Introduction

For many centuries, natural honey has been considered not only as a valuable food but also as a medicinal product with significant antimicrobial activity. This activity is primarily attributed to the presence of monosaccharides, enzymes, organic acids, and phenolic compounds in the product.^1–5 According to some literature sources, the antimicrobial activity of honey depends on the content of hydrogen peroxide (H₂O₂) in it.^6–8

The beneficial properties of honey are preserved for a long time, but only if certain storage conditions are followed. According to the requirements of the Interstate Standard “Natural honey. Technical conditions”, this product should be stored in places inaccessible to direct sunlight, at a temperature not exceeding 20 °C in tightly sealed containers for 12 months from the date of examination, in hermetically sealed containers for 24 months from the date of packaging and 12 months after opening hermetically sealed packaging.⁹

There is data on the storage of honey at low temperatures.^10–13 Most often, this approach is used to suppress fermentation that can occur in honey with a moisture content of more than 20%, as well as to prevent crystallization.^10,14–16 It is known that exposure to low temperatures (from 0 to -20 °C) for a long time leads to a slowdown in chemical processes and, as a result, stabilization of physical and chemical indicators of honey such as electrical conductivity, humidity, acidity and content of phenolic compounds.¹⁰ It is also important to note that storing honey at these temperatures slows down the formation and accumulation of hydroxymethylfurfural (HMF), a product of monosaccharide dehydration that has neurotoxic, cytotoxic, genotoxic, and mutagenic effects.^17–19

Thus, the storage of honey at low temperatures might appear promising for practical application. However, it is essential to bear in mind that, while attempting to prevent the formation of HMF, we must also take into account the potential adverse effects of low temperatures on other physicochemical parameters of honey, particularly those that determine its antimicrobial activity.

In Russia, Commonwealth of Independent States (CIS) countries and Europe some of the most common and important honey plants are buckwheat (buckwheat, Fagopyrum esculentum Moench.), linden (lime linden, Tilia cordata Mill.) and sunflower (annual sunflower, Helianthus annuus L.). For example, in Russia (mainly in the forest-steppe zone of the European part of the country, Altai Territory, Transbaikalia and the Primorye-Amur region), as well as in Kazakhstan, buckwheat provides over 50% of marketable honey.²⁰ The honey productivity of buckwheat can reach 60–362 kg/ha.²¹ Another valuable honey plant, small-leaved linden, occupies a large area in the broad-leaved forest zone of the European part of Russia, spreading to the Urals, and is also found in the Crimea and the Caucasus. Its honey productivity averages 700–1000 kg/ha.²² Annual sunflower is a honey-bearing and pollen-bearing crop that provides honey production in Russia, Kazakhstan, Ukraine, as well as in the countries of southern and south-eastern Europe. Honey productivity is at the level of 40–50 kg/ha.²³

In connection with the above, the purpose of this work was to study the influence of different temperature storage conditions of honey of different botanical origins on its physicochemical and biological parameters during storage for 12 months.

Materials and Methods

Samples collection

The samples of sunflower Helianthus annuus L. (n = 36), linden Tilia cordata Mill. (n = 31) and buckwheat honey Fagopyrum esculentum Moench. (n = 34) admitted to the laboratory of veterinary sanitation and environmental safety in beekeeping of Russian Research Institute of Experimental Veterinary Medicine from the following regions of Russia: Rostov, Volgograd, Kursk, Voronezh and Saratov regions, as well as from the Krasnodar Territory in period 2021-2022. The samples were stored in climatic test chambers M-60/100-500 KTVH (JSC LOIP, Russia).

Palynological analysis

The palynological composition of honey was studied in accordance with Interstate Standard 31769-2012.²⁴ Trinocular microscope AmScope T390C (AmScope, China) with digital camera Levenhuk M1000 PLUS (Levenhuk, USA) was used to perform optical light microscopy.

Pollen grains in the samples were calculated using the following formula:

X_p (%) = G · 100n^-1                                               (1)

where G = ΣG_i is the number of pollen grains of linden, buckwheat or sunflower in all examined fields; n = Σn_i – total number of pollen grains; 100 – conversion factor to percentage.

Physicochemical studies

Physicochemical studies of honey were carried out in accordance with current Interstate Standards, the requirements of which are no less stringent than those of the Codex Alimentarius and Council Directive 2001/110/EU.^25,26 Also, it should be noted that the methods of some analyzes in the above standards are for the most part similar to the methods of Harmonized methods of the international honey commission.²⁷

Determination of moisture

The moisture (mass fraction of water) of the studied samples was determined by the refractometric method according to Interstate Standard 31774-2012²⁸ using an IRF-454 B2M refractometer (JSC Kazan Optical-Mechanical Plant (KOMZ), Russia).

Determination of HMF concentration

HMF was determined by reverse phase HPLC in accordance with the Interstate Standard.²⁹ The study used Shimadzu LC-20 Prominence chromatograph with DAD (simultaneously detectable wavelength range 210-400 nm, working wavelength 283 nm), as well as Eclipse XDB-C18 column (150 × 4.6 mm, 5 µm). The mode used was gradient, the flow rate – 1.0 mL/min, and the peak retention time – 3.83 min. Standard solutions were prepared from the analytical standard HMF (purity no less than 99%, Sigma-Aldrich, USA). The calibration curve was constructed using the following concentrations of HMF: 150.0; 100.0; 50.0; 30.0; 25.0; 20.0; 15.0; 10.0; 5.0; 1.0 µg/mL using the formula:

M_5-HMF = C_HMF ·V_sample/ m_honey                    (2)

where C_HMF – the HMF concentration, determined from the calibration graph (µg/mL); V_sample – volume of analyzed sample (mL); m_honey – mass of the sample (g).

Carrez solutions I and II for precipitation of proteins and stabilization of HMF in aqueous solution were also prepared according to the specified Interstate Standard.

Determination of total mass fraction of reducing sugars

Determination of the glucose and fructose (in %) was carried out according to Interstate Standard 32167-2013 using the colorimetric method.³⁰ The essence of this method is to determine the optical density of a solution of potassium iron sulfide – K₃[Fe(CN)₆] (≥99.5%, LenReaktiv, Russia) after its interaction with reducing sugars of honey. The analysis was carried out on KFK-2 photocolorimeter (JSC Zagorsk Optical-Mechanical Plant, Russia) at a wavelength λ = 440 nm.

Determination of free acidity

To determine free acidity, aqueous solutions of honey samples were titrated with a 0.1 M solution of sodium hydroxide – NaOH (99.2%, LenReaktiv, Russia) to pH 8.30. The pH value was monitored using a potentiometric analyzer – Hanna edge with pH electrode HI11310 (Hanna Instruments, USA). The results were expressed in milliequivalents of HCl in 1 kg of honey – meq/kg.³¹

Free acidity (K) in milliequivalents of HCl in 1 kg of honey was calculated using the formula:

K = V · 10                                                                                            (3)

where V – the volume of sodium hydroxide solution of concentration c(NaOH) = 0.1 mol/dm³, consumed for titration, cm³; 10 – conversion factor for the mass of honey 1 kg.

Determination of the diastase activity

Diastase activity (Gothe units) was determined by the colorimetric method according to the Interstate Standard. Diastasis matched to the amount of starch digested down by honey enzymes.³²

The following formula was used for calculation:

X = 100 · 80(D_C – D_test ) · ^D_C^-1 · (100 – W)^-1                                     (4)

where 80 – the conversion factor; ^D_C  – optical density of the control solution; D_test – optical density of the test solution; W – mass fraction of water in honey (%).

Determination of catalase activity

Catalase activity was determined in accordance with the method developed by Aganin A.V.,³³ according to which the activity was expressed in mm³ of oxygen released when catalase contained in 1 g of honey is exposed to 10 mL of 1% (wt.) H₂O₂ solution for 24 h.

Determination of D-glucose-1-oxidase activity

D-glucose-1-oxidase activity was determined according to the method developed by Flanjak I. et al.³⁴ Under the influence of peroxidase, H₂O₂ is reduced to water. The 3,3′-dimethoxybenzidine used produces a colored product that can be detected colorimetrically. The results were expressed in µg H₂O₂/h g honey.

Determination of hydrogen peroxide content

H₂O₂ in honey was detected using iodometry and spectrophotometry. The potassium iodide used in the reaction reacts with the H₂O₂ contained in the honey.³⁵ The molecular iodine released as a result of the reaction can be detected using spectrophotometer (PE5400UF, Ekroskhim, Russia). Analytical solutions of honey samples were obtained by weighing 1.0±0.01 g of each sample, after which 2.0 mL of HPLC water (Khimiya XXI Vek, Russia) was added to them and mixed for 3-5 min on Vortex mixer VM-300S (Joan Lab Equipment, China) or ultrasonic homogenizer Bandelin Sonopuls HD 2200 (Bandelin, Germany) until completely dissolved. From the resulting analytical solutions, 1.0 mL was taken and 1.0 mL of 0.2 M sulfuric acid (H₂SO₄, ≥93.6%, LenReaktiv, Russia) was added, after which carbon dioxide was passed through. At the last stage of sample preparation, a 5% solution of potassium iodide (KI, 99.5%, Rushim, Russia) was added to the analytical solutions, through which carbon dioxide was previously passed. The prepared analytical solutions of honey samples were incubated at room temperature for 24 h in a dark place. The absorption maximum was observed at λ = 351 nm.

The H₂O₂ concentration was calculated using the formula:

С = R · (D ˗ D₀) / ε,                                                                            (5)

where C – H₂O₂ concentration in the analytical solution (µmol/L); D – optical density of the analytical solution; D₀ – optical density of the control solution; R – dilution coefficient; ε –extinction coefficient (ε₃₅₁= 26400 L ∙ mol^-1 ∙ cm^-1).

Physicochemical studies of honey samples were carried out at room temperature (22±2 °С).

Determination of antimicrobial activity

Microorganism strains

In this work the following strains of microorganisms were used: Staphylococcus aureus 209-P, Escherichia coli 1257 and Bacillus cereus 96 (Table 1).

Table 1: Some characteristics of microorganism strains.

Microbial test

To determine the antimicrobial activity of honey samples before and at the 12th month of storage at the indicated temperatures, the method of inoculating microorganisms (S. aureus, E. coli, B. cereus) on MPA was used. Samples in amount of 100 µg were added to a well made in the center of a Petri dish with MPA seeded with a microorganism. The antimicrobial activity of honey was manifested in the size of the growth inhibition zone.

Statistical analysis

Statistical processing of the results was carried out using MS Excel 2010 software. All measurements were performed in triplicate. The significance of differences in mean values was established using Student’s t-test at a significance level of p < 0.05. Results are presented as means (M) and standard errors of the means (±SEM).

Results and discussion

Results of palynological analysis

According to Interstate Standard “Monofloral honeys. Technical conditions”³⁶ natural linden and buckwheat honey must contain at least 30% of dominant pollen grains, sunflower honey – at least 45%. Moreover, the total number of pollen grains during palynological analysis cannot be less than 500.

The studied samples fully complied with the specified requirements: amount of pollen grains of linden honey exceeded 600, of which 42±2% were identified as pollen grains of linden (Tilia). Palynological analysis of buckwheat and sunflower honey also showed their compliance with the Standard: amount of pollen grains was 620 and 680, respectively. Of these, the dominant grains were 45±3% (Fagopyrum) and 63±2% (Helianthus). Figure 1 shows photographs of pollen grains of these honey plants (×400). 

Figure 1: Photographic images of pollen grains (×400): a – linden (Tilia cordata Mill.), b – buckwheat (Fagopyrum esculentum Moench.), c – sunflower (Helianthus annuus L.) Click here to view Figure

As you can see, the grain of small-leaved linden is three-furrowed, spherical-flattened, color is light yellow. Buckwheat has an ellipsoidal shape, a reticulate structure, and the grain color is dark yellow. The grain of the annual sunflower is golden, spherical in shape with a characteristic prickly sculpture, the arrangement of the thorns is uniform. This description completely coincides with the characteristics of the pollen of these honey plants given in literary sources.^37,38

Results of physicochemical analysis

Before storing samples of freshly pumped linden, buckwheat and sunflower honey, their physicochemical parameters were assessed (Table 2).

Table 2: Analysis of the chemical composition of linden, buckwheat and sunflower honey samples

Note. Interstate Standards requirements for: ^*linden honey, ^**buckwheat honey, ^***sunflower honey; p < 0.05

As can be seen from the table, all parameters regulated by Interstate Standards met their requirements. Currently, the activity of catalase and D-glucose-1-oxidase, as well as the H₂O₂ content, are not regulated. Therefore, we were guided by data from literature sources, which provide positive results of using these indicators to determine the quality of honey.^{33, 34, 39}

In order to study changes in physicochemical and biological properties of honey during prolonged storage under different temperature conditions, initial values obtained were used as a control.

The equal weight of honey samples (100±0.1 g) were placed in plastic containers with hermetically screwed lids and stored in climatic chambers at temperatures of 18, 10, 5, 0, -5, -10 and -18 (±2) °C, humidity 55 (±3)% for a period of 12 months. The choice of temperatures was due to the fact that 18±2 °C is the maximum temperature limit for honey storage according to Interstate Standard requirements (no more than 20 °C), storage at 10, 5, 0, -5, -10 and -18 (±2) °C – modeling the effect of low temperatures at which changes in physicochemical parameters were observed. The resulting dynamics are presented in Figure 2.

Figure 2: Changes in the physicochemical parameters of linden (a), buckwheat (b) and sunflower (c) honey samples during storage for 12 months at temperatures: Click here to view Figure

The mass fraction of HMF in all analyzed samples increased gradually at all temperatures, expect for -18 °C. This process began to occur most intensively after the 3rd month of storage. The dependence of the increase in HMF on storage temperature is monitored in the figure 2. Samples stored at 18 °C exhibited the highest HMF content. At the 12th month, its increase relative to the initial value was found to be 472.5% in samples of linden honey and by 484.0 and 488.1% for buckwheat and sunflower honey. However, these values did not exceed the maximum permissible concentration (MPC) of 25 mg/kg. Throughout the entire study period, the HMF content was stable at -18 °C. In samples of linden and sunflower honey during storage, a slight decrease was noted: in the 12th month – by 5.9 and 2.5%, respectively. The data obtained are consistent with the results of research by I.N. Pasias et al.¹¹ and M. Kędzierska-Matysek et al.,¹⁰ which demonstrated the stability of HMF in honey samples of various botanical origins stored at sub-zero temperatures, and an increase in the level of this indicator in samples stored at room temperature.

A decrease in the diastase activity of honey samples occurred already in the first month of storage under all temperature conditions (Fig. 2). However, for buckwheat honey samples, stored under all specified temperatures for the entire study period, this parameter remained within the limits set by the Interstate Standard. A similar trend was observed for linden honey, except for samples stored for 12 months at 18 °C (average deviation from the standard – 0.9 Gothe units). The diastase of sunflower honey was below the permitted norm in all experimental options, except for storage at 0 °C and 5 °C for 3 months.

It should be noted that as a result of storage at 18 °C, the diastase activity of all samples decreased significantly by the end of the study. Thus, in the 12th month of the experiment, in samples of linden honey it decreased by 42.3%, in samples of buckwheat honey – by 66.4%, in sunflower honey – by 55.8%. Temperature -18 °C also had an unfavorable effect: at the 12th month in linden honey, the diastase number was 11.9±0.96 Gothe units, which is 32.0% less than before storage; in buckwheat – 33.4±0.7 Gothe units (difference from the initial value of 16.9%) and in sunflower – 8.9±0.7 Gothe units (the difference was 46.1%). We noted that minimal changes in this parameter occurred at 0, 5 and 10 °C. I.N. Pasias et al.¹¹ when studying samples of pine, eucalyptus, cotton and thyme honey (Lamia, Greece) showed a decrease in diastase by 33.0-44.4% as a result of storage at room temperature for 12 months and by 5.6-9.1% at 0 °C, as well as no significant changes in this indicator at -18 °C. Due to the fact that the researchers recorded the results once every three months, it is quite difficult to track at what stage the decrease in diastase began to occur. M. Kędzierska-Matysek et al.¹⁰ reported a decrease in diastase activity of rapeseed honey (Brassica napus L.), stored for a year at -20 °C, by more than 7% relative to the control. Thus, there is no data on this parameter that completely coincides with each other. This may be due to differences in the resistance of the enzyme to low temperatures, depending on the botanical origin of the honey.

The D-glucose-1-oxidase activity began to fall in the first month of storage at all temperature conditions just like the diastase number (Fig. 2). The intensity of the decrease in enzyme activity depended on the storage temperature: the maximum was observed in the 12th month at -18 °C: by 64.0, 59.1 and 81.6% (compared to the control) for samples of linden, buckwheat and sunflower honey, respectively. Also, significant changes (by 34.0-76.9% depending on the botanical origin of honey) occurred as a result of storage at -10, -5 and 18 °C. The most suitable storage condition was found to be at temperatures between 5 and 0°C, as the enzyme activity in the 12-month study period decreased by only 14.6%-23.5%.

Analysis of catalase enzyme content in stored honey samples demonstrated its greater resistance to both low and room temperatures compared to the enzymes described above. Despite the fact that, as in the case of diastase and D-glucose-1-oxidase, a gradual decrease in its activity was noted throughout the entire period of research, this process did not occur so intensely at all temperature conditions (Fig. 2). It should be noted that in samples of sunflower honey, characterized by an initially high content of catalase (850±22 mm³ O₂), minimal changes in this indicator were noted: in the 12th month, catalase decreased by 10.0-17.4%, depending on the storage temperature. At the same time, for samples of linden and buckwheat honey in which the initial level of catalase activity was significantly lower (330±14 and 485±19 mm³ O₂, respectively), the activity decreased by 12.7-35.5 % and 11.3-29.5 %, respectively, by the end of the experiment. Overall, it can be noted that regardless of botanical origin, minimal changes in enzyme activity were observed at temperatures of 5 and 0 °C.

The content of H₂O₂ in honey samples was quite stable at temperatures of 5 and 0 °C: in the 12th month, its concentration in linden honey samples decreased by 5.3 and 2.9%, respectively, in buckwheat samples – by 15.5 and 12.2%, respectively, sunflower – by 5.1 and 2.5%, respectively. In samples stored at 18 °C, a significant decrease in H₂O₂ began at the 6th month, which coincides with our previously obtained data. The same trend was observed for samples stored at 10 and -5 °C. At the same time, storage at -10 and -18 °C led to a decrease in this indicator already in the first month (Fig. 2).

It was found that after 12 months of storage at the specified temperatures, the moisture content, total mass fraction of reducing sugars, and free acidity of honey samples did not change significantly (the difference from the control was less than 5%), and they remained within the limits regulated by Interstate Standards. In this regard, Table 3 presents the data obtained at the end of the experiment (after 12 months) as well as the initial values of these parameters (control).

Table 3: Content of moisture, reducing sugars and free acidity in linden, buckwheat and sunflower honey samples stored at different temperatures for 12 months

Note: p < 0.05

The maximum decrease in the moisture content in linden honey samples was 4.9% at a temperature of 18 °C (relative to the control). Buckwheat and sunflower honey had a decrease of 4.5% and 4.8%, respectively, at the same temperature. Minimal deviations from the initial values were observed at 0 °C and negative temperatures. The results obtained are consistent with the data obtained by Kędzierska-Matysek M. et al.¹⁰ Тhe stability of the moisture of samples stored at low temperatures can be explained by the faster transition of water molecules into a bound form.⁴⁰

The same trend was observed in relation to reducing sugars: the smallest amount of them was noted in the analyzed samples stored at 18 °C. Thus, for samples of linden and buckwheat honey the deviation was 3.3%, and for sunflower honey – 1.7%. According to G.P. Ribeiro et al.¹² the content of reducing sugars in honey samples stored at -18 °C for 180 days changed by 4.9%. The results presented in the work of S.N. Esenkina,⁴¹ demonstrated a deviation of this indicator by 4.2% after 3 months of storage at a similar temperature.

Free acidity for 12 months at all temperatures, on the contrary, it increased slightly. The following pattern was identified: the higher the storage temperature, the higher the acidity value. At 18 °C this parameter increased by 4.5% (for linden honey), 3.8% for buckwheat honey, and 2.7% for sunflower honey. F. Braghini et al. noted the same trend when studying honey stored at 22 °C.¹³

The changes that occurred in the content of reducing sugars and free acidity are due to the speed of the dehydration process, as a result of which simpler compounds are formed from monosaccharides, including organic acids, as well as HMF (which explains its stabilization at low temperatures). This is consistent with the findings of Kędzierska-Matysek,¹⁰ who studied rapeseed honey. It showed that when the storage temperature of this product is lowered, chemical reactions proceed much more slowly in it.

Results of antimicrobial analysis

As previously mentioned, according to the Interstate Standard,⁹ honey should be stored at a temperature not exceeding 20 °C. This requirement applies to all types of honey, whether it is monofloral or polyfloral, regardless of botanical origin. There is literature on the antimicrobial activity of honey stored at room temperature and 4 °C, as well as on heat-treated honey. Researchers gave noted that various components contribute to honey’s inhibitory effect of against gram-positive and gram-negative microorganisms, such as sugar content, polyphenolic compounds, acids, 1,2-dicarbonyl compounds, bee defensin-1, and H₂O₂. These elements are present in varying concentrations in honey depending on the source of the nectar and type of bees, so the antimicrobial activity of different types of honey is not uniform.^{4, 42–47} There is evidence of high activity of Manuka honey from the nectar of Leptospermum scoparium flowers. Manuka honey is a source of phenolics and unique chemical compounds, such as methylglyoxal, dihydroxyacetone, leptosperin glyoxal, methylsyringate and leptosin.^{48, 49} Among Russian monofloral honeys, linden honey is considered one of the most highly active, which may be due to its rather high H₂O₂ content.⁵⁰

However, it is not yet clear how the antimicrobial effect of honey is affected by storing it at different temperature conditions for a long time. To investigate this, we conducted the experiment on Escherichia coli (strain 1257), Staphylococcus aureus (strain 209-P) and Bacillus cereus (strain 96, vegetative form). These microorganisms are commonly used as test cultures in microbiological studies and for investigating various biochemical mechanisms. Additionally, E. coli and S. aureus are known as contaminants of the mucous and skin epithelium of humans and animals, therefore, under certain conditions, they can cause inflammatory processes and food poisoning. B. cereus, although a representative of soil bacterium, can also be a cause of toxic infections.^51–54

The studied samples of freshly pumped honey from various botanical origins had a clear inhibitory effect on the growth of all test cultures (Fig. 3).

Figure 3: Inhibition of the growth of E. coli (a), S. aureus (b) and B. cereus (c) by samples of linden, buckwheat and sunflower honey: Click here to view Figure

The antimicrobial activity of linden honey against S. aureus (with an average inhibition zone diameter is 38 mm) was superior to the effect on E. coli (32 mm) and on B. cereus (23 mm). Buckwheat honey also had a greater inhibitory effect on S. aureus (31 mm) than on E. coli (26 mm) and B. cereus (19 mm). Sunflower honey similarly suppressed the growth of Staphylococcus aureus (20 mm) to a greater extent than E. coli (18 mm) and B. cereus (12 mm).

Staphylococcus aureus exhibited the least resistance to honey, regardless of its botanical origin. This is explained by the varying resistance of microorganisms to external factors.

In all honey samples, a decrease in antimicrobial activity was observed after 12 months of storage at different temperatures. The level of this decrease was greatest at -18 °C, followed by -10 °C, -5 °C, 18 °C, 10 °C and 5 °C. This trend was observed for all honey types examined.

Thus, the average degree of decrease in the antimicrobial activity of linden honey (compared to the control) against S. aureus at -18 and 0 °C was 71% and 21%, respectively; for E. coli – 72% and 12.5%; B. cereus – 69.5% and 21.7%. The inhibitory effect of buckwheat honey against S. aureus decreased by 61.3% and 22.6% at -18 and 0 °C, respectively; for E. coli – by 57.7% and 15.4%; B. cereus – by 57.9% and 15.8%. The same trend was noted for sunflower honey: 60.0% at -18 °C and 10.0% at 0 °C for S. aureus; 66.7% and 5.5% for E. coli; 50.0% and 0.0% (not including deviation from the mean) for B. cereus.

To a large extent, these changes were associated with the level of H₂O₂ in honey samples, which is known to be a factor in oxidative stress.^{55, 56} However, the dependence was not direct, which is explained by the influence of other factors on it as well.

Therefore, storage of all types of honey at temperatures outside the range of 0–5 °C resulted in a decrease in H₂O₂ levels, enzymes activity, an increase in HMF, and a reduction in the inhibitory effect on test cultures.

H₂O₂ is synthesized in honey in a reaction with β-D-glucose, catalyzed by the enzyme D-glucose-1-oxidase. This reaction is a two-stage oxidation-reduction process. In the first stage, D-glucose-1-oxidase catalyzes the oxidation of β-D-glucose by transferring two electrons and a proton from the hydroxyl group of β-D-glucose to flavin adenine dinucleotide (FAD). In this case, glucose is oxidized to glucono-1,5-lactone. Then, the resulting glucono-1,5-lactone is hydrolyzed to gluconic acid. In the second stage, the reduced FADH₂ is oxidized by molecular oxygen. Oxygen accepts two electrons from FADH₂, turning into H₂O₂. Excess H₂O₂ is decomposed into water and oxygen by the iron containing enzyme catalase in two stages.^{7, 57} In the first stage, H₂O₂ oxidizes Fe^III to oxyferryl Fe^IVO. In this process, the O–O bond in the H₂O₂ molecule is broken, which ultimately leads to the formation of water and oxygen. In the second stage, the intermediate oxyferryl Fe^IVO is reduced by a second H₂O₂ molecule to the original catalase. Thus, the relationship between the H₂O₂ content and the activity of D-glucose-1-oxidase and catalase is obvious. The decrease in the activity of D-glucose-1-oxidase can be explained by its high temperature sensitivity.⁵⁸ At the same time, catalase is a thermostable enzyme.⁵⁹

HMF is a low-melting cyclic aldehyde. The mechanism of its formation in honey is that as a result of exposure to high temperatures, as well as during long-term storage, one or two water molecules are split off from sugars (dehydration). In this case, anhydrides are formed, which can combine either with each other or with an unchanged sugar molecule and form so-called condensation (reversion) products. Then the third water molecule is split off and HMF is formed. Subsequently, it decomposes with the destruction of the carbohydrate skeleton and the formation of formic and levulinic acids. The most intensive processes described occur when honey is heated.^{60, 61} In our studies, during storage of honey samples at low temperatures, we observed minor changes in sugar content and acidity, as well as no excess of the permissible concentration of HMF.

It should be noted that physicochemical parameters can be used as indicators of honey quality, since their significant change signals inadequate storage conditions for this product. However, honey is a complex biological system, therefore, to obtain more accurate information about possible negative factors, a comprehensive analysis should be carried out taking into account the main criteria. For example, the detection of a high HMF content in combination with a low H₂O₂ concentration and reduced enzymatic activity often indicates heat treatment or adulteration of honey.^62–64

As already mentioned, the inhibitory effect of honey is due to the various compounds present in it. Considering the absence of significant changes in the content of sugars and acidity in the studied honey samples, it can be assumed that H₂O₂ is the leading factor in their antimicrobial activity against E. coli, S. aureus and B. cereus. The effectiveness of H₂O₂ is explained by its strong oxidizing property. H₂O₂ affects several biomolecules within the cell, causing peroxidation and destruction of membrane layers, oxidation of thiol groups, inhibition of enzymes, oxidation of nucleosides, disruption of energy production, disruption of protein synthesis, and ultimately leading to cell death.^65–67

Conclusion

As a result, it can be concluded that the physicochemical and biological analysis of samples of linden, buckwheat, and sunflower honey, which was carried out for 12 months and stored at different temperatures, made it possible to identify certain changes in the physicochemical parameters and biological activity of the studied samples.

A positive result of using negative temperatures (-5, -10 and, in particular, -18 °C) for long-term storage of honey was a slowdown in the rate of chemical processes, which ultimately led to minimal changes in the content of the toxic compound – HMF. However, such temperature storage conditions had a negative impact on the enzymatic activity and H₂O₂ concentration, which, along with the content of sugars and acids, are factors in the antimicrobial activity of honey. Similar changes occurred with samples stored at 18 and 10 °C, in which, in addition, a higher content of HMF was noted. Chemical processes occurred less intensely at 5 and 0 °C. The HMF content in samples stored at these temperatures for 12 months ranged from 7.37 to 11.97 mg/kg, depending on the botanical origin of honey, which is significantly below the MPC.

At the 12th month of storage, all honey samples met the requirements of Interstate Standards for all indicators. The exception was diastase activity in samples of sunflower honey. Nevertheless, a test conducted to study the antimicrobial activity of honey against E. coli, S. aureus and B. cereus demonstrated that samples stored at 5 and 0 °C had the greatest inhibitory effect. Therefore, these temperatures seem to be optimal for storing honey for a long time. The results of this study can be used as additional recommendations when organizing honey storage.

Acknowledgement

The author would like to thank, (Insert university name and Dept. name) for their guidance and support to complete this article.

Funding Sources

This work was carried out within the framework of the Basic Scientific Research Program of the Russian Federation.

Conflict of Interest

There are no conflicts to declare.

Authors’ contribution

The authors contributed equally to the research and the manuscript.

Data availability statement

The manuscript incorporates all datasets produced or examined throughout this research study.

Ethics statement

The document accurately and thoroughly presents the authors’ original research and analysis. 

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