop (Rheodyne, Cottati, CA, USA); an electrochemical 8-channel CoulArray 5600A detector (ESA, Chelmsford, MA, USA) and a PC with a CoulArray software for data acquisition, processing and analysis (ESA, Chelmsford, MA, USA). Chromatographic column Gemini C18 (150 3 mm I.D., 3 lm particle size) was obtained from Phenomenex (Torrence, CA, USA). Hold-up volume (0.59 mL) was determined using uracil as the non-retained marker solute. 5-Hydroxymethylfurfural analysis was carried out using a HPLC-UV system Hewlett-Packard model HP 1090M (Waldbronn, Germany) equipped with a diode array detector and an autosampler. Chromatographic column LiChrospher 60 RP-select B (250 4 mm I.D., 5 lm particle size) was obtained from Merck (Darmstadt, Germany). Hold-up volume (3.18 mL) was determined using uracil as the non-retained marker solute. Antioxidant activity was measured using single beam VIS spectrophotometer Thermo model Spectronic Helios d (Madison, WI, USA) equipped with 10-mm optical path length glass cuvettes. 2.5. HPLC analysis of phenolic compounds All chromatographic separations were carried out at 35 ± 0.1 °C using a gradient elution with mobile phases A and B. The mobile phase A was 5 mM ammonium acetate in water, the pH value of which was adjusted to 3.0 by adding formic acid (1000 lL/L). The mobile phase B was a mixture of mobile phase A and acetonitrile at 1:2 (v/v) and pH value was adjusted to 3.0 by the addition of formic acid (600 lL/150 mL). The mobile phases were filtered through the 0.45 lm filter and degassed (for 10 min) by ultrasonication before use. The applied gradient programme was: 0–27 min, 2% B isocratic; 27–72 min, linear gradient from 2% to 7% B; 72–108 min, linear gradient from 7% to 25% B; and finally, washing and reconditioning of the column was done. The flow rate was 0.45 mL/min and the injection volume was 10 lL. Electroactive compounds were monitored and quantified using eight electrochemical cells with applied potentials from 200 to 900 mV (100 mV increment). Mead samples were diluted with the initial mobile phase at 1:1, filtered through the 0.2 lm PTFE filter only and 10 lL was immediately injected to HPLC. No other sample pre-treatments were used. Results were expressed as an amount (mg) of an analyte per volume (L) of a sample. 2.6. HPLC analysis of 5-hydroxymethylfurfural All chromatographic separations of 5-hydroxymethylfurfural were carried out at laboratory temperature using the isocratic elution with the mobile phase consisting of water and methanol (9:1 v/v). The mobile phase was filtered through the 0.45 lm filter and degassed (for 10 min) by ultrasonication before use. The flow rate was 1.0 mL/min and the injection volume was 10 lL. 5-Hydroxymethylfurfural was determined using a diode array UV detector at 285 nm. Mead samples were diluted with the initial mobile phase at 1:1, filtered through the 0.2 lm PTFE filter only and 10 lL was immediately injected on to the HPLC. No other sample pre-treatments were used. Results were expressed as an amount (mg) of 5-hydroxymethylfurfural in a volume (L) of a sample. 2.7. Determination of antioxidant activity Antioxidant activity was determined using the ABTS method. A tablet of ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) containing 10 mg of ABTS was dissolved in a 10 mL
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volumetric flask with 5 mL of water and then 100 lL of aqueous solution of 17.24 mg/mL potassium dichromate was added. A volumetric flask was wrapped with aluminium foil and kept at laboratory temperature in darkness for 16 h. Before analysis, this ABTS stock solution was diluted with the acetate buffer (pH 4.3) in a 10 mL volumetric flask at 1:39 (v/v) and the ABTS working solution was immediately used for measurement. At first, the absorbance (AABTS) of this ABTS working solution at 734 nm was measured. Afterwards, 2 mL of ABTS working solution and 25 lL of a sample solution (or a Trolox calibration solution) were mixed in a vial, and then the absorbance (Asample) at 734 nm was measured. The decrease in absorbance (DA, %) was calculated according to the equation: DA = 100 ((AABTS Asample)/AABTS). Results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC, lmol/mL). This index describes the amount (lmol) of Trolox in which the antioxidant capacity is equivalent to the antioxidant capacity of a volume (mL) of a sample. 2.8. Methods validation All chromatographic measurements were done using our optimized and validated analytical methods (Kahoun et al., 2008). Validation of the ABTS method used for the determination of antioxidant activity includes the assessment of the following validation parameters. Linearity was assessed from the calibration curves obtained at 7 concentration levels. Decreases in absorbance (DA, %) were plotted versus theoretical amounts of Trolox (x, lmol) and the calibration curve DA = (1658 ± 17)x + (2.50 ± 0.47) was obtained from a least-squares regression analysis. The limit of detection (0.0043 lmol Trolox) and limit of quantification (0.0067 lmol Trolox) were determined according to ISO 11843-2 by statistical software QC Expert. All measurements were performed in triplicate and results are expressed as an arithmetic mean ± 2 standard deviation. 3. Results and discussion 3.1. Effect of heat treatment 3.1.1. Effect of heat treatment on electroactive compounds content Electroactive compound content was evaluated by comparing the peak areas of all detected compounds in chromatograms of the fresh mead and the mead after 100 °C/60 min heat treatment (Fig. 1). Changes in peak areas were registered in 16 peaks and the trend of each change is marked with appropriate arrows. All 7 identified phenolic compounds (gallic acid, protocatechuic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, syringic acid and p-coumaric acid) and two unidentified compounds (B and X) were chosen for more detailed evaluation. Concentrations of phenolic compounds in fresh mead ranged from 0.159 ± 0.002 to 0.466 ± 0.007 mg/L which is consistent with previously reported ˇ ová & Hájek, 2010; Kahoun et al., 2008; Švecová results (Ben et al., 2015). Gallic acid content increased by 32%, changes in protocatechuic acid content were almost negligible (decrease of 5%), 4-hydroxyphenylacetic acid content after 100 °C/60 min heat treatment could not be evaluated because of coelution with an unidentified compound, hereafter refer to as compound X, vanillic acid content decreased by 16%, caffeic acid content decreased by 22%, p-coumaric acid content decreased by 30%. p-Coumaric acid was also evaluated within research focused on the effect of 2 different heat treatments (60 °C and boiling) of honey must on mead composition (Wintersteen et al., 2005). The influence of hightemperature heat treatment was observed only in the case of buckwheat mead (concentration increased from 1.94 ± 0.20 mg/L to 2.49 ± 0.16 mg/L), but in the case of soya mead concentration
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at 100 °C for 1 h and at 100 °C for 8 h and the peak areas of compound X were compared. Both analyses gave almost the same peak area (difference 6%) and this result increased the probability that compound X is a degradation product of compound B. On the condition that compound B is a naturally occurring compound always present in honey, or that this compound is always formed during mead production, it could potentially be used as a marker of the heat treatment (temperature over 70 °C) of mead. However, since the chemical composition of a honey, even from the same floral source, depends on the year of its production, and on the region, due to different climatic and geographical factors, respectively (Al et al., 2009; Juan-Borrás, Domenech, Hellebrandova, & ˇ eksteryte˙, 2010), the Escriche, 2014; Kaškoniene˙, Venskutonis, & C suitability of the marker needs to be proven using either fresh, cold made, pure mead from one producer from different years, or using mead obtained from various producers.
Fig. 1. HPLC-ECD chromatogram of the fresh mead (A) and the mead after 100 °C/60 min heat treatment (B). Symbol " represents content increase and symbol ; represents content decrease.
1.06 ± 0.03 mg/L was not changed (1.06 ± 0.08 mg/L). The results of the measurements of the fresh mead and the mead after all 7 heat treatments are listed in Table 1. A detailed evaluation of heat treatment on two unidentified compounds B and X showed interesting findings. Heat treatment with the range 90–100 °C/60 min caused total degradation of the unidentified compound marked as B and the formation of the new unidentified compound marked as X, so only these two compounds could be hypothesized to be markers of heat treatment. Changes in the content of these two compounds start at 70 °C/60 min heat treatment. At 90 °C/60 min and 100 °C/60 min heat treatment compound B was totally eliminated and the peak area of compound X achieved invariable maximum values (Fig. 2). We hypothesized that compound X is a degradation product of compound B. To confirm this fact, the mead was treated
3.1.2. Effect of heat treatment on 5-hydroxymethylfurfural content 5-Hydroxymethylfurfural is formed during the heat treatment and long-term storage of carbohydrate rich foodstuffs and this compound is used as one of the most important quality parameter of honey in particular. Its maximum allowed amount in honey is normalised at 40 mg/kg (Council Directive, 2001), but no regulation for mead has yet been established. However, in view of the fact that 5-hydroxymethylfurfural is used for honey quality evaluation, it could also be used as a quality parameter of mead because its amount in mead depends on honey quality, mead production technology, and storage conditions etc. 5-Hydroxymethylfurfural content in the mead used for all experiments was 6.40 ± 0.38 mg/L, which is consistent with the study of Kahoun et al. (2008) who found contents ranging from 2.74 to 157 mg/L (Kahoun et al., 2008). In another study, the concentration range started at a 10 times higher concentration (27 mg) (Švecová et al., 2015). An extraordinary low content of 5-hydroxymethylfurfural in some mead samples in the study of Kahoun et al. (2008) may have been caused by two factors – the honey used for mead production was of a very good quality and/ or the meads were produced using cold-made technology (without the heating or boiling of honey must). The two papers mentioned above provide extensive overview of the occurrence of 5-hydroxymethylfurfural in various types of mead, so further research was focused on the effect of heat treatment and storage conditions, because this topic had not previously been studied. No significant changes in 5-hydroxymethylfurfural content were observed until the 80 °C/60 min heat treatment (increase of only 10% from 6.40 ± 0.38 mg/L to 7.06 ± 0.36 mg/L). At 90 °C/60 min heat treatment 5-hydroxymethylfurfural content was 8.73 ± 0.36 mg/L (+36%) and finally, at 100 °C/60 min heat treatment, 5-hydroxymethylfurfural content increased to up to 12.4 mg/L (+93%). With regard to these results it is evident that the maximum
Table 1 Effect of heat treatment on phenolic compounds content. No.
1 2 8 11 13 18 19
Electroactive compound
Gallic acid Protocatechuic acid 4-Hydroxyphenylacetic acid Vanillic acid Caffeic acid Syringic acid p-Coumaric acid
Phenolic compounds content [mg/L] Fresh mead
40 °C/60 min
50 °C/60 min
60 °C/60 min
70 °C/60 min
80 °C/60 min
90 °C/60 min
100 °C/60 min
0.455 ± 0.004 0.182 ± 0.006 0.159 ± 0.002 0.220 ± 0.023 0.466 ± 0.007
0.452 ± 0.012 0.170 ± 0.010 0.158 ± 0.003 0.210 ± 0.009 0.474 ± 0.009
0.445 ± 0.018 0.166 ± 0.008 0.151 ± 0.003 0.212 ± 0.010 0.452 ± 0.032
0.451 ± 0.017 0.164 ± 0.011 0.153 ± 0.002 0.205 ± 0.007 0.448 ± 0.032
0.447 ± 0.021 0.164 ± 0.009 Coel.a 0.202 ± 0.005 0.438 ± 0.026
0.443 ± 0.042 0.161 ± 0.009 Coel.a 0.193 ± 0.009 0.419 ± 0.019
0.499 ± 0.014 0.171 ± 0.002 Coel.a 0.192 ± 0.006 0.383 ± 0.019
0.603 ± 0.021 0.173 ± 0.005 Coel.a 0.184 ± 0.012 0.365 ± 0.023
All measurements were performed in triplicate and results are expressed as arithmetic mean ± 2 standard deviation. a Not evaluated due to coelution with unidentified compound X. b Detected, but not quantified.
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Fig. 2. Effect of heat treatment on two unknown electroactive compounds content.
temperature for warming up mead (e.g. served as a drink) should not exceed 80 °C. 3.1.3. Effect of heat treatment on antioxidant activity Antioxidant activity expressed as TEAC measured by ABTS method is a complex parameter often used for the characterization of the antioxidant properties of beverages e.g. beer (Zhao, Chen, Lu, & Zhao, 2010), wine (Garaguso & Nardini, 2015)or fruit juices (Moreno-Montoro, Olalla-Herrera, Gimenez-Martinez, NavarroAlarcon, & Rufián-Henares, 2015). The antioxidant activity of mead was studied by Wintersteen et al. (2005) using the Oxygen Radical Absorbance Capacity (ORAC) method. Antioxidant activity was measured in 2 home-brewed meads from soya honey (3.53 ± 0.56 lmol TEAC/mL and 7.12 ± 1.11 lmol TEAC/mL), 3 experimental meads (1.11 ± 0.25 lmol TEAC/mL; 3.47 ± 0.05 lmol TEAC/mL and 3.79 ± 0.16 lmol TEAC/mL) and 2 mead analogs (0.54 ± 0.13 lmol TEAC/mL and 0.74 ± 0.09 lmol TEAC/mL), but no significant differences in the antioxidant capacity of experimental meads were found due to the heat treatment of its honey must. Unfortunately, research of Tafulo, Queriós, Delerue-Matos, and Sales (2010) focused on the control and comparison of the antioxidant capacity of beers showed that the results of the ORAC method provide multiple higher values of antioxidant activity than the ABTS method and so these methods are not mutually comparable. Changes in the antioxidant activity of the tested mead, ranging from 0.567 ± 0.012 lmol TEAC/mL (fresh mead) to 0.552 ± 0.020 lmol TEAC/mL (100 °C/60 min heat treatment), were not significant at any heat treatment. These results are in harmony with the results of Wintersteen et al. (2005) and with the results of our HPLC-ECD analyses of electroactive compounds, because changes were registered in only a few compounds. 3.2. Effect of storage conditions 3.2.1. Effect of storage conditions on electroactive compounds content Electroactive compound content was evaluated comparing peak areas of all detected compounds in the chromatograms of the fresh mead, the mead stored for 12 weeks in a refrigerator and the mead stored for 12 weeks at room temperature (Fig. 3). Changes in peaks areas were determined for 16 compounds and the trend of each change was marked with appropriate arrows. Detailed evaluations were again focused on all identified phenolic compounds (gallic acid, protocatechuic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, syringic acid and p-coumaric acid) and two unidentified compounds B and X. The most significant differences were observed in gallic acid content. Storage in a refrigerator caused an increase of 10% only, but storage at room temperature caused an increase of up to
Fig. 3. HPLC-ECD chromatogram of the fresh mead (A), the mead stored for 12 weeks in a refrigerator at 4 °C in darkness (B) and the mead stored for 12 weeks at room temperature (20–25 °C) on daylight without direct sunlight (C). Symbol " represents content increase and symbol ; represents content decrease.
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72%. Protocatechuic acid content was not changed during storage in a refrigerator but storage at room temperature caused an increase of by 26%. The content of 4-hydroxyphenylacetic acid was evaluated for samples stored in a refrigerator only because samples stored at room temperature provided chromatograms wherein this compound again coelutes with unidentified compound X (as already mentioned in Section 3.1.1) and due to this fact, it could not be evaluated. Changes of 4-hydroxyphenylacetic acid content evaluated under storage in a refrigerator were not significant. Vanillic acid content evaluated under storage in a refrigerator was also without significant change and storage at room temperature caused only a slight increase of 20%. The content of caffeic acid and p-coumaric acid decreased under both means of storage. Caffeic acid content decreased under storage in a refrigerator by 6%, p-coumaric acid content decreased under the same conditions by 20%. More significant changes were found under storage at room temperature caffeic acid content decreased by 22%, p-coumaric acid content decreased under the same conditions by 33%. All the results of measurements of the fresh mead and meads after all storage conditions are listed in Table 2. All identified phenolic compounds mentioned above were evaluated within research focused on the maturing of barrique red wines, but instead of a trend (increase or decrease) a co-sinusoidal timedependence of concentration was found (Mateˇjícˇek et al., 2005). This could be caused by the fact that the wooden barrels used for wine maturing also contain some of these phenolic compound representatives (gallic acid, 4-hydroxyphenylacetic acid, vanillic acid and syringic acid) which influence the chemical composition of wines. Obvious trends were presented in a study of García-Falcón et al. (2007) which was focused on the influence of bottle storage of young red wines on their evolution. A decrease in levels or else no change was observed in the case of gallic acid, protocatechuic acid 4-hydroxyphenylacetic acid and syringic acid. An increase in levels or else no change was observed in the case of vanillic acid only. In comparison to our results, the same trends were achieved only in the case of vanillic acid and 4-hydroxyphenylacetic acid. An important output of these measurements is the total degradation of the unidentified compound marked as B and the formation of the new unidentified compound marked as X during 12 weeks of storage at room temperature (Fig. 4), but only 20% degradation of the unidentified compound marked as B during 12 weeks of storage in darkness in a refrigerator at 4 °C. With respect to this findings, these two compounds could be also hypothesized to be markers of storage at room temperature. Comparing results from heat treatment and storage condition evaluation highlights another interesting fact. The peak area of the unidentified compound marked as X from the chromatogram of the mead sample after 100 °C/60 min (1993 ± 93 nC) did not differ from the chromatogram of the mead sample after 12 weeks’ storage at room
Fig. 4. Effect of storage conditions on two unknown electroactive compounds content.
temperature (1965 ± 30 nC). Because of this insignificant difference (only 1.4%) it could be stated that 90–100 °C/60 min heat treatment causes the same effect as 12 weeks’ storage at room temperature. 3.2.2. Effect of storage conditions on 5-hydroxymethylfurfural content 5-Hydroxymethylfurfural content increases not only with heat treatment, but also during storage at improper conditions (da Silva, Gauche, Gonzaga, Costa, & Fett, 2016). Taking into consideration the fact that the absolute majority of meads are stored for months or years at room temperatures, we assumed a significant influence for this means of storage. Thus, mead was stored under 2 different storage conditions – in a refrigerator and at room temperature. No significant changes in 5-hydroxymethylfurfural content were observed during 12 weeks of storage in darkness in a refrigerator at 4 °C (increase from 6.40 ± 0.38 to 6.45 ± 0.36 mg/L), but 12 weeks of storage at room temperature in daylight caused a gradual increase of up to 8.92 ± 0.36 mg/L (+46%). With regard to these results it is evident that only storage at room temperature influences hydroxymethylfurfural content and this effect is almost the same as 90 °C/60 min heat treatment (8.73 ± 0.36 mg/L). 3.2.3. Effect of storage conditions on antioxidant activity Changes in the antioxidant activity of mead stored under both storage conditions were, again, not significant. Results ranging from 0.585 ± 0.020 lmol TEAC/mL (fresh mead) to 0.573 ± 0.014 lmol TEAC/mL (12 weeks’ storage in a refrigerator at 4 °C in darkness) and 0.571 ± 0.022 lmol TEAC/mL (12 weeks’ storage at room temperature in daylight). These results are also
Table 2 Effect of storage conditions on phenolic compounds content. No. Electroactive compound
Phenolic compounds content [mg/L]
1 2 8
0.429 ± 0.029 0.428 ± 0.025 0.429 ± 0.032 0.426 ± 0.025 0.474 ± 0.004 0.494 ± 0.009 0.508 ± 0.011 0.530 ± 0.030 0.737 ± 0.007 0.170 ± 0.007 0.166 ± 0.005 0.165 ± 0.014 0.160 ± 0.007 0.166 ± 0.004 0.180 ± 0.007 0.188 ± 0.004 0.190 ± 0.011 0.215 ± 0.009 0.162 ± 0.005 0.154 ± 0.007 0.153 ± 0.011 0.153 ± 0.006 0.153 ± 0.005 Coel.a Coel.a Coel.a Coel.a
Fresh mead
Refrigerator (4 °C, darkness) Week 1
11 13 18 19
Gallic acid Protocatechuic acid 4-Hydroxyphenylacetic acid Vanillic acid Caffeic acid Syringic acid p-Coumaric acid
0.216 ± 0.005 0.475 ± 0.008
0.219 ± 0.004 0.468 ± 0.020
Week 2
0.222 ± 0.009 0.457 ± 0.010
Room temperature (20–25 °C, daylight) Week 4
0.215 ± 0.014 0.454 ± 0.015
Week 12
0.218 ± 0.021 0.449 ± 0.034
Week 1
0.227 ± 0.005 0.496 ± 0.041
All measurements were performed in triplicate and results are expressed as arithmetic mean ± 2 standard deviation. a Not evaluated due to coelution with unidentified compound X. b Detected, but not quantified.
Week 2
0.230 ± 0.008 0.465 ± 0.011
Week 4
0.232 ± 0.007 0.418 ± 0.010
Week 12
0.259 ± 0.011 0.372 ± 0.009
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in agreement with the results of HPLC-ECD analyses of electroactive compounds, because only a minority of changes in a few compounds were found again. 4. Conclusions The HPLC method with coulometric-array detection provides very good selectivity and sensitivity well suited to the evaluation of small changes in electroactive compound content. The phenolic compound profile of meads is not significantly influenced by heat treatment or storage conditions in general, but an unknown electroactive compound was found to be useful as a possible marker of heat treatment and/or storage conditions. Identification of this unknown electroactive compound is an objective for further research. Considerable changes in 5-hydroxymethylfurfural content were observed at 90–100 °C/60 min heat treatment (an increase of +36% and +93% respectively), so it is evident that maximum temperature for the warming up of mead (e.g. served as a drink) should not exceed 80 °C. Storage at room temperature in daylight also causes a significant increase of 5-hydroxymethylfurfural content (an increase of +46%), thus we recommend the storage of bottles of mead in dark places at lower temperatures (the manufacturer recommends 11–15 °C) if possible, especially in the case of long-term storage. Antioxidant activity was not significantly influenced by either heat treatment or the storage conditions tested. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the grant project No. MSM0021627502 sponsored by the Ministry of Education, Youth and Sports of the Czech Republic and project 203/08/1536 sponsored by the Czech Science Foundation. Authors thank to Dr. Karel Bojda (owner of JANKAR PROFI company) for valuable advice and mead samples. References Al, M. L., Dezmirean, D., Moise, A., Bobis, O., Laslo, L., & Bogdanov, S. (2009). Physicochemical and bioactive properties of different floral origin honeys from Romania. Food Chemistry, 112, 863–867. http://dx.doi.org/10.1016/j.foodchem. 2008.06.055. ˇ Benová, B., & Hájek, T. (2010). Utilization of coulometric array detection in analysis of beverages and plant extracts. Procedia Chemistry, 2, 92–100. http://dx.doi.org/ 10.1016/j.proche.2009.12.015. Council Directive (2001). Council Directive of 20 December relating to honey 2001/ 110/EC. Off. J. Eur. Commun. da Silva, P. M., Gauche, C., Gonzaga, L. V., Costa, A. C., & Fett, R. (2016). Honey: Chemical composition, stability and authenticity. Food Chemistry, 196, 309–323. http://dx.doi.org/10.1016/j.foodchem.2015.09.051.
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